CN114984233B - Local drug delivery system based on giant salamander skin secretion and application thereof - Google Patents

Abstract

The invention relates to the field of biological materials, in particular to a local drug delivery system based on giant salamander skin secretion and application thereof. The invention provides the use of giant salamander skin secretion in the manufacture of a topical delivery system for dynamic wounds, characterised in that the topical delivery system comprises: (a) The giant salamander skin secretion freeze-dried powder has granularity of 14-300 meshes; (b) at least one drug; (c) At least one solvent that gels the lyophilized powder of giant salamander skin secretion; wherein the at least one drug and the at least one solvent are not protein denaturants. The local drug delivery system provided by the invention is beneficial to promoting the regeneration and healing of a wound part (especially a dynamic wound) to a certain extent, and has wide application fields.

Description

Local drug delivery system based on giant salamander skin secretion and application thereof

Priority application

The present application claims priority to "application of giant salamander skin secretion in the preparation of topical drug delivery systems" from chinese patent application filed on 11 months 05 2021 [ CN2021113071734 ], which is incorporated by reference in its entirety.

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a local drug delivery system based on giant salamander skin secretion and application thereof.

Background

Traditional delivery methods based on systemic administration can lead to side effects and low bioavailability. Thus, in recent years, topical administration has received increasing attention because it can improve the bioavailability of topical drugs and reduce systemic toxicity caused by the drugs. However, this approach is still limited by several drawbacks, such as complex wound sites in the oral cavity or wound surfaces after tumor resection.

Periodontitis (periodontis) and diabetes (diabetes mellitus) are common chronic diseases that are highly associated. In the hyperglycemic environment caused by diabetes, advanced glycation end products (AGEs) may accumulate in large amounts in periodontal tissues, which subsequently induce apoptosis, reduce cell proliferation and migration associated with wound healing, and inhibit stem cell differentiation, ultimately compromising the wound healing process. Although systemic administration of aminoguanidine (AG, an AGE inhibitor) has been shown to accelerate palate (palate) wound healing in diabetic rats following free gingival flap implantation (FGG), there are also some safety issues reported with the use of high concentrations of AG. In addition, the moist condition of the mouth and the constant chewing prevent the drug from remaining locally.

Malignant melanoma, on the other hand, is considered one of the most aggressive and highly metastatic skin tumors, and surgical excision is still the most common treatment in the clinic. Melanoma cells that may remain after surgical resection are prone to cancer recurrence. Therefore, local excision in combination with systemic chemotherapy is a common treatment. However, the nonspecific distribution and fluctuating blood concentration of chemotherapeutic agents always lead to serious side effects and place a great burden on the patient. Therefore, topical chemotherapy is increasingly used to minimize drug exposure to healthy tissue while reducing the risk of local recurrence. However, most chemotherapeutic agents, such as Doxorubicin (DOX), are relatively poorly specific and cannot differentiate between normal and cancerous cells.

Ideally, a topical drug delivery system for wound healing should be cost effective, safe, and have multiple functions such as controlled release (controlled release), local maintenance (local maintenance), biodegradability and healing promoting capabilities.

Disclosure of Invention

In one aspect, the invention provides the use of giant salamander skin secretion in the manufacture of a topical delivery system for dynamic wounds, characterised in that the topical delivery system comprises:

(a) The giant salamander skin secretion freeze-dried powder has granularity of 14-300 meshes;

(b) At least one drug;

(c) At least one solvent that gels the lyophilized powder of giant salamander skin secretion; wherein the at least one drug and the at least one solvent are not protein denaturants.

In one embodiment, the agent is an advanced glycation end product inhibitor or a chemotherapeutic agent.

In one embodiment, the drug has a molecular weight of 100 to 600.

In one embodiment, the drug comprises one or more of doxorubicin hydrochloride, aminoguanidine, doxorubicin, benzoyl thiazole bromide, doxycycline, streptomycin, penicillin, paclitaxel, homoharringtonine, cyclophosphamide, nedaplatin.

In one embodiment, the giant salamander comprises one or more of the genera giant salamander, cryptobranchia, mangrove, andrias, and polar.

In one embodiment, the topical drug delivery system is applied to a wound site.

In one embodiment, the wound site is located on the skin and/or mucous membrane.

In one embodiment, the wound site is a diabetic wound or a wound following tumor resection.

In one embodiment, the topical delivery system is for promoting cell proliferation and/or cell migration and/or recruitment of endogenous stem cells and/or hemostasis at the wound site.

In one embodiment, the topical delivery system is for promoting re-epithelialization and/or angiogenesis and/or extracellular matrix remodeling and/or collagen deposition of the wound site.

In one embodiment, the topical drug delivery system is for promoting mucosal healing and/or salivary gland regeneration at the wound site, wherein the wound site is in the oral cavity.

In one embodiment, the topical delivery system is for inhibiting tumor cells.

In one embodiment, the topical delivery system is in the form of a dressing.

In another aspect, the present invention also provides a topical drug delivery system for dynamic wounds, characterized in that the topical drug delivery system comprises:

(a) The giant salamander skin secretion freeze-dried powder is 20-200 meshes;

(b) At least one drug, wherein the dry weight mixing ratio of the drug and the giant salamander skin secretion freeze-dried powder is less than or equal to 400%, and the molecular weight of the drug is 100-600;

(c) At least one solvent in an amount effective to gel the lyophilized powder of the giant salamander skin secretion;

wherein the at least one drug and the at least one solvent are not protein denaturants.

In one embodiment, the drug comprises one or more of aminoguanidine, doxorubicin, and pharmaceutically acceptable salts thereof.

As used herein, "topical drug delivery system" includes hydrogels and drugs of biological origin. The hydrogel of biological origin is a hydrogel generated by freeze-dried powder of skin secretion of giant salamander and a solvent, and is preferably giant salamander Skin Secretion (SSAD) hydrogel. The drug may be dissolved in a solvent or may be partially dissolved or insoluble. The drug and the solvent do not denature the protein (i.e., are not protein denaturants). Exemplary protein denaturants include strong acids, strong bases, heavy metal salts, guanidine hydrochloride, urea, acetone, TCEP, proteases, and some reducing agents (e.g., cysteine, ascorbic acid, beta-mercaptoethanol, and DTT), and the like. Exemplary drugs include AGE inhibitors and chemotherapeutic agents. The molecular weight of the drug may be 100-600. The topical drug delivery system achieves controlled release of the drug through degradation of the hydrogel. The giant salamander skin secretion freeze-dried powder can be any liquid capable of stabilizing the giant salamander skin secretion freeze-dried powder into gel when meeting water to form gel. That is, the common reagents in clinic can gel the freeze-dried powder of the skin secretion of the giant salamander. Exemplary solvents may be buffers such as physiological saline, purified water, fluids such as wound fluids, and cell culture media.

As used herein, "biologically derived" refers to organisms and parts of organisms that are derived or obtained from naturally occurring organisms. In other words, the major component of the topical drug delivery system of the present invention, the biologically derived hydrogel, is not obtained by genetic recombination techniques.

As used herein, "wound" refers to any defect in the skin or mucosa of an individual. The continuity or integrity of the tissue structure at the wound site is compromised. Wounds may be due to abrasion, avulsion, laceration, puncture, cancer, diabetic ulcers or lesions, burns, surgery, or other injuries. "wound healing" refers to the partial or complete restoration of tissue integrity. In one embodiment, the wound site is located on the skin. In another embodiment, the wound is located on a mucous membrane. By "promoting wound healing" is understood restoring or partially restoring the skin or mucous membrane from a disruption of continuity or integrity.

As used herein, "dynamic wound" refers to a wound in which the wound bed is constantly or often in a state of change, such that it is more difficult to heal, due to the continued exposure to other interfering factors. The interfering factors may be pressure (e.g., a wound in the mouth that is susceptible to pressure from chewing), active pulling (e.g., the wound bed is in a position where it is susceptible to movement, and thus susceptible to stretching forces due to movement). The interfering factors may also be due to extreme circumstances that may lead to the wound bed being constantly in an unstable state, and the wound bed being subject to pathological changes (e.g., swelling, infection, ulceration, pus, necrosis, etc. at the wound bed due to diseases or symptoms such as hyperglycemia, tumors, etc.).

As used herein, "diabetic wound" refers to a wound that is initiated by diabetes (particularly hyperglycemia) and that delays healing or is difficult to heal in the hyperglycemic environment of diabetes, etc.

As used herein, "AGE inhibitor" refers to a substance that inhibits AGE (advanced glycation end product) production and/or activity. Exemplary AGE inhibitors include benzoyl thiazole bromide, pyridoxamine, aminoguanidine.

As used herein, "chemotherapeutic agent" refers to an agent used to treat a tumor (or cancer), such as an alkylating agent (e.g., cyclophosphamide), a metabolic antagonist (e.g., 5-fluorouracil), an anticancer antibiotic (e.g., doxorubicin), a plant-based anticancer agent (e.g., paclitaxel), a platinum-based agent, and the like.

As used herein, "swelling ratio" refers to the portion of the hydrogel that increases in weight due to liquid absorption, or decreases in weight due to dissolution.

As used herein, "synergistic effect" is a result that indicates a significantly greater effect than would be expected from each component applied to a wound alone. In one embodiment, the present invention provides a topical drug delivery system wherein the SSAD hydrogel has a synergistic effect with the drug AG or DOX loaded.

As used herein, "regeneration" is a repair process in which a tissue or organ is partially lost by external action, and on the basis of the remainder, a structure morphologically and/or functionally identical to the lost portion is grown.

As used herein, "promoting" refers to a further increase in the described subject at an existing level, including one or more of a quantitative level, an expression level, a functional level, an competence level.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from them without inventive faculty.

FIG. 1 is a schematic representation of the possible mechanisms by which the drug loaded giant salamander skin exudate (SSAD) hydrogel of the present invention may promote wound healing;

FIG. 2 illustrates structural features of SSAD and its sustained drug release characteristics;

FIG. 3 shows SSAD-induced cell migration;

FIG. 4 shows an evaluation of the rate of wound healing of the palate;

FIG. 5 shows immunofluorescent-stained images of a palate wound at 18 days post-surgery;

FIG. 6 shows the therapeutic mechanism of SSAD for wound healing;

FIG. 7 shows in vivo anticancer efficacy of DOX loaded SSAD hydrogels;

FIG. 8 shows microscopic anticancer efficiency in vivo;

FIG. 9 shows the swelling ratio and degradation rate of SSAD hydrogels in PBS and saliva;

FIG. 10 shows the relative cell viability values of L929 cells and HUVECs cultured for 24h with different concentrations of SSAD;

FIG. 11 shows relative cell viability values of L929 and HUVECs cultured with SSAD (0.1 mg/mL) for 24 h;

FIG. 12 is a graph showing the results of a scoring experiment with L929 cells and HUVECs cultured with SSAD, IGF-1, SDF-1 or SDF-1+IGF-1;

FIG. 13 shows the establishment of an in vivo tail vein injury hemostasis model to verify hemostasis characteristics of different size SSAD powders;

FIG. 14 illustrates the creation of an in vivo liver injury hemostasis model to verify the hemostatic capabilities of SSAD;

FIG. 15 shows a model of hemostasis for liver injury in vivo demonstrating that SSAD can effectively adhere to a wound site and stop bleeding;

fig. 16 is an HE image of normal palate tissue (epithelium (yellow arrow), lamina propria (black arrow), submucosa (blue arrow), mucosal glands ();

Fig. 17 is an HE image of the boundary between normal tissue and regenerated tissue (normal tissue (black arrow), regenerated tissue (blue arrow), cell infiltration (x));

FIG. 18 is a representative HE image of the thickness of the palate mucosa measured 8 days post-operatively;

figure 19 shows the re-epithelialization quantification for each group (black arrow indicates distance of wound at baseline, red arrow indicates distance of no epithelialization 18 days post-surgery);

FIG. 20 is a green fluorescence image of FIG. 5a alone (to assess collagen deposition and inflammatory cell infiltration);

figure 21 is a red fluorescence image of figure 5a alone (to assess collagen deposition and neovascularization).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Detailed description of the drawings

Fig. 2: (a) SEM images of the size of the 20, 60 and 200 mesh SSAD powder and the porous structure of the corresponding formed hydrogels are shown. (b) i) AG and DOX, ii) AG loaded SSAD hydrogels of different pore sizes (20, 60 and 200 mesh) and iii) DOX loaded SSAD hydrogels of different pore sizes (20, 60 and 200 mesh). (c, d) cumulative release profile of (c) AG and (d) DOX in SSAD hydrogels of different pore sizes (20, 60 and 200 mesh).

Fig. 3: (a) Scratch test for L929 cells and HUVECs with or without SSAD culture. (b) The ability of SSAD treated L929 cells and HUVECs to migrate was further demonstrated by a Transwell assay. (c, d) (c) relative migration area in scratch test and (d) quantitative analysis of the number of cells migrating from upper to lower chamber in Transwell test. * P <0.05, < P <0.01, < P <0.001.

Fig. 4: (a) macroscopic observation of a wound. (b) the wound closure rate calculated by equation (3). (c) HE staining image of the wound site 18 days post-operative. (d) 8 days post-operative, different groups of neo-formed tissue thickness. (e) Re-epithelialization (re-epithelialization) rates for different groups 18 days post-surgery. (f) Masson's trichromatic images of wound sites at day 8 and day 18, with dashed lines showing the boundary between normal and regenerated tissue. Normal tissue (black arrow), regenerated tissue (blue arrow), re-epithelialization (yellow arrow), glandular regeneration (+). * P <0.05, < P <0.01, < P <0.001.

Fig. 5: (a) Immunofluorescent staining of vascular endothelial cells (CD 31, red) and collagen I (Col-1, green). (b) Immunofluorescent staining of myofibroblasts (α -SMA, red) and macrophages (CD 68, green). Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI, blue) and doubly stained tissues were identified with yellow fluorescence. (c, d) shows quantitative histograms of the percentages of (c) cd31+ cells and Col-1 and (d) myofibroblasts (α -sma+) and macrophages (cd68+) cells on day 18.

Fig. 6: (a) Venn diagram of transcriptional profile between SSAD group and control group. (b) Heat maps (|log|gtoreq 2, p < 0.05) of genes significantly altered after SSAD treatment. (c) KEGG pathway. (d) 20 biological processes that are most significantly enriched. (e) Enrichment analysis of the protein-protein interaction network based on the identified differentially expressed genes of the KEGG database. (f) Immunofluorescent staining of KSCs (α6+, green/CD 71-, red) and proliferating cells (PCNA+, red) at day 7. (g) A histogram representing the quantification of the percentage of KSCs (α6+/CD 71-) and PCNA+ cells.

Fig. 7: (a) Macroscopic observation of tumor-bearing wounds was performed under 4 different treatment regimes. (b, c) photograph of excised tumor (b) and (c) weight 18 days after the different treatments. (d) Growth curves for different groups of tumors after different treatments. (e-g) a (e) tumor recurrence profile, (f) a body weight profile, and (g) a survival profile (n=9) for mice of different treatment groups. * P <0.05, < P <0.01, < P <0.001.

Fig. 8: (a) Images were made of HE staining, TUNEL staining and Ki67 staining (red: apoptotic cells; green: ki67+ cells; blue: nuclei) of different groups of tumor tissue. (b, c) (b) expression of Ki67 and (c) quantification of TUNEL assay results (number of cells per field). (d) representative histological images of HE staining of the major organ. * P <0.05, < P <0.01, < P <0.001.

Fig. 13: (a-c) (a) post-operative bleeding of 20 mesh SSAD, (b) 60 mesh SSAD, and (c) 200 mesh SSAD. (d, e) shows histograms of (d) bleeding time or (e) post-operative blood loss for SSAD groups of different granularity.

Fig. 14: (a) skin disinfection. (b) excision of the abdominal cavity. (c) liver exposure. An enlarged view of (d) (c). (e) Liver injury (diameter=5 mm) with a disposable biopsy punch.

Fig. 15: (a, b) (a) SSAD or (b) post-operative bleeding in the control group. (c, d) represents a histogram of (c) bleeding time and (d) post-operative blood loss.

Embodiment one: materials and methods

Preparation and characterization of ssad

Preparation of ssad, SEM and FT-IR measurements

As previously described (reference 17), back mucus of chinese giant salamanders was collected to obtain giant salamander skin secretions (also understood as "giant salamander mucus extract" or "giant salamander skin mucus extract"). The reason for selecting Skin Secretions (SSAD) of Chinese giant salamanders in the embodiments of the present invention is that they can represent mucous secreted by an amphibian such as a giant salamander by stimulation. In the invention, the giant salamander skin secretion is in the form of powder of freeze-dried powder. Other kinds of giant salamanders can be selected according to actual conditions. Briefly, giant salamanders begin to secrete mucus under mechanical stimulation (gently scraping their skin). After mucus was collected into a clean tube, washed with sterilized PBS, shaken and centrifuged. Next, the mucus was freeze-dried for 24h, ground into powder with a cryo-ball mill at 0 ℃ for different times and finally separated into different particle sizes (particle sizes) with corresponding mesh sizes (20, 60 and 200 mesh).

The invention particularly needs to be explained, and the experiment of the invention selects giant salamander skin secretions with 20 meshes, 60 meshes and 200 meshes to verify the drug release performance of hydrogel formed by freeze-dried powder (powder for short) of the giant salamander skin secretions with different mesh sizes. When a faster release of drug is desired, a smaller mesh powder (e.g., 20 mesh) may be selected; where a slower release of drug is desired, a smaller mesh powder (e.g., 200 mesh) may be selected. Thus, depending on the actual drug and degradation rate requirements, powders of different mesh sizes (even powders of 14-300 mesh sizes may be selected) may be selected to achieve controlled release of the drug.

Aminoguanidine is an AGE inhibitor with nucleophilic hydrazine functional group-NH ₂ NH ₂ And an α -dicarbonyl-oriented guanidine functional group-NH-C (=nh) NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Doxorubicin (also known as doxorubicin), an anthracycline, inhibits the synthesis of DNA and RNA, thereby achieving antitumor efficacy. The molecular weight, structure and application of the two medicines are different, and the porous structure of the giant salamander skin secretion hydrogel is not damaged. That is, the range of drugs that can be loaded in practice by giant salamander skin exudate hydrogels (e.g., SSAD hydrogels) is wide, so long as the proteins in the SSAD hydrogels are not denatured. Methods for denaturing proteins are known to those skilled in the art, such as chemical methods (e.g., protein denaturants, including strong acids, strong bases, and the like, Heavy metal salts, guanidine hydrochloride, urea, acetone, TCEP, proteases and some reducing agents (e.g. cysteine, ascorbic acid, beta-mercaptoethanol and DTT)) and physical methods (heat (high temperature), uv and X-ray irradiation, ultrasound, vigorous shaking or stirring, etc.). The topical drug delivery system provided by the invention is suitable for temperatures of 65 ℃ and below.

Thus, the drugs that SSAD hydrogels can carry may also include aminoguanidine and doxorubicin and pharmaceutically acceptable salts thereof, AGE inhibitors (e.g., benzoylthiazole bromide, pyridoxamine), antibiotics (e.g., penicillin, streptomycin, doxycycline), chemotherapeutic drugs (e.g., paclitaxel, homoharringtonine, cyclophosphamide, nedaplatin). SSAD hydrogels can also be loaded with lyophilized powders (e.g., protein lyophilized powders, polypeptide lyophilized powders), epidermal growth factor, and bioactive substances.

Similarly, solvents that gel the lyophilized powder of giant salamander skin exudate should not denature proteins, such as PBS (phosphate buffer), distilled water, deionized water, physiological saline (NaCl buffer), tris Buffer (TBS), citrate buffer, chlorhexidine aqueous solution, human whole blood, and human blood extract containing water. Wherein the human blood extract containing moisture comprises one or more of plasma, serum, blood cells, platelet Rich Plasma (PRP) and platelet rich plasma fibrin (PRF).

In some embodiments, SSAD hydrogels are loaded with drugs by physical encapsulation or adsorption of non-covalent interactions. In some embodiments, the drug is linked to the SSAD hydrogel by at least one of hydroxyl, carboxyl, amino.

SSAD powder and drug loaded (AG or DOX) hydrogels with different pore sizes were examined by SEM and FT-IR. SSAD powder and lyophilized SSAD hydrogels of different sizes (20, 60 and 200 mesh) were mounted on the scaffold. After gold sputter-coating (gold sputter-coating), all samples were imaged with SEM (SU 8010, ZEISS MERLIN Compact, japan) at 10kV and 5 kV. FT-IR spectra (4000-400 cm) were recorded in transmission mode for AG and DOX and drug loaded SSAD hydrogels using FT-IR spectrometer (Nicolet 670, USA) ^-1 ) And the spectral data is in terms of absorbanceAnd (5) recording units.

SSAD hydrogel swelling and degradation experiments

The swelling ratio and degradation rate of SSAD hydrogels of different particle sizes (20, 60 and 200 mesh) were evaluated in PBS and human saliva. Briefly, 100mg SSAD was gelled with PBS. Thereafter, the hydrogel was immersed in PBS or human saliva (5 mL). For SSAD hydrogel swelling experiments, at predetermined time points, the mass of SSAD hydrogel was weighed and the same volume of fresh PBS or human saliva was added back to the tube. For SSAD hydrogel degradation experiments, SSAD hydrogels were collected at predetermined time points and lyophilized for 24h. The swelling ratio was calculated according to formula (1):

.

In the formula, W _t Representing the weight, W, of SSAD hydrogels after swelling in PBS or human saliva at different time intervals _b Representing the weight of SSAD hydrogel at baseline.

The degradation rate is calculated according to the formula (2):

.

in the formula, W _t Representing the weight of SSAD hydrogel lyophilized after swelling in PBS or human saliva at different time intervals, and W _b Representing the weight of SSAD powder at baseline.

1.1.3. Controlled release of drugs in vitro

The release kinetics of SSAD hydrogels with different particle sizes (20, 60 and 200 mesh) were evaluated using AG or DOX. Briefly, 50mg AG or 10mg DOX were mixed with 500mg SSAD or 1000mg SSAD (20, 60 or 200 mesh), respectively, to form drug loaded SSAD hydrogels in PBS. Thereafter, the drug-loaded hydrogel was immersed in PBS (5 mL, pH 7.4). At predetermined time points, samples were collected and tested at a wavelength of 230nm or 480nm to determine the concentration of AG or DOX. At the same time, the same volume of PBS was added back to each tube to maintain a constant volume.

1.2 cell proliferation and migration assays

1.2.1.CCK-8 detection

200. Mu.L of the medium was plated with all 5,000 cells in each well of a 96-well plate. In incubator (37 ℃,5% CO) ₂ ) After 24h of culture, 200. Mu.L of SSAD-containing hydrogel was used instead of the culture medium for 12h and 24h. At the indicated time points, 100. Mu.L of fresh Dulbecco's Modified Eagle Medium (DMEM) and 10. Mu.L of CCK8 solution were added to each well, incubated at 37℃for 2 hours and absorbance at 450nm was measured with a microplate reader (EnSpire, perkinElmer, singapore).

1.2.2. Scratch wound healing test

For scratch test, 6X 10 cells were seeded in each well of a 6-well plate ⁵ Individual cells. And after they reached confluence (conflux), they were scratched with 200 μl of pipette tips, followed by 3 washes with PBS and incubation with DMEM containing SSAD for 24h. Cells were photographed under a microscope at 0 and 24h.

1.2.3 Transwell migration test

Based on the results of the scratch wound healing, a Transwell migration test was then performed. Briefly, 100. Mu.L of the solution contains 2X 10 ⁵ DMEM of individual cells (L929 or HUVECs) was inoculated into the upper chamber of the insert chamber, and the lower chamber was loaded with 800 μl DMEM supplemented with SSAD. After incubation for 24h, the insert cells were removed and washed, and cells that had migrated to the underside of the membrane were stained with 0.1% crystal violet and counted under a microscope as previously described (reference 50).

1.3 animal study

Hard palate mucosa wound healing model under 1.3.1. Hyperglycemia

Animals were purchased from the university of Chongqing medical science laboratory animal center and fed to the university of Chongqing medical university affiliated oral hospital animal laboratory center without Specific Pathogen (SPF).

40 (8-12 weeks) male Sprague-Dawley (SD) rats with average body weight of 200-220g were used in the present invention, and type 2 diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 60 mg/kg). Only rats with blood glucose levels greater than or equal to 250mg/dL were enrolled in subsequent studies 1 week after STZ injection. The method of inducing damage to the mucosa of the hard palate is as described in First and colleagues (ref.51). Briefly, all animals were anesthetized with 1% sodium pentobarbital (30 mg/kg) for intraperitoneal injection. The mouth was then opened with a blepharostat (blepharostat) and the palate mucosa was disinfected with iodine and 75% ethanol. Next, a full-thickness hard palate mucosal defect was formed in the rat palate (centrally located) with a disposable biopsy punch (diameter = 3 mm). All animals were randomly divided into the following four groups and treated according to their group (n=10) (these treatments were repeated daily):

-control group: blank control, without any treatment

-AG group: AG (0.4 mg/part)

SSAD group: SSAD hydrogel (5 mg SSAD powder per site)

Ssad+ag group: AG-loaded SSAD hydrogel (5 mg SSAD powder+0.4 mg AG per site)

The dose of the lyophilized powder of the skin secretion of the giant salamander is related to the size of the wound for which administration is expected. The dry weight ratio of the medicine to the giant salamander skin secretion lyophilized powder is less than or equal to 4:1, preferably 1:1. If too much drug is loaded (i.e., too little lyophilized powder of the giant salamander skin secretion) it can affect the formation of hydrogels and their viscosity (i.e., difficult to gel and less viscous) in the topical delivery system. This affects the degree of adhesion of the topical drug delivery system to the wound and thus does not allow for a dressing to cover the wound.

The dosage of topical AG (2 mg/kg) used in the present invention is negligible for systemic administration-only 1/50 of the systemic administration AG (100 mg/kg) used in some reports to promote palate wound healing.

Photographs of the wounds were taken with a digital camera (Nikon, JY67ON, japan) at the indicated time points and transmitted to a computer for analysis of closure of the palate incision with ImageJ software (National Institutes ofHealth, NIH, usa). Wound closure rate was calculated according to equation (3):

.

In the formula, S _{Initial initiation} Is the initial wound size, S _{Currently, the method is that} Is the current wound size. Each wound was measured 3 times and the average wound size was recorded. At 8 and 18 days post-surgery, 5 rats per group were euthanized to obtain the hard palate for histological analysis.

Samples were fixed in 4% paraformaldehyde for 24h and decalcified in 10% ethylenediamine tetraacetic acid (EDTA, pH 8.0) for 2-3 months. Thereafter, it was dehydrated with ethanol (60-100%) of different concentrations. Subsequently, the samples were embedded with paraffin and cut into 5- μm sections. For each sample, more than 10 slides were prepared and sampled perpendicular to the midline of the palate at the wound area of each sample. Then, a section of a relatively wide wound was selected for histological analysis and stained with HE (Solarbio, china) and Masson's trichromatism (Solarbio).

Detecting re-epithelialization rate 18 days after operation, and calculating according to a formula (4):

.

in the formula D _b Distance (3 mm) representing mucosal defect at baseline, D _n Representing the distance without epithelialization at 18 days post-surgery (fig. 19).

Furthermore, immunofluorescent staining was performed. After blocking the sections with 3% bovine serum albumin (BSA, a8020, solarbio), immunostaining was performed with primary antibody (Abcam, usa) overnight at 4 ℃. The nuclei were then counterstained with DAPI (Goodbio technologies, china) by incubation with the corresponding secondary antibody (Abcam) for 1h at room temperature. All images were obtained using an inverted fluorescence microscope (Nikon, nikon Eclipse Ti-SR, japan).

To further elucidate the mechanism by which SSAD promotes wound healing, cell proliferation of repaired tissues was assessed using PCNA staining, and transcriptomic analysis was performed. SD rats (diameter=3 mm) with a model of palate mucosal defects were randomized into SSAD groups (receiving SSAD dressing, n=16) and control groups (no treatment, n=16): on days 3 and 7, half of the rats in each group (n=8) were sacrificed and the palate mucosa was collected for transcriptome analysis. RNA extraction, purification, reverse transcription, library construction and sequencing were all performed by Shenzhen BGI Co., ltd (Shenzhen, china).

After sequencing, bioinformatics analysis was performed on the basis of the obtained database, and expression levels of genes were calculated using RSEM (v 1.2.12). Differential expression analysis was performed using PossionDis with False Discovery Rate (FDR). Ltoreq.0.001 and |Log2ratio|gtoreq.1. The Venn diagram was used to detect genes co-expressed in both groups. To gain insight into the change in phenotype, GO and KEGG enrichment analysis (http:// www.geneontology.org/and https:// www.kegg.jp /) was performed on genes co-expressed in both groups by Phyper based on the hypergeometric test (https:// en. Wikipedia. Org/wiki/hypergeometry_distribution). The significance level of the term and pathway was corrected by Bonferroni correction at a Q value of 0.05 or less. In addition, PPI network analysis of co-expressed genes was also performed by the gene/protein interaction search tool algorithm (Search Tool for the Retrieval of Interacting Genes/Proteins algorithm).

1.3.2. model of anti-tumor efficacy and wound healing

36 female C57BL/6 mice (8-10 weeks) were used in the present invention. After removal of the hair at the injection site, 1X 10 ⁶ The B16F10 cells were injected into the back of each C57BL/6 mice to form tumors. When the tumor reaches about 50mm ³ At this time, tumor-bearing C57BL6 mice were randomly grouped as follows (n=9):

-control group: blank control, without any treatment

DOX group: DOX (per part, 4 mg/kg)

SSAD group: SSAD hydrogel (8 mg SSAD powder per site)

Ssad+dox group: DOX-loaded SSAD hydrogels (8 mg SSAD powder per site+4 mg/kg DOX)

Then, a 10mm circular full-thickness skin defect was created at each tumor site, and the corresponding sets of material were used to cover the wound resulting from tumor resection. Whereas the tumor-bearing wounds of the present invention will develop crusting in a relatively dry environment, these tumor-bearing wounds are only treated with a reasonable dose of drug on day 0 to avoid irritating the wound. It should be emphasized that the principle of the dosage of the skin secretions and medicines of the giant salamander is as described above. After treatment, the body weight, tumor size and survival of the mice were recorded every 2 days. The tumor size was calculated according to equation (5):

.

wherein L represents the length of the tumor and W represents the width of the tumor.

At 18 days post-treatment, mice were sacrificed and tumors were collected, weighed and photographed. After a series of conventional treatments (tissue fixation, dehydration, embedding, sectioning), 4 groups of tissue sections were selected and histologically analyzed by HE staining. Ki67 immunohistochemistry was used to assess proliferation of tumor cells. The experimenter also performed TUNEL assays to detect any apoptotic tumor cells.

1.5 statistical analysis

All data are expressed as mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism (GraphPad software, usa). Statistical significance was assessed by using one-way analysis of variance (ANOVA) or unpaired t-test. P values of at least <0.05 are significant.

Embodiment II: structural features and drug release profile of SSAD

SSAD powders of different mesh sizes are readily available and result in hydrogels with different porous characteristics, as shown in fig. 2 a. The hydrogel formed means that the size of the SSAD particles and the associated changes in the hydration process directly affect the pore size of the hydrogel formed. Upon mixing SSAD particles with water, they will hydrate and swell through hydrogen bonds and S-S bonds between amino acid residues of SSAD proteins to form hydrogels with a porous structure. The pores on the hydrogel surface are circular and interconnected (interconnected), which may be the result of two factors: micropores are formed from the hydration of SSAD powder, while macropores are determined by the size of SSAD. This observation confirmed that denser mesh and smaller Kong Youguan and showed that the corresponding hydrogel network might be more stable.

Fourier transform infraredThe (FT-IR) spectrum further indicates the successful loading of AG or DOX molecules in SSAD as shown in FIG. 2 b. For pure AG or DOX molecules, it can be at 3440cm ^-1 And 3326cm ^-1 The absorption bands of the stretching vibrations attributable to the main alkyl chain were observed respectively (fig. 2 b-i). Before loading the SSAD with drug, the N-H stretching vibration peak is 3435cm ^-1 Appear (FIGS. 2b-ii and 2 b-iii). However, after integration with AG or DOX, 2 peaks (3274 cm ^-1 Or 3500cm ^-1 ) As shown in fig. 2b-ii and fig. 2b-iii, respectively. Given the lack of active chemical groups in drug molecules, their loading in SSAD hydrogels can be attributed primarily to physical encapsulation (physical entrapment) or adsorption by a variety of non-covalent interactions.

Drug release characteristics of SSAD hydrogels prepared from powders of different particle sizes (20, 60 and 200 mesh) were evaluated. AG (M) _w :111.55 DOX (M) _w :543.52 Selected as model drug to load into SSAD hydrogel. During the evaluation (24 h). The cumulative release of AG from 20, 60 and 200 mesh SSAD hydrogels was 64%, 56% and 50%, respectively (fig. 2 c). The cumulative release of DOX from 20, 60 and 200 mesh SSAD hydrogels was 73%, 70% and 51%, respectively (FIG. 2 d). As the size of the SSAD powder increases, the drug release rate of the corresponding SSAD hydrogel also increases. In addition, to analyze the swelling and degradation behavior of SSAD hydrogels in vitro, they were immersed in Phosphate Buffered Saline (PBS) or human saliva at 37 ℃ for 24h. At various time intervals, the swelling ratio and degradation rate were measured (fig. 9). SSAD hydrogels gradually degrade in both PBS and saliva. At the early 8 hours, the 20 mesh SSAD hydrogel absorbed water in a heavier weight than degradation. After 8 hours, all hydrogels (20, 60, 200 mesh) were gradually degraded and after 36 hours an equilibrium state was reached (fig. 9 a-b). In addition, biodegradation of SSAD hydrogels was also evaluated. The results (FIGS. 9 c-d) show that as the size of the SSAD increases, the degradation rate of the SSAD hydrogel increases, because the larger the pore size of the SSAD, the faster the degradation rate.

Furthermore, SSAD hydrogels of the same particle size appear to be more stable in saliva, with a flatter swelling rate than in PBS. This may be because certain substances (e.g., proteins contained in saliva) result in a relatively saturated (saturated) swelling environment. As a result, SSAD hydrogels, due to their stability, are a potential new dressing for the microenvironment of wounds in the oral cavity and other related wounds.

Third embodiment: in vitro cytology study

.SSAD-induced cell proliferation:

the biocompatibility of SSAD with L929 fibroblasts and Human Umbilical Vein Endothelial Cells (HUVECs) was assessed using a cell counting kit-8 (CCK-8) assay, both of which play an important role in skin and mucosal regeneration. In preliminary experiments of the present invention (FIG. 10), a range of concentrations (from 5 to 0.005 mg/mL) of SSAD were tested for cytotoxicity against L929 cells and HUVECs. Of these various concentrations, 0.1mg/mL SSAD appears to significantly promote cell proliferation and was selected for subsequent cell experiments. The CCK8 assay results showed that after 24h exposure to SSAD conditioned medium (0.1 mg/mL), L929 cells and HUVECs still showed relatively high cell viability values (105% and 110% of control medium, respectively), indicating that SSAD showed good biocompatibility (fig. 11).

.SSAD-induced cell migration:

the effect of SSAD on migration of L929 cells and HUVECs was verified in vitro using scratch (fig. 3 a) and Transwell migration (fig. 3 b) assays. SSAD conditioned medium significantly promoted lateral (lateral) migration of L929 cells (1.5 fold of control) and HUVECs (1.7 fold of control) compared to control (fig. 3 c). SSAD conditioned medium also significantly promoted transmembrane migration of L929 cells (2.1 fold of control) and HUVECs (3.0 fold of control) compared to control (fig. 3 d). Since cell migration is a critical step in wound healing, the above results indicate that SSAD can greatly increase migration of vascular endothelial cells (HUVECs) and fibroblasts (L929).

It is well known that the expression of some growth factors may be beneficial in the process of wound healing. Bioassay (biological) results show that SSAD contains a large amount of growth factors such as Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor 1 (IGF-1), stromal cell derived factor 1 (SDF-1), and the like (table 1). Based on the amount of each growth factor contained in the SSAD, the present inventors selected the maximum of two growth factors contained in the SSAD, SDF-1 and IGF-1. SSAD performed better in promoting cell behavior than SDF-1 or IGF-1 alone or both (fig. 12), indicating that this is a combined result of SSAD in promoting cell proliferation and migration.

TABLE 1 bioassay results for SSAD

Fourth embodiment: hard palate mucosa injury model for in vivo animal study

Patients receiving FGG suffer from complications associated with defects in the palate mucosa, such as pain, infection and eating discomfort, especially diabetic patients. Although diabetic ulcers have received much attention in recent years, little research has been done on healing of intraoral wounds in diabetics. To the inventors' knowledge, the present solution for the first time employs AG-loaded topical application to promote palate wound healing in the presence of hyperglycemia. SSAD hydrogels cannot be left in place stably for long periods of time due to the moist and unstable environment of the oral cavity. Thus, a relatively rapid release of AG may be a better choice for in vivo applications, which may maximize bioavailability. Furthermore, as particle size increases, the same weight of SSAD powder requires more liquid to gel, indicating that SSAD powder with larger particle size may have better hemostatic effect (fig. 13). All of these data form the basis for selecting a 20-mesh SSAD to treat the palate mucosal lesion model.

No abnormal physiological symptoms were observed throughout the experiment of the present invention. Mice treated with SSAD and AG-loaded SSAD (ssad+ag) showed less bleeding than the other two groups (fig. 4 a). Furthermore, the classical model of liver injury-hemostasis (fig. 14) suggests that SSAD can effectively adhere to wound sites, seal and stop bleeding (fig. 15). As shown in fig. 4b and table 2, animals of ssad+ag group (61.2±4.3%), AG group (55.1±9.8%) and SSAD group (56.8±13.1%) were significantly faster in wound closure than control group (37.7±9.9%) at 4 days post-operation, with ssad+ag group healing fastest. At 8 days post-surgery, the wound closure rate (78.3±5.1%) was still significantly higher for ssad+ag animals than for the control group (53.7±15.3%). At 18 days post-surgery, the wounds of ssad+ag group almost healed, with an average wound closure rate of 97.4±3.3%, which was also higher than that of the control group (88.3±9.8%), SSAD group (92.5±7.1%) and AG group (92.5±9.0%) and the difference between ssad+ag group and control group was significant (P < 0.05). Thus, ssad+ag greatly accelerates healing of oral mucosal defects in a relatively short period of time.

TABLE 2 wound closure rates for each group calculated according to equation (3)

In the present invention, the full-thickness hard palate mucosal defect is 3mm in diameter, and the inventors have also attempted to create a wound in the middle of the hard palate, which is constant for each rat. From this, the position of the wound relative to the upper jaw can be roughly calculated. More importantly, normal oral tissue consisted of epithelium, lamina propria and submucosa with small salivary glands (+) present (fig. 16). In addition, during healing, the thickness of the regenerated palate mucosa is significantly higher than normal tissue, and collagen arrangement in regenerated tissue is relatively disordered and cell infiltration is more (fig. 17). Thus, hematoxylin-eosin (HE) staining was performed to observe mucosal thickness, epithelial integrity and gland regeneration in the treatment group, with the black dashed line representing the boundaries of normal tissue (black arrow) and regenerated tissue (blue arrow) (fig. 4 c). Although there was no statistically significant difference in the regenerated palate mucosal thickness between 4 groups at 18 days post-surgery, the regenerated palate mucosal thickness (847.4 + -17.8 μm) of the SSAD+AG group was significantly higher than that of the SSAD group (285.4 + -5.1 μm), AG group (299.6+ -11.4 μm) and control group (248.8 + -29.8 μm) (FIGS. 18 and 4d, P < 0.001) at 8 days post-surgery, which could be attributed to tissue remodeling during wound healing. Specifically, in the early recovery stage, a large amount of extracellular matrix (ECM) is secreted by keratinocytes and fibroblasts at the wound margin, which migrate and proliferate, and then, the granulation tissue becomes mature to form a scar, characterized by sustained collagen synthesis and collagen catabolism.

Epithelial spikes (yellow arrows, fig. 4 c) are indicative of mucosal healing. The re-epithelialization rate of each group was calculated according to the criteria shown in fig. 19 (99.5±0.5%) significantly higher than that of SSAD group (93.7±3.3%), AG group (91.4±0.8%) and control group (72.3±1.9%) (fig. 4 e). More interestingly, little saliva has previously been available for regeneration in the area of full-thickness mucosal defects. In the present invention, some salivary glands (+) were observed in the ssad+ag group, whereas none in AG, SSAD and control groups. Accessory (appage) regeneration is one of the key indicators of scar-free healing.

Collagen is the main component of the lamina propria, and the balance of collagen synthesis and degradation plays an important role in wound regeneration. Masson's trichromatography was performed to observe collagen deposition (fig. 4 f). At 8 days post-surgery (i.e., the relatively early stages of wound healing), the rapidly increasing collagen fibers in the ssad+ag group were densely arranged in a coarse bundle-like morphology. At 18 days post-surgery, regenerated collagen from ssad+ag and SSAD groups gradually rebuild to normal levels with a more dense fibrous matrix arrangement (fig. 4f,40×); however, the collagen fibers of AG and control groups were still in a relatively disordered arrangement (fig. 4 f). The above data show that matrix collagen deposition in ssad+ag group gradually rebuilds to normal level at a relatively late stage of wound healing, suggesting that AG-loaded SSAD can significantly promote tissue regeneration under hyperglycemia by accelerating collagen synthesis and maturation. The above combination effect was superior to SSAD or AG alone and far superior to the blank. Thus, AG-loaded SSAD can be considered as an effective method of promoting wound healing in diabetes.

Immunofluorescent staining of related biomarkers related to tissue regeneration was also performed to compare the regeneration effect between 4 groups. CD31 (red) is used to stain vascular endothelial cells, which demonstrates neovascularization; col-1 (green) was used to stain collagen, an important marker for ECM deposition (fig. 5 a). Alpha-smooth muscle actin (alpha-SMA, red) was used to stain myofibroblasts, which is one of the causes of contraction of mature blood vessels, while macrophage marker CD68 (green) was used to assess inflammation levels (fig. 5 b).

Cd31+ and Col-1+ cells expressed at 18 days post-surgery in each microscopic field of ssad+ag treated group (38.6±6.5%, or 33.0±4.2%) were significantly higher than control group (1.2±0.5%, or 3.4±1.2%), SSAD group (16.1±4.0%, or 11.8±4.3%) and AG group (8.9±2.6%, or 7.4±2.1%) (fig. 5 c). The expression density of α -sma+ cells (2.1±0.8%) was significantly higher in each microscopic view of ssad+ag treated group than in control group (0±0%), SSAD group (0.5±0.4%) and AG group (0.3±0.3%) (fig. 5 d). Cd68+ cells were expressed at a significantly lower density (0.6±0.2%) in each microscopic field of ssad+ag treated group than in control group (11.5±2%), SSAD group (1.3±0.3%) and AG group (1.3±0.3%) (fig. 5 d). Separate fluorescence images of fig. 5a are provided to assess collagen deposition and neovascularization (fig. 20 and 21). These results further indicate that AG-loaded SSAD can enhance SSAD's performance in promoting angiogenesis, enhancing ECM remodeling, and reducing inflammation.

Overall, by releasing AG in a controlled manner and promoting proliferation and migration of L929 cells and HUVECs, AG-loaded SSAD is believed to promote granulation tissue formation and neovascularization through interactions between keratinocytes during the proliferative phase (proliferative phase).

To further elucidate the basic mechanism of SSAD in promoting wound healing, transcriptomic analysis of regenerated oral mucosa was performed. The Venn diagram (FIG. 6 a) shows that 994 Differentially Expressed Genes (DEGs) are co-expressed in both groups. A thermal diagram of the DEGs after SSAD treatment is shown in fig. 6 b. The protein-protein interaction (PPI) network corresponding to co-expressed DEGs was analyzed and the color intensity (blue to red) and size of each node clearly represented their association with other protein molecules. As shown in fig. 6c, PPI analysis revealed that the major relevant signal molecules are collagen-related proteins, such as Col1a1, col3a1 and Col6a1. Analysis of the kyoto gene and genome encyclopedia (KEGG) pathway enrichment of upregulated DEGs (fig. 6 d) showed that these genes were significantly enriched in collagen digestion and uptake, ECM-receptor interactions and Cell Adhesion Molecules (CAMs), suggesting that SSAD may accelerate cell adhesion, collagen deposition and ECM production. Furthermore, although not significant, some genes are also enriched in other signaling pathways, such as signaling pathways that regulate stem cell pluripotency, suggesting that SSAD may also promote wound healing by recruiting stem cells. Functional enrichment analysis was performed based on KEGG database and the first 20 Gene Ontology (GO) terminology is shown in fig. 6e, where the biological processes of significant enrichment are positive regulation of ECM formation, cell adhesion and migration, ECM organization (organization), collagen fibrosis, multicellular organism development and cell-matrix adhesion associated with cell-matrix adhesion. All transcriptome results above were confirmed by immunofluorescent staining of the corresponding antibodies. The results in FIG. 5 show that positive staining rates for Col-1, CD31 and α -SMA were high in the SSAD-related group following SSAD treatment, consistent with transcriptome data. The above results indicate that by SSAD treatment, pathways and biological processes associated with wound healing are activated, such as ECM-receptor interactions, cell-matrix adhesion and ECM organization.

Wound healing can generally be divided into 4 phases: hemostasis, inflammation, cell proliferation, and tissue remodeling. Activation of stem cells and efficient recruitment to the wound area are key to promoting wound re-epithelialization. Homing of Keratinocyte Stem Cells (KSCs) to the wound site plays an important role in wound healing and can maintain and repair epithelial tissue and preserve the proliferative potential of the tissue due to its ability to differentiate into various functional cells. Bioassay results show that ssay contains a large amount of growth factors such as VEGF, IGF-1, SDF-1, etc. (Table 1). Most growth factors are thought to promote cell proliferation and migration, thereby promoting wound healing. In particular, SDF-1 can recruit stem cells to a wound site; at the wound site, stem cells differentiate into endothelial cells and fibroblasts, which become myofibroblasts by cell activation/proliferation or mesenchymal transformation (mesenchymal transition), which play a key role in soft tissue regeneration. In addition, IGF-1 expression also plays an important role in mediating wound healing. In the present invention, to further verify whether SSAD can recruit endogenous stem cells and activate cell adhesion and migration pathways, experimental staff assessed stem cell recruitment and cell proliferation in regenerated tissues by immunofluorescence, as demonstrated by transcriptome analysis described above.

Immunofluorescent staining of KSCs (. Alpha.6+/CD 71-) and staining of Proliferating Cell Nuclear Antigen (PCNA) were performed on SSAD treated and control blanks. The results (fig. 6f and 6 g) show that at 7 days post-surgery, SSAD treated groups had significantly more KSC recruited to the wound site than control groups (P < 0.001). At the same time point, the number of pcna+ keratinocytes was also significantly greater in SSAD treated group than in control group.

Based on the above results, it can be concluded that: SSAD can promote KSC recruitment to the wound site and maintain a larger pool of regenerated epithelial keratinocytes that accelerate wound healing. In addition, SSAD can promote cell adhesion and deposition of ECM and collagen, making it a promising dressing for soft tissue regeneration.

Fifth embodiment: tumor-bearing wound healing model for in vivo animal research

Encouraged by the excellent performance of in vitro drug delivery and controlled release in the above experiments, further proposed the concept of synergistic combination of local chemotherapy and tissue regeneration to treat skin tumors. For the tumor-bearing wound healing model, a relatively low release rate of DOX may be advantageous for melanoma-bearing wound healing given the strong irritation of topical application of DOX, at the same total dose of DOX. From this, a 200 mesh SSAD hydrogel was ultimately selected that could release drug more stably and continuously. The incidence of malignant melanoma is rising in many areas, and melanoma can occur in the skin or mucous membranes. Among them, cutaneous malignant melanoma is most common in caucasians, while mucosal malignant melanoma is most common in xanthogenic people (e.g., japanese). Malignant melanoma in the oral cavity mostly occurs in the upper gums and hard palate. Thus, melanoma is selected as a tumor model.

Randomly dividing the B16F10 tumor-bearing mice into 4 groups: control, SSAD, DOX, and ssad+dox. Fig. 7a shows a photograph of tumor-bearing wound healing at the indicated time points. The photograph of resected tumor (fig. 7 b) can further intuitively illustrate the therapeutic effect of each group after 18 days of each treatment.

The resected tumors of each group were also weighed (fig. 7 c) and measured for size (fig. 7 d). The average tumor size of the control group increased rapidly and exceeded 4,000mm at 18 days ³ And 2.8g, with the highest tumor recurrence rate (up to 67%) (fig. 7 e). Interestingly, tumor volume of SSAD hydrogel groups increased slowly and the average tumor size (1,500 mm ³ 1.8 g) was smaller than the control group, and the tumor recurrence rate was 44%, which proves that SSAD itself has exhibited antitumor activity to some extent. Slightly more pronounced tumor inhibition (1000 mm) was also observed in the DOX group than in the control group ³ 0.78 g) and the recurrence rate was 22%. However, remarkably, mice in ssad+dox group did not show tumor recurrence during the evaluation period (fig. 7 e). Meanwhile, no significant weight abnormality was found in all 4 groups (fig. 7 f). The survival rates of tumor-bearing mice in the control, DOX and SSAD groups were 78%, 89% and 100%, respectively, throughout the treatment period; however, mice in ssad+dox treated group not only survived 100% but also remained tumor-free (fig. 7 g).

These encouraging results indicate that: in the ssad+dox group, tumor cells can be effectively eliminated at an early stage by the DOX released by SSAD hydrogel, and the SSAD's ability to heal full-thickness skin defects is not significantly affected by relatively short DOX treatment. While normal skin cells in the vicinity of the DOX-loaded SSAD may also be cleared by the released DOX, SSAD may induce multiple types of cells of relatively healthy tissue to migrate to the wound site where they may participate in the subsequent wound healing process. This synergy plays an important role in the subsequent tissue healing process. More importantly, SSAD contains a large number of growth factors that recruit stem cells during the wound healing process and activate the wound healing-related signaling pathways and biological processes, thereby promoting neovascularization, ECM production, and collagen deposition. At the same time, SSAD itself mildly inhibits tumor effects, probably due to immunomodulation of the recruited stem cells. In addition, SSAD hydrogels composed of 200 mesh fines can limit the exchange of nutrients with metabolites. While this limitation may be very weak and have no significant effect on normal tissue, solid tumors that require more nutrition and oxygen and produce more waste during rapid growth may be better inhibited.

Finally, tumor tissue on day 18 was subjected to HE, terminal deoxynucleotidyl transferase mediated notch end labeling (TUNEL) and Ki67 staining (fig. 8 a). HE images revealed that ssad+dox group induced the most tumor cell destruction. As shown by TUNEL staining, the highest expression of apoptotic fluorescence was seen in ssad+dox groups compared to the other three groups. In addition, ki67+ tumor cell expression was hardly seen in ssad+dox group, indicating that the DOX loaded SSAD wound dressing significantly reduced proliferation of tumor cells. Quantitative analysis of apoptotic cells and ki67+ tumor cells is shown in fig. 8b and 8 c. Overall, it is evident that ssad+dox has the best in vivo inhibition of melanoma compared to the other groups. Furthermore, there were no significant histopathological changes in the major organs after the different treatments in the present invention (fig. 8 d), suggesting that SSAD could be a safe vehicle for local drug delivery.

Summarizing:

in recent years, local drug delivery has received increasing attention. However, in certain settings (e.g., wound surfaces in the oral cavity or after tumor resection), the therapeutic effect of local drug delivery remains limited. The topical delivery system provided by the present invention has proven to be an effective topical sustained release system for a variety of drugs (e.g., AG and DOX).

The aminoguanidine-or doxorubicin-loaded topical drug delivery system of the present invention demonstrated controlled drug release and healing-promoting properties and was validated in diabetic wounds (diabetic rat palatine mucosal defect model) or wounds after tumor resection (C57 BL/6 mouse back melanoma model), respectively, as shown in fig. 1. Both wounds are dynamic wounds, wherein the intraoral diabetic wound is a wet wound under hyperglycemia of diabetes, which is disturbed by pressure and bacteria such as chewing; the wound after back skin tumor resection is a dry wound in the tumor environment that is stretched and at risk of developing a tumor again. The local drug delivery system provided by the invention can stably adhere to a dynamic wound for a long time and keep good fit even in an environment which is easily disturbed and changeable, and the viscosity and the integrity are not lost due to the influence of the environment in which the dynamic wound is positioned; this demonstrates the excellent adhesion (wet and dry bonding) and flexibility (flexible bonding) of the topical drug delivery system provided by the present invention and ensures stable and controlled drug release. That is, the topical delivery system of the present invention performs the dual functions of drug topical delivery and promoting wound healing to promote healing of the aforementioned dynamic wound in a difficult-to-heal state. On the other hand, in the topical delivery system of the present invention, the giant salamander skin exudate hydrogel and the loaded drug act synergistically in promoting wound healing (especially dynamic wound healing), and promote wound healing while the drug is delivered locally. Its role in promoting wound healing is in promoting cell migration and proliferation, accelerating ECM deposition (up-regulating pathways associated with cell adhesion and extracellular matrix deposition), and recruiting endogenous stem cells to the wound site.

According to the actual requirements (such as the requirement on the drug release rate and the type of wound), the hydrogel with targeted pore structure and loaded with the drug can be prepared by controlling the granularity of the corresponding giant salamander skin secretion lyophilized powder.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are all within the protection of the present invention.

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Claims (10)

1. Use of giant salamander skin secretions in the manufacture of a topical delivery system for dynamic wounds, the topical delivery system comprising:

(a) The giant salamander skin secretion freeze-dried powder is characterized in that the granularity of the giant salamander skin secretion freeze-dried powder is 14-300 meshes, and the giant salamander is giant salamander;

(b) At least one drug, the molecular weight of the at least one drug comprising 100-600;

(c) At least one solvent that gels the lyophilized powder of giant salamander skin secretion;

Wherein the at least one drug and the at least one solvent are not protein denaturants, the at least one solvent comprising one or more of pure water, distilled water, deionized water, phosphate buffer, physiological saline, tris buffer, citrate buffer, body fluid, cell culture medium, aqueous chlorhexidine solution, human whole blood, and a human blood extract containing moisture; the dry weight mixing ratio of the at least one drug and the giant salamander skin secretion freeze-dried powder is 400% or less; the at least one drug is loaded in the gelled giant salamander skin secretion lyophilized powder by physical encapsulation or adsorption of non-covalent interactions or is linked with the gelled giant salamander skin secretion lyophilized powder by at least one of hydroxyl, carboxyl, amino groups.

2. The use of claim 1, wherein the at least one drug is an advanced glycation end product inhibitor or a chemotherapeutic agent.

3. The use of claim 2, wherein the at least one drug comprises one or more of doxorubicin hydrochloride, aminoguanidine, doxorubicin, benzoyl thiazole bromide, doxycycline, streptomycin, penicillin, paclitaxel, homoharringtonine, cyclophosphamide, nedaplatin.

4. The use of claim 1, wherein the topical drug delivery system is applied to a wound site; the wound site is optionally located on the skin and/or mucous membrane; the wound site is optionally a diabetic wound or a wound after tumor resection.

5. The use according to claim 4, wherein the topical drug delivery system is for promoting cell proliferation and/or cell migration and/or recruitment of endogenous stem cells and/or hemostasis at the wound site.

6. The use according to claim 4, wherein the topical delivery system is for promoting re-epithelialization and/or angiogenesis and/or extracellular matrix remodeling and/or collagen deposition of the wound site.

7. The use according to claim 4, wherein the topical drug delivery system is for promoting mucosal healing and/or salivary gland regeneration at the wound site, wherein the wound site is in the oral cavity.

8. The use according to claim 1 or 2, wherein the topical delivery system is for inhibiting tumor cells.

9. The use according to claim 1, wherein the topical drug delivery system is in the form of a dressing.

10. A topical drug delivery system for a dynamic wound, the topical drug delivery system comprising:

(a) Giant salamander skin secretion freeze-dried powder, wherein the giant salamander skin secretion freeze-dried powder is 20-200 meshes, and the giant salamander is giant salamander;

(b) At least one drug, wherein the dry weight mixing ratio of the at least one drug and the giant salamander skin secretion freeze-dried powder is 400% or less, and the molecular weight of the at least one drug is 100-600;

(c) At least one solvent in an amount effective to gel the lyophilized powder of the giant salamander skin secretion;

wherein the at least one drug and the at least one solvent are not protein denaturants, the at least one solvent comprises one or more of pure water, distilled water, deionized water, phosphate buffer, physiological saline, tris buffer, citrate buffer, body fluid, cell culture medium, chlorhexidine aqueous solution, human whole blood, and human blood extract containing moisture, the at least one drug comprises one or more of aminoguanidine and pharmaceutically acceptable salts thereof, doxorubicin and pharmaceutically acceptable salts thereof, benzoylthiazole bromide, doxycycline, streptomycin, penicillin, paclitaxel, homoharringtonine, cyclophosphamide, and nedaplatin.

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