CN111793018B - PHTrp-Cu complex and preparation method thereof, low-molecular-weight hydrogel preparation and preparation method thereof - Google Patents
PHTrp-Cu complex and preparation method thereof, low-molecular-weight hydrogel preparation and preparation method thereof Download PDFInfo
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Abstract
The invention provides a PHTrp-Cu complex and a preparation method thereof, a low-molecular-weight hydrogel preparation and a preparation method thereof, and belongs to the technical field of hydrogels. The PHTrp-Cu complex provided by the invention has a structure shown in a formula I. The PHTrp-Cu complex provided by the invention is added into 3,3' - (octadecyl aza dialkyl) dipropionic acid low molecular weight hydrogel to obtain a low molecular weight hydrogel preparation, the 3,3' - (octadecyl aza dialkyl) dipropionic acid is changed into a fiber network structure from a layered structure, the fiber network structure captures water molecules through capillary action, and the change of the 3,3' - (octadecyl aza dialkyl) dipropionic acid on a microscopic level leads to the increase of the mechanical strength of the C18ADPA hydrogel; and the low molecular weight hydrogel preparation can accelerate the wound healing.
Description
Technical Field
The invention relates to the technical field of hydrogel, and particularly relates to a PHTrp-Cu complex and a preparation method thereof, a low-molecular-weight hydrogel preparation and a preparation method thereof.
Background
The hydrogel has high form resistance and flexibility, and the three-dimensional structure of the hydrogel can inhibit the movement of drug molecules, so that the release rate of the drug is controlled by adjusting the diffusion process, and the hydrogel is frequently used as a controlled release preparation of a drug preparation. Various dosage forms can use hydrogels as sustained release modulators, e.g.
A polyacrylic acid hydrogel for suspension, biological adhesive, and wet granulation of solid agent (see Avinash H Hos)mani,YS Thorat,PV Kasture,et al.Carbopol and its pharmaceutical significance:areview.Pharmaceuticalreviews,4(1),2006)。
Low Molecular Weight Gels (LMWG), also known as molecular gels, are a new class of gels that self-assemble into ordered structures by intermolecular interactions from gels with molecular weights below 3000 Da. The LMWG has similarities with extracellular matrix in structure and viscosity, and is widely applied to the aspects of tissue engineering, enzyme immobilization, cell culture, drug delivery and the like. In terms of drug carriers, LMWG exhibits advantages of controlled release, biocompatibility, and biodegradability. Nilsson et al used Fmoc phenylalanine to deliver diclofenac in vivo for anti-inflammatory effects (Danielle M Raymond, Brittany L Abraham, Takumi Fujita, Matthew J Watorus, Ethan S Toriki, Takahiro Takano, and Bradley L Nilsson.Low-molecular-weight supra hydrogels for stabilized and localized in vivo drive delivery. ACS applied BioMaterials,2(5): 2116. 2124, 2019); cao et al demonstrated that supramolecular phenylalanine-derived hydrogels released salicylic acid in vitro experiments (Shuqin Cao, Xinjian Fu, Ningxia Wang, Hong Wang, and Yajiang Yang. Release catalysts of salicylic acid in Suspolcarboxylic acids for used by l-phenylalkane derivatives as hydrogels. International patent medicines, 357(1-2):95-99,2008); xun et al use disodium pamidronate, Fmoc-Leu and Fmoc-Lys to compose a hydrogel to alleviate UO2 2+The inflammatory response and toxicity of (Zhimou Yang, Keming Xu, Ling Wang, Hongwei Gu, Heng Wei, Mingjie Zhang, and Bing Xu. self-assembly of small molecules, enzymes, chemical communications, (35): 4414-. However, due to the poor mechanical stability of the above-mentioned gel systems, hydrogels are easily separated from drugs when drug delivery is performed in vivo using these hydrogels.
The gelation of the low molecular weight gel is formed by the equilibrium of the interaction between the low molecular weight gel and the supramolecular component. At present, common supramolecular components mainly comprise macrocyclic main bodies such as crown ether, cryptate, cyclodextrin, calixarene and porphyrin, and the like, and some functional small molecules, however, the supramolecular components are added into low-molecular-weight gel to cause poor mechanical stability of the low-molecular-weight gel.
Disclosure of Invention
In view of the above, the present invention provides a PHTrp-Cu complex and a method for preparing the same, and a low molecular weight hydrogel formulation and a method for preparing the same, which can improve the mechanical stability of a hydrogel when added to a low molecular weight hydrogel.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a PHTrp-Cu complex, which has a structure shown in a formula I:
the invention provides a preparation method of the PHTrp-Cu complex, which comprises the following steps:
mixing L-tryptophan, D-glucose, an alkaline reagent and a mixed solvent under a protective atmosphere, and carrying out a substitution reaction to obtain a substitution product;
mixing the substitution product with a reducing agent to carry out reduction reaction to obtain a reduction product;
adjusting the pH value of the reduction product to 1-5 to obtain PHTrp;
and mixing the PHTrp, a divalent copper source, an alkaline reagent and water, and carrying out a complexing reaction to obtain a PHTrp-Cu complex.
Preferably, the molar ratio of the L-tryptophan to the D-glucose is 1 (1-10);
the molar ratio of the L-tryptophan to the alkaline reagent for substitution reaction is 1 (0.5-2.5);
the mixed solvent comprises an organic solvent and water, and the organic solvent comprises methanol, ethanol, propanol or acetonitrile;
the temperature of the substitution reaction is 45-85 ℃, and the time is 4-12 h.
Preferably, the reducing agent comprises one or more of sodium borohydride, lithium aluminum hydride and sodium cyanoborohydride;
the molar ratio of the L-tryptophan to the reducing agent is 1 (1-10);
the temperature of the reduction reaction is 70-120 ℃, and the time is preferably 6-48 h.
Preferably, the molar ratio of the PHTrp to the divalent copper source is 1 (0.1-4);
the temperature of the complex reaction is 50-80 ℃, and the time is 2-20 min.
The invention provides a low molecular weight hydrogel preparation, which comprises a 3,3'- (octadecyl aza dialkyl) dipropionic acid hydrogel and PHTrp-Cu complex loaded on the 3,3' - (octadecyl aza dialkyl) dipropionic acid hydrogel;
the PHTrp-Cu complex is the PHTrp-Cu complex in the technical scheme or the PHTrp-Cu complex obtained by the preparation method in the technical scheme.
Preferably, the pH value of the low-molecular-weight hydrogel preparation is 3-7;
the mass concentration of 3,3' - (octadecyl aza dialkyl) dipropionic acid in the low-molecular-weight hydrogel preparation is 2-10%;
preferably, the mass concentration of the PHTrp-Cu complex in the low-molecular-weight hydrogel preparation is 1-10%.
The invention provides a preparation method of the low-molecular-weight hydrogel preparation in the technical scheme, which comprises the following steps:
mixing 3,3' - (octadecyl aza dialkyl) dipropionic acid, an alkaline reagent, a PHTrp-Cu complex and water, and carrying out self-assembly reaction to obtain the low-molecular-weight hydrogel preparation.
Preferably, the mass ratio of the 3,3' - (octadecyl azadialkyl) dipropionic acid to the PHTrp-Cu complex is 1: 0.05-0.25;
the temperature of the self-assembly reaction is 50-70 ℃, and the time is 10-30 min.
The invention provides a PHTrp-Cu complex which has a structure shown in a formula I. The PHTrp-Cu complex provided by the invention is added into the low molecular weight 3,3'- (octadecyl aza dialkyl) dipropionic acid hydrogel as a supermolecular component, so that the 3,3' - (octadecyl aza dialkyl) dipropionic acid is changed into a fiber net structure from a lamellar structure, and the mechanical property of the low molecular weight hydrogel is improved.
The preparation method of the PHTrp-Cu complex provided by the invention is simple to operate and suitable for industrial production.
The invention provides a low-molecular-weight hydrogel preparation which comprises a 3,3'- (octadecyl azadialkyl) dipropionic acid hydrogel and a PHTrp-Cu complex loaded on the 3,3' - (octadecyl azadialkyl) dipropionic acid hydrogel. In the invention, 3,3' - (octadecyl azadialkyl) dipropionic acid (C18ADPA) has two layered structures formed by bilayer crystals and interlaced alkyl chains, wherein the interlayer spacing is 4.1nm and 3.1nm respectively, the special layered structure endows the C18ADPA hydrogel with high strength, after PHTrp-Cu complex is added, electrostatic interaction exists between the PHTrp-Cu complex and the C18ADPA hydrogel, the interlayer spacing is increased from 4.1nm to 4.3nm, the C18ADPA is changed into a fiber network Structure (SAFiN) from the layered structure, the SAFiN captures water molecules through capillary action, and the change of the C18ADPA on the microscopic level leads to the increase of the mechanical stability of the C18ADPA hydrogel; the low molecular weight hydrogel preparation provided by the invention is similar to biological tissues, has amino acid and sugar, is good in biocompatibility and good in antibacterial effect, and after the low molecular weight hydrogel preparation is used, collagen deposition is early in the regeneration process of skin tissues, and the wound healing speed is high.
The preparation method provided by the invention comprises the following steps: mixing 3,3' - (octadecyl aza dialkyl) dipropionic acid, an alkaline reagent, a PHTrp-Cu complex and water, and carrying out self-assembly reaction to obtain the low-molecular-weight hydrogel preparation. The preparation method of the low-molecular-weight hydrogel preparation provided by the invention is simple to operate and suitable for industrial production.
Drawings
FIG. 1 is a diagram of the transformation process of hydrogels of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1;
FIG. 2 is a graph of frequency strain sweep data for PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1;
FIG. 3 is a DSC of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1;
FIG. 4 is a cryo-SEM image of the PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and the C18ADPA hydrogel prepared in comparative example 1;
FIG. 5 is an L-XRD plot of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1;
FIG. 6 is an FTIR chart of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1;
FIG. 7 is a graph of the process of wound healing in mice;
FIG. 8 is a graph of wound healing rate data for mice;
figure 9 is an omics map of wound healing in mice.
Detailed Description
The invention provides a PHTrp-Cu complex, which has a structure shown in a formula I:
the invention provides a preparation method of the PHTrp-Cu complex, which comprises the following steps:
mixing L-tryptophan, D-glucose, an alkaline reagent and a mixed solvent under a protective atmosphere, and carrying out a substitution reaction to obtain a substitution product;
mixing the substitution product with a reducing agent to carry out reduction reaction to obtain a reduction product;
adjusting the pH value of the reduction product to 1-5 to obtain PHTrp;
and mixing the PHTrp, a divalent copper source, an alkaline reagent and water, and carrying out a complexing reaction to obtain a PHTrp-Cu complex.
In the present invention, all the raw material components are commercially available products well known to those skilled in the art unless otherwise specified.
In the invention, L-tryptophan, D-glucose, an alkaline reagent and a mixed solvent are mixed under a protective atmosphere to carry out substitution reaction, thus obtaining a substitution product.
In the present invention, the molar ratio of L-tryptophan to D-glucose is preferably 1: (1-10), more preferably 1: (2-8), most preferably 1: (5-6).
In the present invention, the alkaline agent preferably comprises NaOH, Na2CO3、NaHCO3、KOH，NaH、K2CO3And KHCO3One or more of NaOH and Na are more preferable2CO3、NaHCO3、KOH、NaH、K2CO3Or KHCO3. In the present invention, the molar ratio of the L-tryptophan and the alkaline agent is preferably 1: (1-10), more preferably 1: (2-8), most preferably 1: (4-6).
In the present invention, the mixed solvent preferably includes an organic solvent and water, and the organic solvent preferably includes methanol, ethanol, propanol or acetonitrile; the volume ratio of the organic solvent to water in the mixed solvent is preferably 1: (1-20), more preferably 1: (1-10), most preferably 1:1, 1:2, 1:3, 1:4 or 1: 5. In the present invention, the ratio of the amount of the substance of the alkaline reagent to the volume of the mixed solvent is preferably (1 to 100) mmol: 3mL, more preferably (5 to 50) mmol: 3mL, most preferably 10 mmol: 3 mL.
In the present invention, the mixing is preferably stirring mixing, and the speed of stirring mixing is not particularly limited in the present invention, and the raw materials can be uniformly mixed; the temperature of the mixing is preferably room temperature. In the present invention, the order of mixing preferably includes first mixing the alkaline agent and the mixed solvent to obtain an alkaline agent solution; adding L-tryptophan into the alkaline reagent solution for second mixing to obtain a mixed solution; adding D-glucose into the mixed solution for third mixing. In the present invention, the time for the first mixing is not particularly limited, and the alkaline agent may be dissolved in the mixed solvent. In the present invention, the time for the second mixing is preferably 10 to 60min, more preferably 20 to 50min, and most preferably 30 to 40 min. In the present invention, the time for the third mixing is not particularly limited, and L-tryptophan may be dissolved in the mixed solution.
In the invention, the temperature of the substitution reaction is preferably 45-85 ℃, more preferably 50-80 ℃, and most preferably 60-70 ℃; the time is preferably 4 to 12 hours, more preferably 6 to 10 hours, and most preferably 8 hours. In the invention, L-tryptophan and D-glucose react under the action of an alkaline reagent to generate Schiff base in the substitution reaction process.
After the substitution reaction, the present invention preferably further comprises cooling the system of the substitution reaction to room temperature to obtain a substitution product. The cooling method of the present invention is not particularly limited, and a cooling method known to those skilled in the art may be used, specifically, natural cooling.
After the substitution reaction, the substitution product is mixed with a reducing agent for reduction reaction to obtain a reduction product.
The present invention preferably adds a reducing agent to the substitution product. In the present invention, the reducing agent preferably includes one or more of sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride and potassium borohydride, and more preferably, sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride or potassium borohydride. In the present invention, the molar ratio of the L-tryptophan and the reducing agent is preferably 1: (1-10), more preferably 1: (2-6), most preferably 1: 3.
In the present invention, the mixing is preferably stirring mixing, and the speed and time of the stirring mixing are not particularly limited in the present invention, and the raw materials may be uniformly mixed.
In the invention, the temperature of the reduction reaction is preferably 70-120 ℃, more preferably 80-110 ℃, and most preferably 90-100 ℃; the time is preferably 6 to 48 hours, more preferably 10 to 40 hours, and most preferably 20 to 30 hours. In the present invention, the Schiff base, which is a substitution product, is reduced to a C-N single bond during the reduction reaction.
In the invention, after the reduction reaction, the method preferably further comprises cooling a system of the reduction reaction to-10-5 ℃ to obtain a reduction product. The cooling method of the present invention is not particularly limited, and a cooling method known to those skilled in the art may be used. In the present invention, the temperature after cooling is more preferably-5 to 2 ℃, and more preferably 0 ℃.
After the reduction reaction, the pH value of the reduction product is adjusted to 1-5 to obtain PHTrp. In the present invention, the pH is further preferably adjusted to 2 to 4, and more preferably 2.5 to 3. The pH regulator used for adjusting the pH value is not particularly limited in the present invention, and can be an acid or a base known to those skilled in the art, such as HCl and KHSO4、NaOH、Na2CO3Or NaHCO3. In the invention, the purpose of adjusting the pH value to 1-5 is to free amino acid carboxyl.
In the invention, after the pH is adjusted to 1-5, the method preferably further comprises the steps of performing solid-liquid separation on the obtained system to remove precipitates, sequentially concentrating the obtained liquid components, adding an organic solvent to dissolve the obtained concentrate, performing secondary solid-liquid separation to remove the precipitates, repeating the steps of performing solid-liquid separation on the obtained liquid components to remove the precipitates, sequentially concentrating the obtained liquid components, adding an organic solvent to dissolve the obtained concentrate, performing secondary solid-liquid separation to remove the precipitates for 5-6 times, and dissolving the finally obtained liquid components in water after concentration to perform acidic ion exchange resin separation to obtain PHTrp. The solid-liquid separation and the secondary solid-liquid separation are not particularly limited in the present invention, and a solid-liquid separation method known to those skilled in the art, such as filtration, may be employed. In the present invention, the concentration is preferably performed by concentration under reduced pressure; in the present invention, the conditions for the concentration under reduced pressure are not particularly limited, and the concentration may be carried out in the form of syrup. In the present invention, the organic solvent preferably includes methanol, ethanol, propanol or acetonitrile. The amount of the water is not particularly limited, and the concentrate can be dissolved; in the examples of the present invention, the ratio of the amount of the substance of L-tryptophan to the volume of water is preferably 1 mmol: 1 mL. In the present invention, the acidic ion exchange resin is preferably a strong-acid cation exchange resin of type 721. In the present invention, the eluent used for the separation of the acidic ion exchange resin is preferably an alkaline solution, and the alkaline solution preferably comprises an aqueous solution of N-methylmorpholine, an aqueous solution of triethylamine, ammonia water, an aqueous solution of ammonium carbonate or an aqueous solution of ethylenediamine; the mass percentage concentration of the alkaline solution is preferably 1-10%, more preferably 2-8%, and most preferably 3-5%.
After obtaining the PHTrp, mixing the PHTrp, a divalent copper source, an alkaline reagent and water, and carrying out a complex reaction to obtain a PHTrp-Cu complex.
In the present invention, the divalent copper source preferably includes one or more of copper chloride, copper sulfate, copper nitrate and copper hydroxide. In the present invention, the mass ratio of the PHTrp to the divalent copper source is preferably 1: (0.1 to 4), more preferably 1: (1-2), most preferably 1: 1.
In the present invention, the alkaline agent preferably comprises NaOH, Na2CO3、NaHCO3、KOH、NaH、K2CO3And KHCO3And more preferably NaOH or KOH. In the present invention, the molar ratio of PHTrp and the alkaline agent is preferably 1: (1 to 4), more preferably 1: (1-2), most preferably 1: 1.
In the present invention, the water is preferably distilled water or deionized water. In the present invention, the ratio of the amount of substance of PHTrp to the volume of water is preferably 1 mmol: (2-20) mL, more preferably 1 mmol: (5-15) mL, most preferably 1 mmol: 10 mL.
In the present invention, the mixing is preferably stirring mixing, and the speed of stirring mixing is not particularly limited in the present invention, and the raw materials can be uniformly mixed; the mixing temperature is preferably 0-10 ℃, and more preferably 5 ℃; the invention can prevent the racemization of the chiral structure of the amino acid by mixing under the temperature condition. In the present invention, the order of mixing is preferably that PHTrp, the divalent copper source and water are fourth mixed, and an alkaline agent is added to the resulting mixed solution for fifth mixing. In the present invention, the time for the fourth mixing is preferably 10 to 30min, more preferably 15 to 25min, and most preferably 20 min. In the present invention, the time for the fifth mixing is not particularly limited, and the alkaline agent may be dissolved in the mixed solution.
In the invention, the temperature of the complexation reaction is preferably 50-80 ℃, more preferably 55-75 ℃, and most preferably 60-70 ℃; the time is preferably 2 to 20min, more preferably 5 to 15min, and most preferably 10 min. In the present invention, during the complexation reaction, the carboxyl group is complexed with Cu ion.
After the complexing reaction, the method preferably further comprises the steps of carrying out solid-liquid separation on a system of the complexing reaction, and purifying the obtained liquid component to obtain the PHTrp-Cu complex. The solid-liquid separation method is not particularly limited, and a solid-liquid separation method known to those skilled in the art, such as filtration, may be employed. In the present invention, the purification is preferably performed by size exclusion chromatography (Sephadex G10). In the present invention, the eluent used for the size-exclusion chromatography purification is preferably pure water.
The invention provides a low molecular weight hydrogel preparation, which comprises a 3,3'- (octadecyl aza dialkyl) dipropionic acid hydrogel and PHTrp-Cu complex loaded on the 3,3' - (octadecyl aza dialkyl) dipropionic acid hydrogel; the PHTrp-Cu complex is the PHTrp-Cu complex in the technical scheme or the PHTrp-Cu complex obtained by the preparation method in the technical scheme.
In the invention, the pH value of the low molecular weight hydrogel preparation is preferably 3-7, more preferably 4-6, and most preferably 5.
In the invention, the concentration of 3,3' - (octadecyl azadialkyl) dipropionic acid in the low molecular weight hydrogel preparation is preferably 2-10%, more preferably 3-8%, and most preferably 4-6%.
In the invention, the mass concentration of the PHTrp-Cu complex in the low molecular weight hydrogel preparation is preferably 1-10%, more preferably 2-8%, and most preferably 3-5%.
The invention provides a preparation method of the low-molecular-weight hydrogel preparation in the technical scheme, which comprises the following steps:
mixing 3,3' - (octadecyl aza dialkyl) dipropionic acid, an alkaline reagent, a PHTrp-Cu complex and water, and carrying out self-assembly reaction to obtain the low-molecular-weight hydrogel preparation.
In the invention, the 3,3' - (octadecyl azadialkyl) dipropionic acid has the structure as follows:
in the invention, the mass ratio of the 3,3' - (octadecyl azadialkyl) dipropionic acid to the PHTrp-Cu complex is preferably 1: 0.05-0.25, more preferably 1: 0.1-0.2, and most preferably 1: 0.15-0.2.
In the present invention, the alkaline agent preferably comprises NaOH, Na2CO3、NaHCO3、KOH、NaH、K2CO3And KHCO3And more preferably NaOH or KOH. In the present invention, the mass ratio of the 3,3' - (octadecyl azadialkyl) dipropionic acid to the basic agent is preferably 1: (0.05 to 0.5), more preferably 1: (0.1 to 0.4), most preferably 1: (0.2-0.3).
In the present invention, the water is preferably distilled water or deionized water. In the present invention, the ratio of the mass of the PHTrp-Cu complex to the volume of water is preferably (2 to 10) g: 1L, more preferably (4-8) g: 1L, most preferably (5-6) g: 1L of the compound.
In the present invention, the mixing is preferably stirring mixing, and the speed and time of the stirring mixing are not particularly limited in the present invention, and the raw materials may be uniformly mixed. In the present invention, the mixing order is preferably that the PHTrp-Cu complex and water are mixed in the sixth order to obtain a PHTrp-Cu aqueous solution; and mixing 3,3' - (octadecyl azadialkyl) dipropionic acid, an alkaline reagent and the PHTrp-Cu aqueous solution for the seventh time. In the present invention, the time for the sixth mixing is not particularly limited, and the PHTrp-Cu complex may be dissolved in water. In the invention, the temperature of the seventh mixing is preferably 50-100 ℃, more preferably 60-90 ℃, and most preferably 70-80 ℃; in the present invention, the seventh mixing time is not particularly limited, and the mixed system may be transparent. In the present invention, the PHTrp-Cu aqueous solution is sufficiently dissolved in the seventh mixing process.
In the present invention, after the mixing, the present invention preferably further comprises mixing the mixtureThe pH value of the system is adjusted to 3-7. In the present invention, the pH is further preferably adjusted to 4 to 6, and more preferably 5. The pH regulator used for adjusting the pH value is not particularly limited in the present invention, and can be an acid or a base known to those skilled in the art, such as HCl and KHSO4、NaOH、Na2CO3、NaHCO3、KOH、NaH、K2CO3Or KHCO3. In the invention, the purpose of adjusting the pH value to 3-7 is to approach neutrality, have low toxicity and facilitate administration.
In the invention, the temperature of the self-assembly reaction is preferably 40-80 ℃, more preferably 50-70 ℃, and most preferably 60 ℃; the time is preferably 10 to 90min, more preferably 30 to 70min, and most preferably 50 to 60 min. In the present invention, a gel having uniform properties and no insoluble particles is formed during the self-assembly reaction.
After the self-assembly reaction, the invention preferably further comprises cooling the system of the self-assembly reaction to room temperature to obtain the low molecular weight hydrogel preparation.
In the present invention, the cooling is preferably still cooling.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
(1) Preparation of N- (2,3,4,5, 6-pentahydroxyhexyl) -L-tryptophan (PHTrp)
Under the protection of argon, 0.40g (10mmol) of sodium hydroxide is dissolved in 3mL of methanol/water solution (the volume ratio of methanol to water is 1:1), 2.04g (10mmol) of L-tryptophan (L-Trp) is added, the mixture is stirred and mixed for 20min at room temperature, 1.80g (10mmol) of D-glucose is added and stirred until the D-glucose is completely dissolved, then the substitution reaction is carried out for 6h at 55 ℃, 1.62g (30mmol) of sodium borohydride is added for reduction reaction for 96h, the obtained reaction liquid is cooled to 0 ℃, the pH value is adjusted to 2.5 by hydrochloric acid, the precipitate is removed by filtration, the filtrate is decompressed and concentrated, the obtained concentrate is dissolved in absolute ethanol, the filter residue is removed by filtration again, the reaction is repeated for 5 times, the obtained concentrate is dissolved in 10mL of water, the concentrate is loaded on an acidic ion exchange resin column and is eluted by 3 wt% of N-methylmorpholine water solution, PHTrp (white powder, 701mg, yield 19%, purity 98%) was obtained.
Structural characterization of PHTrp: the melting point (Mp) of PHTrp is 206-207 ℃; [ alpha ] to]D 20＝+10.0(C＝1.6,H2O)；IR(KBr):3407,3353,3095,2968,2916,1617,1596,1400,1355,1080,1042,742,675,534cm-1；ESI(+)/FT-MS(m/e):369.15620[M+H]+；1H NMR(300MHz,D2O):δ＝7.22(d,J＝7.5Hz,1H),7.20(d,J＝7.5Hz,1H),7.16(t,J＝7.8Hz,1H),7.14(t,J＝7.8Hz,1H),6.92(s,1H),4.16(m,J＝4.8Hz,1H),3.90(t,J＝5.1Hz,1H),3.85(m,J＝5.1Hz,1H),3.83(m,J＝5.1Hz,1H),3.77(m,J＝3.9Hz,1H),3.71(d,J＝5.1Hz,2H),3.27(dd,J1＝3.6Hz,J2＝12.9Hz,1H),3.20(dd,J1＝9.3Hz,J2＝12.9Hz,1H),2.93(d,J＝4.9Hz,2H)。
(2) Preparation of PHTrp-Cu complexes
368mg (1.0mmol) of PHTrp and 170.5mg (1.0mmol) of CuCl are mixed at 5 deg.C2·2H2O and 10mL of water are stirred and mixed for 20min, 40mg (1.0mmol) of NaOH is added and mixed uniformly, complexation reaction is carried out for 10min at the temperature of 60 ℃, then the obtained reaction liquid is filtered, and the obtained filtrate is purified by molecular exclusion chromatography (Sephadex G10) with pure water as eluent, so that PHTrp-Cu complex (502mg, 92% of yield and 95% of purity) is obtained.
Structural characterization of PHTrp: ESI (-)/FT-MS (m/e): 500.01675; [ alpha ] to]D 20＝+18.5(C＝1.0,H2O)；1HNMR(500MHz,D2O)δ＝7.23(d,J＝7.5Hz,1H),7.21(d,J＝7.5Hz,1H),7.18(t,J＝7.5Hz,1H),7.15(t,J＝7.5Hz,1H),6.93(s,1H),4.07(m,1H),3.83(t,J＝5.0Hz,1H),3.75(m,J＝5.0Hz,1H),3.73(m,J＝5.0Hz,1H),3.67(m,J＝3.5Hz,1H),3.61(d,J＝5.0Hz,2H),3.31(dd,J1＝3.5Hz,J2＝12.5Hz,1H),3.21(dd,J1＝9.0Hz,J2＝12.5Hz,1H),2.89(d,J＝5.0Hz,2H)。
Example 2
0.025g of PHTrp-Cu complex prepared in example 1 was dissolved in 5mL of water to obtain a PHTrp-Cu aqueous solution, 0.2g C18ADPA and 0.04g of NaOH were dissolved in the PHTrp-Cu aqueous solution, the solution was heated to 70 ℃ until the solution was clear, the pH was adjusted to 5, the solution was self-assembled at 60 ℃ for 30min, and the solution was allowed to stand and cool to room temperature to obtain a low molecular weight hydrogel formulation (abbreviated as PHTrp-Cu/C18ADPA hydrogel), in which the concentration of C18ADPA was 4g/100mL and the concentration of PHTrp-Cu complex was 0.5g/100 mL.
Comparative example 1
0.2g C18ADPA (0.2g) and 0.04g NaOH were dissolved in 5mL of water, heated to 70 ℃ until the solution became clear, the pH was adjusted to 5, heated at 50 ℃ for 30min, and allowed to stand to cool to room temperature, to give a 3,3' - (octadecyl azadialkyl) dipropionic acid hydrogel (C18ADPA hydrogel) having a C18ADPA content of 4g/100mL in the C18ADPA hydrogel.
Performance testing
(1) The C18ADPA hydrogel prepared in comparative example 1 was translucent at 50 ℃ and cloudy at 25 ℃. The PHTrp-Cu/C18ADPA hydrogel prepared in example 2 is shown in the state of FIG. 1 at different times, wherein the inversion at 12h is an inverted image after standing for 12 h. As can be seen from FIG. 1, PHTrp-Cu/C18ADPA hydrogel shows the same light scattering behavior as copper hydrate blue solution, the PHTrp-Cu/C18ADPA hydrogel is formed very quickly, and the hydrogel can be formed within 30min during standing and cooling at room temperature after self-assembly reaction.
(2) Rheology test
The elastic modulus (G ') and viscous modulus (G') of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1 were measured with a TAdhr1 rheometer (TAinstraction GmbH, Newark) with a plate diameter of 40mm and a default gap value of 1 mm; through the measurement of an oscillation shearing experiment, a frequency scanning test is carried out in a linear viscoelastic region, the frequency range is 0.5-100 rad/s, and the test result is shown in figure 2. As can be seen from FIG. 2, after 0.5G/mL PHTrp-Cu is added to the C18ADPA hydrogel, the storage modulus G 'and the loss modulus G' are improved by nearly 1 order of magnitude, which indicates that the PHTrp-Cu molecules are fused into the C18ADPA hydrogel structure to improve the mechanical strength of the hydrogel.
(3) Differential Scanning Calorimetry (DSC) test
DSC measurements were performed using a TA-dscq2000 (TAinstument GmbH, Newark) instrument, sealing 20mg of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1, respectively, in an aluminum pan, scanning temperature range 15-65 ℃, N2A DSC thermogram was recorded at a heating rate of 2 deg.C/min under ambient conditions, as shown in FIG. 3, in which the solid line is PHTrp-Cu/C18ADPA hydrogel and the dashed line is C18ADPA hydrogel. As can be seen in FIG. 3, the C18ADPA hydrogel showed a temperature of 43.3 deg.C (T)g) A gel-gel transition occurs at a temperature of 48.8 ℃ (T)m) An additional endothermic peak appears; the corresponding peak in PHTrp-Cu/C18ADPA hydrogel is changed to higher temperature, namely 47.6 ℃ (peak 1) and 51.4 ℃ (peak 2), which shows that PHTrp-Cu and C18ADPA hydrogel have interaction and self-assembly; the temperature of PHTrp-Cu/C18ADPA hydrogel was close to 49.5 ℃ of Xerogel xerogel due to melting of the alkyl chains (T)m) (ii) a Except for TgAnd TmIn addition, PHTrp-Cu/C18ADPA hydrogel showed another endothermic peak (peak 3) at 56.6 ℃ due to electrostatic interaction of PHTrp-Cu with the C18ADPA hydrogel structure.
(4) Cryo-scanning electron microscope (cryo-SEM) testing
The morphology of the hydrogel is researched by adopting an ultralow temperature scanning electron microscope high-pressure freezing method, and the damage of ice crystal formation to the hydrogel structure is prevented or reduced to the maximum extent. Placing 5 mu L of PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1 for 12h, placing the PHTrp-Cu/C18ADPA hydrogel and the C18ADPA hydrogel into a low-temperature specimen box, transferring the low-temperature specimen box into a liquid nitrogen tank until the liquid nitrogen is boiled, then transferring the liquid nitrogen tank to vacuum immediately, raising the temperature of a sample to-100 ℃, freeze-drying the surface of the sample for 30min, testing the sample at-146 ℃, 3kV or 5kV acceleration voltage, and imaging by using an analysis mode and a back-scattered electron signal, wherein the test result is shown in figure 4, wherein (a) is C18ADPA hydrogel, and an insertional graph is a C18ADPA hydrogel real graph; (b) the gel is PHTrp-Cu/C18ADPA hydrogel, and the inset picture is a PHTrp-Cu/C18ADPA hydrogel real picture. As can be seen in fig. 4, the three-dimensional structure of the C18ADPA hydrogel and the layered structure that interconnects and surrounds in the liquid in the open space; after the PHTrp-Cu complex is added, the structure of the C18ADPA hydrogel is obviously changed, the complete layered structure is changed into a fiber network Structure (SAFiN), the SAFiN captures water molecules through capillary action, and the change of the C18ADPA hydrogel on the microscopic level after the PHTrp-Cu complex is added leads to the increase of the mechanical strength of the hydrogel.
(5) Low angle X-ray diffraction (L-XRD) testing
XRD measurements were carried out on PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel samples prepared in comparative example 1 by using a Bruker D8 advance diffractometer, wherein the X-ray source was Cu-alpha, the wavelength thereof was 0.15406nm, the diffraction angle range of the samples was 1-10 DEG, and the test results are shown in FIG. 5. As can be seen from FIG. 5, the C18ADPA hydrogel has two lamellar structures ("1" and "2" in the figure), and the d-spacing of the lamellar structure 1 is d1This is equal to the d-spacing observed in the reflection (001) of the lamellar bilayer crystals of octadecyl ammonium crystals, 4.1nm, and thus, like the octadecyl amine crystals, layered structure 1 is a bilayer crystal composed of polar and non-polar; d-spacing d of the layered structure 22A peak was observed at a position very close to the d-spacing (d 3.6nm) of the xenogel xerogel layer structure at 3.1nm, indicating the presence of a lamellar structure formed by alkyl chains interlaced with each other. The PHTrp-Cu/C18ADPA hydrogel still keeps a layered structure as a structural unit of SAFiN, and is similar to the C18ADPA hydrogel, two layered structures can be observed, but the d spacing of the two layered structures is increased, the layered structure 1 is compactly arranged, and the d-spacing is 4.3 nm; the alkyl chains are staggered with each other to form a layered structure 2, and the d-spacing is 3.1 nm. The L-XRD result shows that although the morphology of the hydrogel is changed after PHTrp-Cu is added, the basic construction structure is still a sheet structure from the microscopic view, the two layer structures are very consistent with the DSC analysis result, and the layer structure 1 is consistent with the layer structure 1 because the two layer structures are formed by bilayer crystals and mutually staggered alkyl chainsTmCorresponding to the absorption peak of (2), layer structure TgThe endothermic peaks correspond.
(6) Fourier transform Infrared Spectroscopy (FTIR) testing
FTIR measurements were performed on PHTrp-Cu/C18ADPA hydrogel prepared in example 2 and C18ADPA hydrogel prepared in comparative example 1 by using Thermo fisher Nicole 6700FTIR spectrometer, samples were prepared by KBr tablet pressing method, and the wave number scanning range was 4000-700 cm-1The test results are shown in fig. 6, where S represents symmetry and AS represents asymmetry. As can be seen from FIG. 6, the FTIR spectrum is divided into two parts, and the infrared fingerprint scanning range is 2200-700 cm-1To better identify all peaks, a smaller transmittance is used; functional area (4000-2200 cm)-1): the-OH stretching vibration absorption of sugar in PHTrp-Cu/C18ADPA hydrogel is 3267cm-1Here, the peak is relatively narrow, indicating that-OH is in a non-polar environment. C18ADPA is a zwitterionic surfactant that reacts at the head group, acidic (-COOH) and weakly basic (tertiary) groups to form complex salts, and in C18ADPA hydrogels, carboxylic acids: amine 2:1 the spectral band of the complex indicates that the complex was formed; the formation of the 2:1 complex indicates that the reaction occurred. For the C18ADPA hydrogel, the characteristic peak is 1733cm-1Peak for ν (C ═ O), 1183cm-1At v (C-O) peak at 1391cm-1Is at vs(CO2 -) Peak at 1620cm-1Is at vAs(CO2 -). After addition of PHTrp-Cu complex, 3 changes were observed: (1) at 1733cm-1The v (C ═ O) peak intensity is obviously reduced, and (2) vAs(CO2-) peak shift to 1592cm-1(3) at 1120cm-1A v (C-O) peak appears; and 1183cm-1The peak intensity of v (C-O) of (A) is relatively small. Based on the above results, it is assumed that the interaction between the C18ADPA hydrogel head groups forms an amine: the carboxylic acid number ratio was 1:2 complex, which became a 1:1 complex upon addition of PHTrp-Cu. The compactness of the arrangement of the three groups (amino, carboxylic and hydroxyl) in the 2:1 complex indicates that the effective area of the groups is smaller compared to the groups in the 1:1 complex, which promotes the higher adoption of the gel system after the addition of PHTrp-CuSelf-assembly of the curvature of (a) to form a SAFiN structure.
(7) Animal experiments
Healthy male ICR mice for experiments are purchased from the animal center of Beijing university, the mice are covered by iron wire cages, sterilized wood chips are used as padding, the feeding temperature is 20-25 ℃, the mice are freely drunk and drunk, 20% of urethane solution is injected into the abdominal cavity to anaesthetize the mice, the weight of each mouse is about 20g, the volume of the anesthetic is 0.14mL, hairs on the backs of the mice are removed by an electric hair remover, the back skins are lifted by an index finger and a thumb, and the mice are folded at the midline to form a sandwich-shaped skin fold. The animals were placed in lateral decubitus position and a biopsy drill of 5mm diameter was pressed to remove the superficial skin completely and form a symmetrical wound. The wounds were evaluated and photographed daily until the lesions were completely closed. All procedures were approved by the ethical committee of the university of capital medical sciences, which warrants that animal welfare was in accordance with the requirements of the animal welfare act and the guidelines of the national institutes of health in the united states for the care and use of laboratory animals.
The mice were randomly divided into four groups of not less than six mice each, group I (control group) being a group that did not receive any treatment; group II (matrix group) was treated with the C18ADPA hydrogel prepared in comparative example 1; group III (hydrogel group) was treated with PHTrp-Cu/C18ADPA hydrogel prepared in example 2; group IV (Vaseline group) was treated with Vaseline loaded with PHTrp-Cu complex at the same concentration as in example 2.
Percent wound healing (PWC) was calculated as percent healing of the original wound size, and wound area was calculated using vernier caliper measurements (see Tzu-Wei Wang, Jui-Sheng Sun, Hsi-Chin Wu, Yang-Hwei Tsuang, Wen-Hsi Wang, and Feng-Huei Lin. the effeffect gelatin-collagen sulfate-hyaluronic acid skin collagen binding peptides, biomaterials,27(33):5689-0) And day n wound area (A)n) And (4) calculating. The formula for PWC is calculated as shown in equation 1:
PWC＝(A0-An)/A0x 100% (formula 1).
Animal experiments followed the randomized, double-blind principle, with data expressed as mean ± standard deviation. According to the results of preliminary experiments, the number of animals in each group is determined to be 12, and each animal is coated with 1.5mL of medicine at the wound every day. Statistical methods for all wound healing experimental data were performed using student's t-test. Differences were considered statistically significant when p < 0.05. The process of wound healing on days 0, 2, 5 and 8 after surgery is shown in figure 7, with the wound diameter on the day of surgery being 5 mm. A graph of wound healing rate data for days 0, 2,3, 5 and 7 post-operative is shown in fig. 8.
As can be seen from FIG. 7, the low molecular weight hydrogel formulation prepared in example 2 can accelerate wound repair, and experimental animals in groups III and IV were treated with hydrogel containing PHTrp-Cu complex (57. + -.2 mg) and vaseline paste (1 time per day) at the same dose, respectively. The number of mice, the dose, the wound area and the duration of the experiment were determined from the results of the preliminary experiment. The wound area is observed to begin to shrink on the 1 st day after the operation, most of wound surfaces are closed on the 7 th to 8 th days, and the remaining area is small and difficult to accurately measure; since the environment and the pharmaceutical formulation are not sterile, a small area of redness was observed on day 2 after surgery.
As can be seen from fig. 8, the wound area of the hydrogel group was significantly smaller than that of the other groups from day 2, and the wounds of the gel group and the vaseline group healed faster than those of the other groups, and on day 8, the wound areas of the two groups were reduced by 80 to 88%, and the healing sequence was hydrogel group > vaseline group > matrix group > control group. Results were statistically different for each group by ANOVA analysis.
Histological study: skin tissues on day 2 and day 8 were collected, tissue samples were fixed with 10% formalin solution, dehydrated with 50%, 75%, 90% and 100% alcohol solution in this order, paraffin-embedded after xylene washing, and then hematoxylin-eosin (H & E) staining (H & E) was performed on the tissues (see Raija Tammi, san a Pasonen-Seppanen, Elina kolehmain, and Markku Tammi. hyaluren synthesis and hyaluren acquisition in mouse peptides from down walking and skin healing in mouse wounds, 124(5) (fig. 8, 905,2005), and the results of histological analysis of skin tissues at mouse wounds on day 2 and day 8 after surgery were shown in fig. 9.
As can be seen from fig. 9, in the data of day 2, edema and increased interstitial space were observed in the dermis of group I animals and in the position near the wound, the skin margin was healthy, the wound was deep into the dermis, the surface layer of the wound had a thin outer shell, and the collagen fibers were visible on day 2; group II was still characterized by edema, but the degree of edema was lower than group I, scabbing was seen in the upper epidermal layer, and edema and fibrin bundles were evident; in group III, edema and fiber disappeared significantly, more mononuclear cells were observed in the interstitial layer and dermis, and the wound margins and adjacent healthy skin were clearly visible. Group IV wounds scab extensively, with clear borders, and the wounds extended to the surface of the dermal layer, with a significant increase in hair follicles compared to the other groups. The data of 8 days can show that the wounds of the group I are widely scabbed, the boundary is clear, the collagen fibers are very thick, irregular fiber bundles are easily formed, and the scab phenomenon can be seen everywhere; group II wounds have a lower number of collagen fibers and the wound appears to be substantially healed, but unlike adjacent undamaged skin, inflammation remains the most prominent feature, but edema is less, and fibroblasts and neovascularization are evident; the group III wounds almost completely heal, a keratinocyte layer, fibroblasts and endothelial cells form an inseparable part of a repair tissue, and a collagenous fiber tissue at the wounds is recovered; for group IV, the wound had contracted and the dermal tissue had recovered as compared to day 2, and the dermal collagen density at the wound site was lower as compared to the adjacent healthy dermis, and the healthy tissue was more visibly purplish red in color due to the aligned collagen bundles.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A PHTrp-Cu complex having the structure shown in formula I:
2. the method of preparing PHTrp-Cu complex as set forth in claim 1, comprising the steps of:
mixing L-tryptophan, D-glucose, an alkaline reagent and a mixed solvent under a protective atmosphere, and carrying out a substitution reaction to obtain a substitution product;
mixing the substitution product with a reducing agent to carry out reduction reaction to obtain a reduction product;
adjusting the pH value of the reduction product to 1-5 to obtain PHTrp;
and mixing the PHTrp, a divalent copper source, an alkaline reagent and water, and carrying out a complexing reaction to obtain a PHTrp-Cu complex.
3. The method according to claim 2, wherein the molar ratio of L-tryptophan to D-glucose is 1 (1 to 10);
the molar ratio of the L-tryptophan to the alkaline reagent for substitution reaction is 1 (0.5-2.5);
the mixed solvent is an organic solvent and water, and the organic solvent is methanol, ethanol, propanol or acetonitrile;
the temperature of the substitution reaction is 45-85 ℃, and the time is 4-12 h.
4. The preparation method according to claim 2, wherein the reducing agent is one or more of sodium borohydride, lithium aluminum hydride and sodium cyanoborohydride;
the molar ratio of the L-tryptophan to the reducing agent is 1 (1-10);
the temperature of the reduction reaction is 70-120 ℃, and the time is 6-48 h.
5. The preparation method according to claim 2, wherein the molar ratio of PHTrp to the divalent copper source is 1 (0.1-4);
the temperature of the complex reaction is 50-80 ℃, and the time is 2-20 min.
6. A low molecular weight hydrogel formulation comprising a 3,3'- (octadecyl azadialkyl) dipropionic acid hydrogel and a PHTrp-Cu complex supported on said 3,3' - (octadecyl azadialkyl) dipropionic acid hydrogel;
the PHTrp-Cu complex is the PHTrp-Cu complex according to claim 1.
7. The low molecular weight hydrogel formulation of claim 6, wherein the low molecular weight hydrogel formulation has a pH of 3 to 7;
the mass concentration of 3,3' - (octadecyl aza dialkyl) dipropionic acid in the low-molecular-weight hydrogel preparation is 2-10%.
8. The low molecular weight hydrogel formulation of claim 6 or 7, wherein the mass concentration of the PHTrp-Cu complex in the low molecular weight hydrogel formulation is 1-10%.
9. A process for the preparation of a low molecular weight hydrogel formulation as claimed in any of claims 6 to 8, comprising the steps of:
mixing 3,3' - (octadecyl aza dialkyl) dipropionic acid, an alkaline reagent, a PHTrp-Cu complex and water, and carrying out self-assembly reaction to obtain the low-molecular-weight hydrogel preparation.
10. The preparation method according to claim 9, wherein the mass ratio of the 3,3' - (octadecyl azadialkyl) dipropionic acid to the PHTrp-Cu complex is 1: 0.05-0.25;
the temperature of the self-assembly reaction is 50-70 ℃, and the time is 10-30 min.
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