KR102641530B1 - Hyaluronic acid hydrogel based on supramolecular chemistry and covalent bond - Google Patents

Abstract

The present invention provides an HA-dopamine-PEG conjugate in which dopamine and PEG are bound to hyaluronic acid (HA); cyclodextrin; and a hydrogel composition containing a cation of a pharmaceutically usable salt, and a drug delivery system such as a subcutaneous injection containing the same.
The hyaluronic acid hydrogel composition of the present invention and drug carriers such as subcutaneous injections containing the same have immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, sustained release characteristics of the drug, and slow release after subcutaneous injection. It exhibits biodegradable properties and can be injected into subcutaneous tissue with minimal invasiveness in the fields of medicine, pharmaceuticals, and skin procedures, and can be useful as a formulation that can improve patient compliance by reducing the frequency of administration through sustained drug release.

Description

Hyaluronic acid hydrogel based on supramolecular chemistry and covalent bond}

The present invention relates to a hyaluronic acid hydrogel based on supramolecular chemistry and covalent bonding, and more specifically, to an HA-dopamine-PEG conjugate in which dopamine and PEG are bound to hyaluronic acid (HA); cyclodextrin; and a hydrogel composition containing a cation of a pharmaceutically usable salt, and a drug delivery vehicle containing the same.

Hydrogels are defined as three-dimensional (3D) networks composed of polymers capable of absorbing water, and hydrogels can be used to prepare various types of dosage forms (e.g., films, microparticles, and nanoparticles). Hydrogels enable the loading of hydrophilic drugs due to their high water content. Cross-linked polymer networks can modulate the rheological and mechanical properties of hydrogels and thus contribute to the controlled release of drugs (i.e., small molecule compounds, nucleic acids, peptides, proteins, and cells) with diverse molecular weight ranges. Several physical and chemical cross-linking strategies have been introduced to prevent the rapid degradation of the hydrophilic polymer chains in the hydrogel and to control the immediate drug release from the hydrogel. Chemical crosslinking methods (i.e., addition reactions, condensation reactions, enzymatic reactions, and radical polymerization) and physical crosslinking methods (i.e., hydrogen bonding, ionic interactions, and protein interactions) are each known to have their own unique advantages and disadvantages.

Among the various materials used as hydrogel matrices, hyaluronic acid (HA) (mostly composed of D-glucuronic acid and N -acetyl -D-glucosamine) Due to the biocompatibility of silver hydrogel systems, they are widely used as biodegradable matrices. HA is one of the components of epithelial and connective tissues and a major component of the extracellular matrix (ECM). HA can be decomposed by hyaluronic acid degrading enzyme and can be decomposed in the liver and lymphatic vessels in the body. In addition to the widespread application of HA in drug delivery systems, it is also used commercially as dermal filler and wound dressing due to its biocompatibility and biodegradability.

International open document WO2020/032056 (2020.02.13.)

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The problem to be solved by the present invention is to provide a hydrogel system that can prevent rapid decomposition of the hydrophilic polymer chain in the hydrogel and more efficiently control immediate drug release from the hydrogel in the controlled release of drugs with various molecular weight ranges. is to provide.

The problem to be solved by the present invention is not limited to the problems mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above problem, according to one aspect of the present invention, an HA-dopamine-PEG conjugate in which dopamine and PEG are bound to hyaluronic acid (HA); cyclodextrin; and a cation of a pharmaceutically usable salt, and a hydrogel composition having a pH of 7 to 11 is provided.

In one embodiment, the hyaluronic acid may have an average molecular weight of 10 to 3000 kDa.

In one embodiment, the PEG may have a molecular weight of 100 to 100000 Da.

In one embodiment, the cation of the pharmaceutically usable salt may be one cation selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, barium ions, aluminum ions, and mixtures thereof, and is preferably Examples include sodium hydroxide (NaOH), potassium phosphate monobasic (KH ₂ PO ₄ ), potassium phosphate dibasic (K ₂ HPO ₄ ), potassium chloride (KCl), sodium phosphate monobasic (NaH ₂ PO ₄ ), sodium phosphate dibasic. It may be a cation of one type of salt selected from the group consisting of (Na ₂ HPO ₄ ), sodium chloride (NaCl), and mixtures thereof.

In one embodiment, the hydrogel composition may further include a poorly soluble drug, and the poorly soluble drug includes donepezil, amphotericin B, curcumin, phloretin, paclitaxel, docetaxel, dexamethasone, budesonide, and cyclo It may be one selected from the group consisting of sporin, tacrolimus, rapamycin, fenofibrate, prednisolone, itraconazole, insulin (neutral pH), indomethacin, and mixtures thereof, and the poorly soluble drug is encapsulated in microspheres. It may be.

According to another aspect of the present invention, a drug delivery system comprising the hydrogel composition is provided.

According to another aspect of the present invention, an injection for subcutaneous injection containing the hydrogel composition is provided.

According to another aspect of the present invention, a filler comprising the hydrogel composition is provided.

According to another aspect of the present invention, a dressing containing the hydrogel composition is provided.

According to another aspect of the present invention, combining dopamine and PEG with hyaluronic acid (HA) to obtain an HA-dopamine-PEG conjugate; and mixing the HA-dopamine-PEG conjugate and cyclodextrin and then adjusting the pH to 7 to 11 by mixing a pharmaceutically usable salt.

In one embodiment, the HA-dopamine-PEG conjugate comprises a carboxylic acid group of hyaluronic acid and an amine group of dopamine; and an amide bond formed between the carboxylic acid group of hyaluronic acid and the amine group of PEG.

In one embodiment, the method for producing the hydrogel composition may further include mixing the HA-dopamine-PEG conjugate and cyclodextrin and then mixing a poorly soluble drug.

According to the present invention, an HA-dopamine-PEG conjugate in which dopamine and PEG are bound to hyaluronic acid (HA); cyclodextrin; and a cation of a pharmaceutically usable salt. The hydrogel composition exhibits immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, sustained release characteristics of the drug, and slow biodegradation characteristics after subcutaneous injection, so that the hydrogel composition It has been found that a sustained-release drug formulation (subcutaneous injection formulation, etc.) can be developed that can more efficiently control immediate drug release from the gel.

Therefore, the hyaluronic acid hydrogel composition of the present invention and drug carriers such as subcutaneous injections containing the same can be injected into subcutaneous tissue with minimal invasiveness in the fields of medicine, pharmaceuticals, and skin procedures, and the frequency of administration by sustained drug release It can be usefully used as a formulation that can improve patient compliance by reducing .

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 shows the results of the synthesis and properties of HDP[HA-dopamine-PEG], and (A) is a schematic diagram of the process of synthesizing HDP from HA. (B) H-NMR spectrum of HDP, in which the proton NMR chemical shifts for the N -acetyl group of HA (shift ¹ ), dopamine (shifts 2-4), and methylene groups of PEG (shifts 5 and 6) are shown in the spectrum. It is displayed. (C) Linear regression line between weight ratio (mPEG2K-NH ₂ /HA) and integral ratio (3.6-3.7/1.8-2.0 ppm). (D) ¹³ C-NMR spectra of HD and HDP showing the carbon NMR chemical shifts for the N -acetyl group of HA (shift 1), dopamine (shifts 2-4), and methylene group of PEG (shifts 5 and 6). It appears. (E) FT-IR spectra of HD, mPEG2K-NH ₂ and HDP.
Figure 2 shows particle characteristics of PDM, (A) FE-SEM image and (B) particle size distribution of PDM.
Figure 3 shows results explaining the gel cross-linking mechanism, (A) ¹ H-NMR spectra of aCD and HDP/aCD. (B) 2D NOESY (NMR nuclear Overhauser effect spectroscopy) result of HDP/aCD. (C) UV-Vis absorbance results of HDP and HDP 8.5 groups. (D) XRD profiles of aCD, HDP, and HDP/aCD samples.
Figure 4 shows the rheological properties of the hydrogel systems: (A) HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5, and HDP As a result of the inversion test of the /PDM 8.5 group, the gelation behavior was tested at 0 hours (normal position) and 6 hours (inversion position). (B) Gelation behavior of HDP/aCD/PDM 8.5 hydrogel according to incubation time. An inversion test was performed for 180 minutes to measure the gelation time. (C) Results of injectability test of HDP/aCD/PDM 8.5 hydrogel (containing 0.2 mg/mL methylene blue) through a syringe needle. (D) Rheology results for HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5, and HDP/aCD/PDM 8.5 samples, strain sweep, frequency sweep, step time -Viscoelasticity and shear rate-shear stress results were measured.
Figure 5 shows the physicochemical properties of the cross-linked hydrogel system, (A) FE-SEM images of the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups, and the cross-sectional image of the freeze-dried sample was observed, and the scale bar ( white) is 20 μm in length. (B) Release profile of the drug in donepezil solution and HDP/aCD/PDM 8.5 hydrogel group, with each measurement expressed as mean ± SD (n = 3). (C) In vitro degradation results of HDP/aCD/PDM 8.5 hydrogel in the presence or absence of HAase, with each measurement expressed as mean ± standard deviation (n = 4).
Figure 6 shows the in vivo degradation characteristics in mice of the designed hydrogel systems, (A) control, PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5. This is a photo of an incision made at the gel injection site in the military. (B) This is a picture of a gel excised from the HDP/aCD 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5 groups. (C) Residual gel weight of HDP/aCD 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5 groups, and each measurement value is expressed as mean ± standard deviation (n = 5).

The present invention provides an HA-dopamine-PEG conjugate in which dopamine and PEG are bound to hyaluronic acid (HA); cyclodextrin; and a cation of a pharmaceutically usable salt, and provides a hydrogel composition having a pH of 7 to 11.

In the present invention, various chemical and physical crosslinking approaches were attempted to optimize the rheological and mechanical properties of HA-based hydrogels. Chemical reactions (i.e., click chemistry, disulfide bonds, enzymatic cross-linking, and esterification) and physical interactions (i.e., host-guest interactions and hydrophobic interactions) tune the physicochemical and mechanical properties of the hydrogel. It is being used. In previous studies (Lee et al., 2020; Seo et al., 2020; Yang et al., 2020), HA-dopamine conjugates and mineral or buffer salts (e.g. FeSO ₄ , KH ₂ PO ₄ and Na ₂ SeO ₃ ) Coordination and catechol polymerization were introduced as the main cross-linking mechanisms in the HA hydrogel system. The common goals of these studies are control of gelation for subcutaneous or intratumoral injection, injectability through a single syringe, low biodegradation rate, self-healing ability, and sustained drug release. In the present invention, in order to ensure safety in the use of injections, the use of cross-linking agents that exhibit high toxicity was excluded. Instead, physiologically essential minerals and buffer salts were added as reactants of HA derivatives to crosslink the hydrogel system.

Additionally, in the present invention, supramolecular chemistry (physical interaction) was used to control the rheological properties of HA-based hydrogel. In the field of supramolecular chemistry, cyclodextrins (CDs) are widely used as host moieties due to their molecular recognition properties, which allow other guest molecules to bind to form inclusion complexes. Additionally, instead of incorporating small hydrophobic molecules into the internal cavity of the CD, the “molecular necklace” concept is based on linear polymer chains penetrating the intramolecular cavity of the CD, such as alpha-CD (aCD)-polyethylene glycol (PEG). )) was developed to form a polyrotaxane structure. In another study, in addition to a polyrotaxane system in which the terminal linear chains can be restricted by large plug molecules, a polypseudorotaxane structure that can easily debond under certain conditions was also designed for hydrogel crosslinking. In addition to using linear polymers as guest molecules, branched or grafted polymers are also introduced as counterparts of aCD for polypseudorotaxane hydrogel structures. In particular, as a host-guest inclusion complex assembly structure, alginate, chitosan, dextran, and heparin grafted with a PEG chain penetrating the cavity within the CD molecule have been produced.

In the present invention, PEG (in the form linked to the HA-dopamine-PEG (HDP) polymer) and dopamine (in the form attached to the HDP conjugate) participate in the formation of polypseudorotaxane and catechol polymerization, respectively, forming a HA hydrogel crosslink. provides. Although hydrogel structures based on HA-PEG and aCD have been reported previously, the present invention is the first to introduce polydopamine linkage for chemical crosslinking of HDP hydrogel along with aCD-PEG interaction. By this gel cross-linking strategy, the viscoelasticity of the designed HDP-based hydrogel was controlled to achieve immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, and slow biodegradation after subcutaneous injection, which will be explained later. In addition, donepezil-encapsulated microspheres were incorporated into the cross-linked HA hydrogel to reduce the frequency of administration.

The hyaluronic acid (HA)-dopamine-PEG conjugate contained in the hydrogel composition of the present invention is formed by combining dopamine and PEG with hyaluronic acid (HA), and the hyaluronic acid used herein is commonly used in the technical field to which the present invention pertains. Hyaluronic acid can be used, preferably having an average molecular weight of 10 to 3000 kDa, more preferably hyaluronic acid having a low molecular weight grade of 100 to 1000 kDa.

Dopamine used in the present invention has the chemical structure of the following formula (1), and in the chemical structure, the amine group forms a conjugate by amide bonding with the carboxylic acid group of hyaluronic acid.

<Formula 1>

Polyethylene glycol used in the present invention is a polyether compound produced by polycondensation of ethylene glycol and is usually expressed as H-[O-CH ₂ -CH ₂ ] _n -OH, PEG, poly It is also referred to as ethyl oxide (polyethylene oxide, PEO), polyoxyethylene (POE), etc. Polyethylene glycol (PEG) referred to in the present invention includes polyethylene glycol and polyethylene glycol derivatives whose both ends are partially modified, for example, methoxy polyethylene glycol amine (mPEG2K-NH ₂ ), etc. It can be included. The PEG may have a molecular weight of 100 to 100,000 Da, preferably 500 to 20,000 Da, and more preferably 1,000 to 10,000 Da. The PEG may be included in an amount of 10 to 70% by weight, preferably 20 to 60% by weight, based on the total amount of the hyaluronic acid (HA)-dopamine-PEG conjugate.

Cyclodextrin used in the present invention is cycloamylose or cycloglucan that is formed as cyclic dextrin upon starch decomposition, and forms one cyclus of 6, 7, or 8 α-1,4 linked glucose units. Therefore, they are referred to as α-, β-, or γ-cyclodextrins. These are bound within a crystal lattice to form penetrating internal molecular channels within which hydrophobic guest molecules, such as alcohols or hydrocarbons, can be deposited in variable amounts up to saturation. The cyclodextrin may be included in the hydrogel composition at a ratio of 0.5 to 50 times the weight of the hyaluronic acid (HA)-dopamine-PEG conjugate, and preferably at a ratio of 1 to 10 times.

In this specification, “pharmaceutically usable salt” refers to a salt that can be commonly used in pharmaceuticals in the technical field to which the present invention pertains, and is preferably a salt that can be used in formulations applied to the human body, such as subcutaneous injection. For example, alkali metal salts such as sodium salt, potassium salt, etc.; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts, etc.; Aluminum salts, etc. may be included, but are not limited thereto. Specific examples of the pharmaceutically usable salt include sodium hydroxide (NaOH), potassium phosphate monobasic (KH ₂ PO ₄ ), potassium phosphate dibasic (K ₂ HPO ₄ ), potassium chloride (KCl), sodium phosphate monobasic (NaH ₂ PO). ₄ ), dibasic sodium phosphate (Na ₂ HPO ₄ ), sodium chloride (NaCl), and mixtures thereof may be included, but are not limited thereto. The cation of the pharmaceutically usable salt is a cation selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, barium ions, aluminum ions, and mixtures thereof.

When a pharmaceutically usable salt as described above is used in a hydrogel composition at a concentration that exhibits a sol-gel transition, the hydrogel composition exhibits the characteristic of pH 7 to 11, preferably pH 7.5 to 10, and more preferably pH 7.5 to 10. shows the characteristic of pH 8 to 9.

In one embodiment, the hydrogel composition may further include a poorly soluble drug, and the poorly soluble drug refers to a drug with relatively low water solubility. For example, the poorly soluble drugs include donepezil, amphotericin B, curcumin, phloretin, paclitaxel, docetaxel, dexamethasone, budesonide, cyclosporine, tacrolimus, rapamycin, fenofibrate, prednisolone, itraconazole, and insulin (neutral pH ), indomethacin, and mixtures thereof, but is not limited thereto.

In one embodiment, the poorly soluble drug may be encapsulated in microspheres. The microspheres can be manufactured using commonly used emulsification and solvent evaporation methods, which are summarized as follows. An emulsion is obtained by dissolving poly( lactic-co-glycolic acid) (PLGA) and a poorly soluble drug in an organic solvent and then adding it to an aqueous polyvinyl alcohol solution and homogenizing it. The PLGA used at this time may be PLGA commonly used in the technical field to which the present invention pertains, and is preferably lactic acid:glycolic acid (10:90 to 90:10), more preferably lactic acid:glycolic acid (40:10). 60 ~ 60:40) and may have an average molecular weight of 10 ~ 100 kDa, but is not limited thereto. The obtained emulsion is stirred to evaporate the organic phase, and then the microspheres are separated using an appropriate separation method (e.g., centrifugation, etc.).

Microspheres prepared by the above method may have an average bulk density-related diameter of 1 to 100 μm, preferably 5 to 50 μm, but are not limited thereto.

The present invention also provides a drug delivery system comprising the hydrogel composition. The hydrogel composition of the present invention modulates the viscoelasticity of the HDP-based hydrogel by the gel cross-linking strategy as described above, thereby providing properties such as immediate gelation, injectability through a single syringe, self-healing ability, shear thinning behavior, and slow biodegradation after subcutaneous injection. characteristics can be achieved. Therefore, depending on the drug properties, the drug-microsphere encapsulated in the microsphere can be appropriately designed to be incorporated into the HA-dopamine-PEG (HDP) polymer, so it can be used as a drug carrier and subcutaneous injection with controlled properties. there is.

In addition, the hydrogel composition of the present invention can be designed using the viscoelasticity control properties described above, and therefore can be used as a filler or dressing agent containing the hydrogel composition.

The present invention also includes the steps of combining dopamine and PEG with hyaluronic acid (HA) to obtain an HA-dopamine-PEG conjugate; and mixing the HA-dopamine-PEG conjugate and cyclodextrin and then mixing them with a pharmaceutically usable salt to adjust the pH to 7 to 11.

The method for producing the hydrogel composition of the present invention includes the step of combining dopamine and PEG with hyaluronic acid (HA) to obtain an HA-dopamine-PEG conjugate. In this step, the carboxylic acid group of hyaluronic acid and the amine group of dopamine; And the HA-dopamine-PEG conjugate can be prepared by forming an amide bond between the carboxylic acid group of hyaluronic acid and the amine group of PEG. When amide bonding occurs between hyaluronic acid and dopamine, the amide bonding reaction can be performed using EDC-NHS coupling. To summarize the manufacturing process, hyaluronic acid is dispersed in water, and then the organic solvent solution of EDC and NHS is combined with the hyaluronic acid. The dispersion is mixed to activate the carboxylic acid groups of hyaluronic acid. After acidifying the pH of the mixed solution, a hyaluronic acid-dopamine conjugate can be obtained by adding and reacting with a dopamine solution. In the case of amide bonding between hyaluronic acid and PEG, the hyaluronic acid-dopamine conjugate is dispersed in water, and then the organic solvent solution of EDC and NHS is mixed with the hyaluronic acid-dopamine conjugate dispersion to activate the carboxylic acid group of hyaluronic acid. HA-dopamine-PEG conjugate can be obtained by acidifying the pH of the mixed solution and then adding and reacting with a PEG solution containing an amine group.

The method for producing a hydrogel composition of the present invention includes mixing the HA-dopamine-PEG conjugate and cyclodextrin and then adjusting the pH to 7 to 11 by mixing a pharmaceutically usable salt. In this step, the HA-dopamine-PEG conjugate and cyclodextrin can be dispersed in water to prepare a dispersion, and then a solution of a pharmaceutically usable salt can be added to the dispersion. At this time, a poorly soluble drug can be additionally added to the HA-dopamine-PEG conjugate dispersion to prepare a hydrogel composition into which the poorly soluble drug is introduced. The poorly soluble drug may be added in encapsulated form in microspheres. The poorly soluble drug and microspheres are as described above. Additionally, the pharmaceutically usable salts are as described above.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and experimental methods

1.1. matter

Hyaluronic acid (hyaluronic acid [HA], 150-600 kDa; average Mw measured by gel permeation chromatography: 667 kDa) was purchased from Bioland Co., Ltd. (Cheonan, Korea). 3-Hydroxytyramine hydrochloride (dopamine) was purchased from Tokyo Chemical Industry Co., Ltd. Obtained from (Tokyo, Japan). Methoxy polyethylene glycol amine (molecular weight: 2K) (mPEG2K-NH ₂ ) was purchased from Creative PEGWorks (Chapel Hill, NC, USA). Poly(lactic-co-glycolic acid), PLGA] (lactic acid:glycolic acid=50:50, M n: 45-55 kDa) was obtained from PolySciTech (Akina, Inc., West Lafayette). , IN, USA). Donepezil base was obtained from Chong Kun Dang Pharmaceutical Co., Ltd. (Seoul, Korea). Water-d ₂ ( D ₂ O), hyaluronidase (HAase; derived from bovine testicles, 400∼1000 units/mg solid), N -hydroxysuccinimide (NHS), polyvinyl alcohol (polyvinyl alcohol, PVA; MW: 67 kDa), N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride ( N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride, EDC) and Tween 80 were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

1.2. Synthesis and validation of HDP conjugates

HA-dopamine (HD) polymer derivatives were synthesized by EDC and NHS-mediated amide bond formation as reported in a previous study (Lee et al., 2020). HA (100 mg) was dispersed in distilled water (DW, 10 mL) with magnetic stirring. Dimethyl sulfoxide (DMSO, 10 mL) in which EDC (57.5 mg) and NHS (34.5 mg) were dissolved was mixed with the HA dispersion and 1N HCl was added to adjust the final pH to 5. Then dopamine (37.9 mg) dissolved in DMSO (5 mL) was added to the mixture of HA, EDC, and NHS and stirred for one day. The resulting product was transferred to a tubular dialysis membrane (molecular weight cutoff (MWCO): 7 kDa) and dialyzed against DW for 3 days. The dialyzed material was lyophilized for storage.

To prepare HDP, mPEG2K-NH ₂ was coupled to HD using the amide bond between the carboxylic acid group of HA (in HD) and the amine group of mPEG2K-NH ₂ as follows (Cho et al., 2012). First, a dispersion of HD (96 mg) dispersed in DW (10 mL) was mixed with a mixture of EDC (38.3 mg) and NHS (22.3 mg) in DMSO (10 mL), and the resulting final mixture was adjusted to pH 5 with 1N HCl. It was adjusted to . mPEG2K-NH ₂ (200 mg) in DMSO (5 mL) was added to the reaction mixture of HD, EDC and NHS. This was stirred at room temperature for 15 hours and dialyzed for 36 hours in DW contained in a tubular dialysis membrane (molecular weight cutoff (MWCO): 7 kDa). The dialyzed result was freeze-dried and then washed with acetone to remove unreacted mPEG2K-NH ₂ . The final product was freeze-dried for further storage.

To confirm that the HD polymer derivative was synthesized correctly, the proton nuclear magnetic resonance ( ^1H -NMR) (600 MHz, JNM-ECA-600, JEOL Ltd., Akishima, Japan) spectrum of HDP was measured. To calculate the weight percentage of mPEG2K-NH ₂ in the HDP conjugate, linear regression lines were obtained from standard samples of different weight ratios (mPEG2K-NH ₂ /HA). Additionally, to confirm the synthesis of the HDP polymer, carbon-13 nuclear magnetic resonance ( ¹³ C-NMR) (800 MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) spectra of HD and HDP were measured.

The synthesis of HDP was also verified by Fourier-transform infrared (FT-IR) spectrometer (Frontier™FT-IR spectrometer, PerkinElmer Inc., Buckinghamshire, UK). The spectra of HD, mPEG2K-NH ₂ and HDP were scanned in attenuated total reflectance (ATR) mode in the wavenumber range of 400-4000 cm ^-1 .

1.3. Fabrication and validation of hydrogel structures

PLGA/donepezil microsphere (PDM) was produced in oil-in-water (o/w) as reported in previous studies (Lee et al., 2020; Seo et al., 2020). ) It was prepared using emulsification and solvent evaporation methods. Donepezil base (5 mg) and PLGA (50 mg) were dissolved in dichloromethane (1.5 mL) and mixed with PVA (0.5 %, w/v) in DW (15 mL). The o/w emulsion was then blended at 9,500 rpm using a high-speed homogenizer. The organic phase was removed by stirring for 30 minutes at room temperature, and the particle suspension was centrifuged at 16,100 g for 5 minutes to separate the particle pellet. After removing the supernatant, the particle pellet was redispersed in DW and lyophilized.

Magnified morphological photographs of lyophilized PDM were taken using a field-emission scanning electron microscope (FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany). The bulk density profile of PDM was measured with a particle size analyzer (Mastersizer 3000, Malvern Instruments Ltd., Malvern, UK).

The encapsulation ratio of donepezil in PDM was measured using a high-performance liquid chromatography (HPLC) system (1260 Infinity II, Agilent Technologies) equipped with an automatic sampler (1260 Vialsampler), a pump (1260 Quat Pump VL), and a UV-Vis detector (1260 VWD). , Santa Clara, CA, USA). The mobile phase was prepared with a mixture of potassium phosphate monobasic (10 mM) and acetonitrile (65:35, v/v), and the flow rate was maintained at 1 mL/min. An aliquot (20 μL) of the sample was injected onto a reverse-phase C18 column (Kinetex, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA), and absorbance was detected at 268 nm.

Afterwards, the designed hydrogel system was manufactured by blending and pH control methods. To prepare 1 mL of HDP/aCD/PDM 8.5 sample, HDP (20 mg) was uniformly dispersed in DW (0.79 mL) containing aCD (100 mg) and PDM dispersion (donepezil) in DW (0.1 mL). equivalent to 1 mg). The pH value of the mixture obtained with 1N NaOH was adjusted to 8.5, and DW was added to make the final volume 1 mL.

The formation of polypseudorotaxane structure between HDP and aCD was confirmed by ¹ H-NMR analysis (600 MHz; JNM-ECA-600, JEOL Ltd.). aCD (25 mg/mL) or HDP/aCD (5:25, mg/mL) was dissolved in D ₂ O for ¹ H-NMR analysis.

The host-guest interaction between HDP and aCD was confirmed by two-dimensional (2D) NOESY (NMR nuclear Overhauser effect spectroscopy) (850 MHz, Bruker AVANCE III HD, Bruker, Billerica, MA, USA) experiment. A mixture of HDP (5 mg/mL) and aCD (25 mg/mL) was also prepared by dissolving in D ₂ O.

Polymerization of dopamine molecules in the HDP conjugate was confirmed by UV-Vis spectrometry (Libra S80 dual beam spectrophotometer, Biochrom Ltd., Cambridge, UK). Plain HDP (20 mg/mL in DW) and cross-linked HDP (pH 8.5; 20 mg/mL in DW) samples were prepared and incubated for 6 hours. The absorbance profiles of both samples were monitored in the range of 250 to 900 nm.

The mechanism by which the PEG chains of HDP penetrate the internal cavity of aCD was further investigated using X-ray diffractometry (XRD) (Philips X'Pert PRO MPD diffractometer; PANalytical Corp., Almero, Netherlands). did. The intensity values of aCD, HDP and HDP/aCD (lyophilized form) were scanned in the angle range of 10 to 60°. The generator voltage and tube current were set to 40 kV and 30 mA, respectively, and tested.

1.4. Rheological properties of the prepared hydrogel structure

The gelation properties of the designed hydrogel system were evaluated by inversion test. HA/aCD (20:100, mg/mL), HD/aCD (20:100, mg/mL), HP/aCD (20:100, mg/mL), HP/aCD/PDM (20:100:1 (at donepezil concentration, mg/mL), HDP/aCD (20:100, mg/mL), HDP/aCD 8.5 (20:100, mg/mL; pH 8.5), HDP/aCD/PDM 8.5 (20 :100:1 (at donepezil concentration), mg/mL; pH 8.5) and HDP/PDM 8.5 (20:1 (at donepezil concentration), mg/mL; pH 8.5). Samples were incubated for 6 hours at room temperature. It was later inverted to observe the gelation behavior in the reverse position.

The times required for the sol-gel transition of HDP/aCD/PDM 8.5 (20:100:1 (at donepezil concentration), mg/mL; pH 8.5) are 1, 3, 5, 10, 30, 60 in the reverse position. The behavior was observed and measured at 120 and 180 minutes.

The injectability of HDP/aCD/PDM 8.5 hydrogel (containing 0.2 mg/mL methylene blue) was tested with a single syringe. To evaluate injectability, the hydrogel system was extruded through a syringe needle and its behavior was observed.

The rheological properties of the HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5 and HDP/aCD/PDM 8.5 groups were measured at room temperature using a rheometer (MCR 302, Anton Paar GmbH, Graz, Austria). Evaluation was performed after daily incubation. Storage modulus (G') and loss modulus (G") values were measured over a range of strain changes (1-400%) using a strain sweep test at a fixed frequency of 10 rad/s and a temperature of 37°C. (1-100 rad/s)-dependent G' and G" values were measured at 10% strain and 37°C temperature. G' and G" values over time for three cycles of 100% strain (0-1, 2-3, and 4-5 min) and 0% strain (1-2, 3-4, and 5-6 min) Measured in. A correlation (plot) of shear stress and viscosity according to shear rate was obtained in the range of 0.01-100 s ^-1 .

1.5. In vitro ( in vitro ) Drug release and degradability evaluation

Cross-sectional photographs of lyophilized HDP/aCD 8.5 and HDP/aCD/PDM 8.5 gels were obtained using field-emission scanning electron microscopy (FE-SEM; SUPRA 55VP, Carl Zeiss, Oberkochen, Germany).

The DPZ release profile from the HDP/aCD/PDM 8.5 system was measured in vitro at pH 7.4. Donepezil base solution (1 mg/mL in 50% DMSO) and HDP/aCD/PDM 8.5 (containing 1 mg/mL donepezil) were prepared, and an aliquot (0.6 mL) of each sample was transferred into a dialysis tube (Midi-GeBAflex tube, MWCO:3.5 kDa; Gene Bio-Application Ltd., Yavne, Israel). The dialysis tube was immersed in PBS (pH 7.4) containing 0.3% Tween 80 (30 mL) and incubated in a water bath at 37°C and 50 rpm. A certain amount of release fluid (0.2 mL) was administered at 6 hours and 24 hours for the donepezil solution group and at 6, 24, 48, 72, 168, 192, 216, 336, 504, and 672 hours for the HDP/aCD/PDM 8.5 group. ) was taken. The donepezil concentration of the released solution was analyzed by the HPLC method described above.

The in vitro degradation properties of the HDP/aCD/PDM 8.5 system were evaluated in the absence or presence of HAase. HDP/aCD/PDM 8.5 (0.5 mL) was mixed with DW (0.5 mL) containing or without HAase (1 mg/mL) and placed in a microcentrifuge tube. At each sampling time point (24, 48, 72, 168, 240, 336, and 504 hours), the supernatant was removed and the remaining gel mass was measured. Afterwards, the same volume of new medium was replenished and incubated at 37°C. The relative percentage of remaining gel fraction was calculated compared to the initial value (0 hours).

1.6. biodegradation test

The biodegradation properties of the developed hydrogel system were evaluated by measuring the volume reduction profile in a mouse model. The designed hydrogel system was injected subcutaneously into ICR mice (Institute of Cancer Research mouse, male, 5 weeks old; Orient Bio, Seongnam, Korea). All samples were incubated at room temperature for one day, and then aliquots (0.1 mL for 20 g body weight) of PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5 samples were administered to mice. It was injected subcutaneously into the back. At 14 days after injection, mice were sacrificed under inhalation anesthesia, residual gel was excised, and weight was measured.

1.7. data analysis

For statistical analysis of the obtained experimental data, a two-tailed t -test and one-way analysis of variance (ANOVA) followed by a post hoc test were performed. Each experiment was repeated at least three times, and the obtained data were expressed as mean ± standard deviation (SD).

2. Results and discussion

2.1. Hydrogel system design and HDP polymer synthesis

In order to fabricate a physically interconnected and chemically cross-linked hydrogel system, the present invention uses a polypseudorotaxane structure formed between PEG (in the form linked to the HDP conjugate) and aCD, and a dopamine group (in the form attached to the HDP conjugate). A polydopamine bond was introduced. The aCD can be linked (internally penetrated) by PEG chains (linked to HDP) to bind the HDP structure. Crystalline complexes can be formed through hydrogen bonding between adjacent aCD molecules and their aggregation. Additionally, dopamine polymerization at alkaline pH can provide chemical cross-linking of the HDP network. Through this binding mechanism, the rheological and mechanical properties of the hydrogel can be adjusted to develop an optimized subcutaneous injection formulation. Additionally, donepezil-encapsulated PLGA microspheres (PDM) were incorporated into a hydrogel system designed for sustained drug release.

As the main gel matrix, the HDP conjugate was finally synthesized by covalently linking a dopamine group and a PEG group to the HA skeleton (Figure 1A). Dopamine was linked to the HA scaffold through an amide bond catalyzed by EDC and NHS. Next, PEG was further linked to the synthesized HD polymer based on the amide bond between the amine group of mPEG2K-NH ₂ and the carboxylic acid group of HA (in HD).

The synthesis of HDP was confirmed by NMR analysis (Figures 1B-1D). In ^{the 1H} -NMR spectrum of HDP, the chemical shifts for the N -acetyl group of HA, the phenyl ring of dopamine, and the methylene group of PEG are 1.8 to 2.0 ppm (peak 1), 6.6 to 6.8 ppm (peaks 2 to 4), and 3.6 ppm, respectively. appeared at ~3.7 ppm (peaks 5 and 6) (Figure 1B). As HD synthesis was already confirmed through spectroscopic analysis in a previous study (Lee et al., 2020), relevant analytical data were not included herein. The PEG content of the HDP polymer was determined by plotting a regression line with a standard sample (mixture of mPEG2K-NH ₂ and HD). The average PEG content of the HDP conjugates in the present invention, calculated from the regression line (Figure 1C), was 50.7%. The synthesis of HD and HDP was confirmed by ¹³ C-NMR analysis (Figure 1D). ^{The 13} C-NMR spectrum of HD showed a carbon shift toward the N -acetyl group of HA (22.4 ppm; peak 1) and a carbon shift toward the phenyl ring group of dopamine (116.5 and 121.2 ppm; peaks 2–4) (Ling et al. ., 2015; Zhang et al., 2016). The carbon shifts for the methylene carbon of PEG in the HDP spectrum are 69.6 and 71.0 ppm ( peaks 5 and 6) (Mahou and Wandrey, 2012). These ¹³ C-NMR data also support that HDP was properly synthesized.

The synthesis of HDP was also demonstrated by FT-IR analysis (Figure 1E). Peaks for OH stretching (3286 cm ^-1 ), CH stretching in the pyranose ring (2917 cm ^-1 ), and C=O stretching in NH-Ac (1643 cm ^-1 ) appeared in the spectrum of the HD conjugate. . In the HDP spectrum, peaks for OH stretching (3361 cm ^-1 ), CH stretching in the pyranose ring (2875 cm ^-1 ), and C=O stretching in NH-Ac (1649 cm ^-1 ) were also observed, and peaks at 1466 cm ^-1 were observed in the HDP spectrum. The peak at ¹ indicates the amide bond between HA and mPEG2K-NH ₂ (Chatterjee et al., 2019). From all these FT-IR data, it was confirmed that dopamine and PEG moieties were properly bound to the HA skeleton.

2.2. Fabrication of hydrogel structure and identification of hydrogel cross-linking mechanism

Supramolecular structures and catechol-functionalized chemically-linked hydrogel systems were facilely prepared using a blending method (Lee et al., 2020; Seo et al., 2020). Donepezil base-encapsulated microspheres (PDM) were prepared and mixed with the precursor of the hydrogel in a gel cross-linking process. PDM was incorporated into the hydrogel network with the goal of sustained release of donepezil after subcutaneous injection.

PDM was prepared using the emulsification/evaporation method as reported in previous studies (Lee et al., 2020; Seo et al., 2020). Donepezil base, which has low water solubility, was introduced into PLGA microspheres for sustained release. As shown in Figure 2A, the surface was observed to be a smooth sphere in the FE-SEM image. The bulk density (Dv) frequency according to size was measured, and the Dv(10), Dv(50), and Dv(90) values of PDM were 10, 19, and 34 μm, respectively (Fig. 2B). These particle size distribution profiles were similar to previously reported data performed with similar experimental protocols (Seo et al., 2020). The average encapsulation efficiency of donepezil in PDM was 68%, indicating adequate drug encapsulation in microspheres.

The hydrogel cross-linking mechanism based on supramolecular chemistry and catechol polymerization was identified by NMR, UV-Vis, and XRD analysis (Figure 3). The host-guest interaction between aCD and PEG was investigated by ¹ H-NMR analysis (Figure 3A). A proton shift (H3-H6) was observed at 3.5 to 3.9 ppm in the aCD spectrum, and this shift was also seen in the ¹ H-NMR spectrum of HDP/aCD. In the spectrum of HDP/aCD, proton transfer at the methylene group of PEG was also observed at about 3.6 ppm. These data indicate that HDP and aCD are incorporated into the lyophilized HDP/aCD hydrogel system. The penetration of the PEG chains (in the form linked to the HDP conjugate) into the internal cavity of aCD was further demonstrated by 2D NOESY NMR analysis (Figure 3B). The 2D NOESY NMR spectrum of the HDP/aCD polypseudorotaxane structure showed a crosspeak based on the interaction between the hydrogen atoms of the PEG methylene group and the hydrogen atoms of the aCD unit. This 2D NMR result indicates rotaxation of the designed HDP/aCD structure.

Another hydrogel crosslinking mechanism, referred to as catechol polymerization, was studied by UV-Vis spectroscopy analysis (Figure 3C). The high absorbance value around 280 nm in the HDP spectrum indicates the contribution of the phenyl ring of the dopamine molecule. In the spectrum of HDP 8.5, the absorbance value around 400 nm was measured to be higher than that of HDP itself. Since polydopamine formation by pH control affected the absorbance profile, it can be seen from these data that the hydrogel is cross-linked through polymerization of catechol (in the form linked to the HDP conjugate).

The host–guest chemistry between aCD and PEG and the crystalline complex based on aCD aggregates were further evaluated by XRD analysis (Figure 3D). As reported (Feng et al., 2016), crystalline properties were observed in the spectrum of aCD. PEG chains can penetrate the tunnel structure of aCD, and the inclusion complex of PEG/aCD is known to be insoluble in water due to strong hydrogen bonding between aCD molecules. In the XRD pattern of HDP/aCD, a clear peak appeared at 19.8˚, indicating a channel-type crystal structure. The observed XRD patterns support the formation of supramolecular structures based on PEG/aCD in this study.

2.3. Rheological properties of hydrogels

The gelability, injectability, and viscoelastic properties of the designed hydrogel structure were evaluated as follows (Figure 4). The gelation behavior of HA/aCD, HD/aCD, HP/aCD, HP/aCD/PDM, HDP/aCD, HDP/aCD 8.5, HDP/aCD/PDM 8.5 and HDP/PDM 8.5 groups was evaluated by inversion test (Figure 4A). In the HA/aCD and HD/aCD groups, the proposed cross-linking mechanism (i.e., supramolecular chemistry and polydopamine linkage) was not applied and therefore did not appear to increase the viscoelastic aspect. Although the PEG/aCD-based physical interaction strategy was theoretically applied to the HP/aCD and HP/aCD/PDM groups, these gel samples did not remain in the inverted position in the inversion test due to insufficient viscoelastic behavior. The HDP/aCD group was partially maintained in the reverse state, and the HDP/aCD 8.5 group showed complete gel curing behavior. In the HDP/aCD 8.5 group, a color change occurred due to polydopamine formation compared to the HDP/aCD group. As shown in the HDP/aCD/PDM 8.5 group, the incorporation of PDM into the HDP/aCD 8.5 network did not interfere with the gelation phenomenon. Additionally, the HDP/PDM 8.5 group also showed an increase in viscoelastic properties due to catechol polymerization without supramolecular chemistry (between PEG and aCD).

The gelation time was measured by an inversion test as shown in Figure 4B. The HDP/aCD/PDM 8.5 sample showed that the gel was maintained in the reverse position even after 1 minute, and the gel curing properties were maintained for 360 minutes. The injectability of the HDP/aCD/PDM 8.5 group was tested by extrusion through a syringe needle (Figure 4C). The observed injectability in the HDP/aCD/PDM 8.5 group suggests the possibility of convenient subcutaneous injection.

The viscoelastic properties of the HP/aCD/PDM, HDP/aCD 8.5, HDP/PDM 8.5, and HDP/aCD/PDM 8.5 samples were evaluated by rheometer (Figure 4D). The HDP/PDM 8.5 group remained intact in the inverted position (Figure 4A), but in strain sweep and frequency sweep tests, there was a significant difference in test method and sample incubation time between inversion and rheology tests. By factor, the storage modulus (G') and loss modulus (G") values of this group were significantly lower than other supramolecular hydrogel structures (mainly based on aCD and PEG interactions), as shown in the inversion test results ( Figure 4A), in the frequency change test and viscosity test, the G' and G" values of the HP/aCD/PDM group were lower than those of the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups. Both the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups showed higher G' and G" values than the HP/aCD/PDM and HDP/PDM 8.5 groups in the strain change and frequency change tests. This is consistent with the results of supramolecular chemistry and catechol polymerization. The introduction of a composite strategy means that the viscoelastic properties of hydrogels can be significantly improved. Both the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups have higher G' (elastic behavior) values than G" (representing viscous behavior) values. (represents) value, which suggests that it is mainly a solid state characteristic in the frequency change data. A time-dependent stepwise test was performed to evaluate the destruction and subsequent recovery of the hydrogel structure under high shear. For both HDP/aCD 8.5 and HDP/aCD/PDM 8.5 hydrogels, the same as seen in the first step (0% strain, 1-2 min), during the second and third resting steps (0% strain, 3-4 and 5-6 min) restoration of solid-like properties (G'>G"), suggesting self-healing behavior. These HDP/aCD 8.5 and HDP/aCD/PDM 8.5 hydrogels show a change in the viscosity data line with increasing shear rate. This shear-thinning behavior of the HDP/aCD/PDM 8.5 hydrogel may facilitate its injection through a syringe needle in the subcutaneous tissue. All observed data are consistent with the designed HDP/aCD/PDM 8.5 hydrogel. The PDM 8.5 hydrogel exhibited immediate gelation, injectability through a single syringe, self-healing ability, and shear thinning behavior, suggesting that these properties would be suitable for subcutaneous injection.

2.4. Physicochemical characteristics of hydrogel

The internal structure of the freeze-dried HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups was observed by FE-SEM imaging (Figure 5A). A honeycomb-like structure appeared in both the HDP/aCD 8.5 and HDP/aCD/PDM 8.5 groups, which may be due to the double cross-linking strategy based on the polypseudorotaxane structure and polydopamine linkage. In particular, in the photos of the HDP/aCD/PDM 8.5 group, it was confirmed that PDM exists on the inner surface of the HDP/aCD 8.5 hydrogel system.

The drug release pattern of the HDP/aCD/PDM 8.5 group was compared with the donepezil solution group (pH 7.4) (Figure 5B). In drug release tests, donepezil solution diffused immediately through the polymer membrane. Approximately 94.4% of the loaded drug amount diffused from the dialysis tubing into the release fluid within 24 hours. In contrast, the amount of donepezil released from HDP/aCD/PDM 8.5 hydrogel was only 6.0% in 24 hours. The amount of donepezil released from HDP/aCD/PDM 8.5 hydrogel on days 7 and 14 was 39.9% and 56.7%, respectively. The HDP/aCD/PDM 8.5 hydrogel group showed sustained drug release for 4 weeks at pH 7.4 in this study. The sustained drug release observed in the microsphere-embedded cross-linked hydrogel system may increase patient compliance by reducing the frequency of administration.

The in vitro degradation profile of the designed HDP/aCD/PDM 8.5 hydrogel was evaluated in the presence or absence of HAase (Figure 5C). In the absence of HAase, the relative weight of the hydrogel increased to 1.64 times the initial value over 72 hours, which may be caused by the swelling properties of the HA scaffold. Afterwards, on day 21, the relative weight of the gel decreased to 118.7% of the initial weight. In the case of the group containing HAase, the relative weight of the hydrogel was measured to be 119.9% at 48 hours. Afterwards, a decreasing pattern was observed, finally reaching 61.8% of the initial value on day 21. The lower relative weight of the hydrogel in the HAase presence group compared to the HAase absence group may mean that enzymatic degradation of the HA polymer occurred in the HDP/aCD/PDM 8.5 hydrogel. The remaining hydrogel weight (61.8%) at day 21 in the presence of HAase may represent a portion of the non-degradable inclusion complex between PEG (linked to the HA backbone) and aCD tethered to the HDP network. From the observed in vitro degradation data, it can be seen that the designed hydrogel system can be slowly degraded in biological tissues containing HAase. The sustained drug release from the cross-linked hydrogel and the low degradation rate of the hydrogel even in the presence of HAase may contribute to reducing the frequency of administration and creating a long-lasting drug delivery system.

2.5. biodegradable properties

The biodegradation pattern of the designed hydrogel system was tested after subcutaneous injection in mice (Figure 6). PDM, HDP/aCD 8.5, HDP/PDM 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5 samples were injected subcutaneously into the back of mice and retention characteristics were monitored in vivo . No remaining sample fragments could be obtained from the PDM and HDP/PDM 8.5 groups, which may be because the PDM and HDP/PDM 8.5 hydrogels immediately diffused into the subcutaneous tissue and decomposed. In the HDP/PDM 8.5 group, catechol polymerization was used as a cross-linking method, but the sample dispersion did not remain in the subcutaneous tissue 14 days after injection. As can be seen from the rheology data (Figure 4D), in the strain and frequency change tests, the maximum G' value of the HDP/PDM 8.5 group was only 20.6 and 18.6 Pa, respectively, and these low viscoelastic properties are associated with rapid biodegradation in the subcutaneous tissue and It appears to be related. The order of gel weights measured by collecting the remaining gel portions from the HDP/aCD 8.5, HDP/aCD/PDM, and HDP/aCD/PDM 8.5 groups is: HDP/aCD/PDM 8.5 > HDP/aCD 8.5 > HDP/aCD/PDM It was. The residual gel weight of the HDP/aCD/PDM 8.5 group was significantly higher than that of the HDP/aCD 8.5 group (p <0.05). The difference between the two groups was greater than the weight of PDM contained in the same injection amount, meaning that the PDM dispersed in the hydrogel network contributed to maintaining the hydrogel structure at the injection site and delaying the biodegradation rate. The remaining gel weight of the HDP/aCD/PDM 8.5 group was 5.8 times higher than that of the HDP/aCD/PDM group (p <0.05). The chemical cross-linking strategy based on the linkage of dopamine polymerization and HDP conjugates with PEG/aCD-based supramolecular structures appears to significantly contribute to the low biodegradation rate. The presented data suggest that HDP/aCD/PDM 8.5 hydrogel can be used as a biodegradable long-lasting drug delivery system.

3. Conclusion

Dopamine and PEG-linked HA polymer conjugates (HDP) were synthesized and cross-linked hydrogels were prepared by forming inclusion complexes between aCD and PEG and polydopamine bonds. Supramolecular systems (between aCD and PEG) and catechol polymerization (between dopamine molecules grafted to HA polymers) were introduced as dual physical and chemical hydrogel cross-linking strategies, thereby significantly improving the viscoelastic properties. Additionally, the designed hydrogel system was verified for rapid sol-to-gel conversion, injectability through a single syringe, self-healing ability, and shear thinning behavior. PDM was incorporated into the cross-linked hydrogel network for sustained release, and the HDP/aCD/PDM 8.5 hydrogel showed a low biodegradation rate and negligible toxicity after subcutaneous injection in mice. From all these results, it can be seen that the designed HDP/aCD/PDM 8.5 hydrogel can be safely used for long-term delivery of donepezil through subcutaneous injection.

4. Summary

In the present invention, a cross-linked hyaluronic acid (HA) hydrogel based on polypseudorotaxane structure and polydopamine bond was developed to continuously deliver a drug (donepezil) after subcutaneous injection. Dopamine and polyethylene glycol (PEG) molecules were covalently linked to the HA polymer for amide bonding for catechol polymerization and inclusion complexation with cyclodextrin (CD), respectively. Alpha-cyclodextrin (aCD) is bound to the PEG chain of the HA-dopamine-PEG (HDP) conjugate to create a polypseudorotaxane structure, and the pH is adjusted to alkaline conditions (pH 8.5) to form polymerized dopamine. (in the form attached to the HD conjugate). For sustained release, poly(lactic-co-glycolic acid) [poly(lactic-co-glycolic acid), PLGA]/donepezil microspheres (microspheres, MS) (PDM) were incorporated into the HDP polymer network. . The designed HDP/aCD/PDM 8.5 hydrogel system exhibited an immediate gelation pattern, injectability via a single syringe, self-healing ability, and shear-thinning behavior. Donepezil was released in a continuous pattern from HDP/aCD/PDM 8.5 hydrogel, and HDP/aCD/PDM 8.5 hydrogel showed the property of being degraded by hyaluronic acid degrading enzyme. After subcutaneous injection, a greater amount of HDP/aCD/PDM 8.5 hydrogel remained than the other groups even after 14 days. The observed results suggest that the developed HDP/aCD/PDM 8.5 hydrogel has high clinical feasibility as a subcutaneous injection formulation.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (15)

HA-dopamine-PEG conjugate formed by an amide bond between the carboxylic acid group of hyaluronic acid (HA) and the amine group of dopamine, and an amide bond between the carboxylic acid group of hyaluronic acid and the amine group of a PEG derivative containing an amine group at the terminal; cyclodextrin; And a cation of a pharmaceutically usable salt, the pH is 7 to 11, and the dopamine bound to the hyaluronic acid undergoes catechol polymerization to form polydopamine. The hydrogel composition according to claim 1, wherein the hyaluronic acid has an average molecular weight of 10 to 3000 kDa. The hydrogel composition according to claim 1, wherein the PEG has a molecular weight of 100 to 100,000 Da. The method of claim 1, wherein the cation of the pharmaceutically usable salt is one type of cation selected from the group consisting of sodium ions, potassium ions, calcium ions, magnesium ions, barium ions, aluminum ions, and mixtures thereof. Hydrogel composition. The method of claim 1, wherein the cation of the pharmaceutically usable salt is sodium hydroxide (NaOH), potassium phosphate monobasic (KH ₂ PO ₄ ), potassium phosphate dibasic (K ₂ HPO ₄ ), potassium chloride (KCl), A hydrogel composition characterized in that it is a cation of one type of salt selected from the group consisting of sodium phosphate (NaH ₂ PO ₄ ), dibasic sodium phosphate (Na ₂ HPO ₄ ), sodium chloride (NaCl), and mixtures thereof. The hydrogel composition according to claim 1, further comprising a poorly soluble drug. The method of claim 6, wherein the poorly soluble drug is donepezil, amphotericin B, curcumin, phloretin, paclitaxel, docetaxel, dexamethasone, budesonide, cyclosporine, tacrolimus, rapamycin, fenofibrate, prednisolone, itraconazole, insulin ( A hydrogel composition characterized in that it is selected from the group consisting of neutral pH), indomethacin, and mixtures thereof. The hydrogel composition according to claim 6, wherein the poorly soluble drug is encapsulated in microspheres. A drug delivery system comprising the hydrogel composition of any one of claims 1 to 8. An injection for subcutaneous injection comprising the hydrogel composition of any one of claims 1 to 8. A filler comprising the hydrogel composition of any one of claims 1 to 8. A dressing agent comprising the hydrogel composition of any one of claims 1 to 8. Obtaining an HA-dopamine-PEG conjugate by combining hyaluronic acid (HA) with dopamine and PEG containing an amine group at the terminal; and
After mixing the HA-dopamine-PEG conjugate and cyclodextrin, mixing a pharmaceutically usable salt to adjust the pH to 7 to 11 so that the dopamine bound to the hyaluronic acid undergoes catechol polymerization to form polydopamine.
Method for producing a hydrogel composition comprising. The method of claim 13, wherein the HA-dopamine-PEG conjugate comprises a carboxylic acid group of hyaluronic acid and an amine group of dopamine; and a method for producing a hydrogel composition, characterized in that it is produced by an amide bond formed between the carboxylic acid group of hyaluronic acid and the amine group of a PEG derivative containing an amine group at the terminal. The method of claim 13, further comprising mixing the HA-dopamine-PEG conjugate and cyclodextrin with a poorly soluble drug.