CN114984233B - A local drug delivery system based on salamander skin secretion and its application - Google Patents
A local drug delivery system based on salamander skin secretion and its applicationDownload PDFInfo
- Publication number
- CN114984233B CN114984233BCN202210643002.7ACN202210643002ACN114984233BCN 114984233 BCN114984233 BCN 114984233BCN 202210643002 ACN202210643002 ACN 202210643002ACN 114984233 BCN114984233 BCN 114984233B
- Authority
- CN
- China
- Prior art keywords
- ssad
- drug
- delivery system
- salamander
- drug delivery
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Translated fromChinese
Description
Translated fromChinese优先权申请priority application
.本申请要求2021年11月05日提交的中国发明专利申请【CN2021113071734】、名称为“鲵皮肤分泌物在制备局部给药系统中的应用”的优先权，该优先权发明专利申请以引用方式全文并入。.This application claims the priority of the Chinese invention patent application [CN2021113071734] filed on November 05, 2021, entitled "Application of salamander skin secretions in the preparation of local drug delivery system", which is cited by reference Incorporated in its entirety.
技术领域technical field
.本发明属于生物材料领域，具体涉及一种基于鲵皮肤分泌物的局部给药系统及其应用。. The invention belongs to the field of biological materials, in particular to a local drug delivery system based on salamander skin secretion and its application.
背景技术Background technique
.基于全身给药的传统递送方法会导致副作用和低生物利用度(bioavailability)。因此，近年来，局部给药受到越来越多的关注，因为其可以提高局部药物的生物利用度并减少药物引起的全身毒性。然而，此方法仍受一些不足之处的限制，如口腔内复杂的伤口部位或肿瘤切除后的创面。. Traditional delivery methods based on systemic administration lead to side effects and low bioavailability. Therefore, topical drug delivery has received increasing attention in recent years because it can improve the bioavailability of local drugs and reduce drug-induced systemic toxicity. However, this method is still limited by some inadequacies, such as the complex wound site in the oral cavity or the wound after tumor resection.
.牙周炎(periodontitis)和糖尿病(diabetes mellitus)是高度相关的常见慢性疾病。在糖尿病造成的高血糖环境下，晚期糖基化终末产物(AGEs)可能在牙周组织中大量积累，其随后诱导细胞凋亡、降低与伤口愈合有关的细胞增殖和迁移并抑制干细胞分化，最终损害伤口愈合的过程。尽管已经证明，全身给药氨基胍(AG，一种AGE抑制剂)可以加速糖尿病大鼠在游离龈瓣移植术(FGG)后的腭部(palatal)伤口愈合，但也有一些与使用高浓度AG有关的安全问题被报道出。此外，口腔的湿润条件和不断的咀嚼阻碍药物在局部的保留。.Periodontitis and diabetes mellitus are highly correlated common chronic diseases. Under the hyperglycemic environment caused by diabetes, advanced glycation end products (AGEs) may accumulate in large quantities in periodontal tissues, which subsequently induce apoptosis, reduce cell proliferation and migration related to wound healing, and inhibit stem cell differentiation, Ultimately impairing the wound healing process. Although it has been shown that systemic administration of aminoguanidine (AG, an AGE inhibitor) can accelerate palatal wound healing in diabetic rats after free gingival flap grafting (FGG), there are some studies related to the use of high concentrations of AG. Related security issues were reported. In addition, the moist conditions of the oral cavity and constant chewing hinder the local retention of the drug.
.另一方面，恶性黑色素瘤被认为是最具侵略性和高度转移性的皮肤肿瘤之一，而手术切除仍是临床上最常用的治疗方法。手术切除后可能残留的黑色素瘤细胞容易导致癌症复发。因此，局部切除结合全身化疗是一种常见的治疗方法。然而，化疗药物的非特异性分布和波动的血液浓度总导致严重的副作用并给患者带来很大的负担。因此，局部化疗被越来越多地使用，以最小化对健康组织的药物暴露(drug exposure)，同时降低局部复发的风险。然而，大多数化疗药物如多柔比星(DOX)，特异性相对较差且不能区分正常细胞和癌细胞。. On the other hand, malignant melanoma is considered to be one of the most aggressive and highly metastatic skin tumors, and surgical resection is still the most commonly used treatment in clinic. Melanoma cells that may remain after surgical removal predispose the cancer to relapse. Therefore, local excision combined with systemic chemotherapy is a common treatment. However, the nonspecific distribution and fluctuating blood concentrations of chemotherapy drugs always lead to severe side effects and impose a great burden on patients. Therefore, local chemotherapy is increasingly used to minimize drug exposure to healthy tissue while reducing the risk of local recurrence. However, most chemotherapy drugs, such as doxorubicin (DOX), are relatively poorly specific and cannot distinguish normal cells from cancer cells.
.理想情况下，用于伤口愈合的局部药物递送系统应划算、安全，并具备多种功能，如控释(controlledrelease)、局部保持(local retention)、可生物降解和促愈合能力。.Ideally, a topical drug delivery system for wound healing should be cost-effective, safe, and multifunctional, such as controlled release, local retention, biodegradability, and healing-promoting capabilities.
发明内容Contents of the invention
.一方面，本发明提供了鲵皮肤分泌物在制备用于动态伤口的局部给药系统中的应用，其特征在于，所述局部给药系统包括：. On the one hand, the present invention provides the application of salamander skin secretion in the preparation of the topical drug delivery system for dynamic wound, it is characterized in that, described topical drug delivery system comprises:
(a)鲵皮肤分泌物冻干粉，所述鲵皮肤分泌物冻干粉的粒度为14-300目；(a) lyophilized powder of salamander skin secretion, the particle size of the lyophilized powder of salamander skin secretion is 14-300 mesh;
(b)至少一种药物；(b) at least one drug;
(c)至少一种溶剂，所述至少一种溶剂使所述鲵皮肤分泌物冻干粉凝胶化；其中，所述至少一种药物和所述至少一种溶剂不属于蛋白变性剂。(c) at least one solvent, which gels the lyophilized powder of salamander skin secretion; wherein, the at least one drug and the at least one solvent are not protein denaturants.
.在一个实施方案中，所述药物为晚期糖基化终末产物抑制剂或化疗药物。. In one embodiment, the drug is an inhibitor of advanced glycation end products or a chemotherapeutic drug.
.在一个实施方案中，所述药物的分子量为100-600。. In one embodiment, the drug has a molecular weight of 100-600.
.在一个实施方案中，所述药物包括盐酸多柔比星、盐酸氨基胍、氨基胍、多柔比星、苯酰基噻唑溴化物、多西环素、链霉素、青霉素、紫杉醇、高三尖杉酯碱、环磷酰胺、奈达铂中的一种或多种。.In one embodiment, the drug includes doxorubicin hydrochloride, aminoguanidine hydrochloride, aminoguanidine, doxorubicin, benzoylthiazole bromide, doxycycline, streptomycin, penicillin, paclitaxel, triacine One or more of paclitaxel, cyclophosphamide, and nedaplatin.
.在一个实施方案中，所述鲵包括大鲵属、隐鳃鲵属、山溪鲵属、小鲵属、巴鲵属、爪鲵属、肥鲵属、拟小鲵属、北鲵属、极北鲵属中的一种或多种。. In one embodiment, the salamanders include giant salamanders, cryptobranch salamanders, mountain salamanders, small salamanders, genus genus, clawed salamanders, fat salamanders, pseudobranchus, northern salamanders, polar salamanders, One or more species of salamanders.
.在一个实施方案中，所述局部给药系统被施用于伤口部位。. In one embodiment, the topical drug delivery system is applied to a wound site.
.在一个实施方案中，所述伤口部位位于皮肤和/或粘膜。. In one embodiment, the wound site is located on the skin and/or mucous membranes.
.在一个实施方案中，所述伤口部位为糖尿病性创面或肿瘤切除后的创面。. In one embodiment, the wound site is a diabetic wound or a wound after tumor resection.
.在一个实施方案中，所述局部给药系统用于促进所述伤口部位的细胞增殖和/或细胞迁移和/或内源性干细胞的募集和/或止血。. In one embodiment, said topical delivery system is used to promote cell proliferation and/or cell migration and/or recruitment of endogenous stem cells and/or hemostasis at said wound site.
.在一个实施方案中，所述局部给药系统用于促进所述伤口部位的再上皮化和/或血管生成和/或细胞外基质重塑和/或胶原蛋白沉积。. In one embodiment, said topical delivery system is used to promote re-epithelialization and/or angiogenesis and/or extracellular matrix remodeling and/or collagen deposition at said wound site.
.在一个实施方案中，所述局部给药系统用于促进所述伤口部位的粘膜愈合和/或唾液腺再生，其中所述伤口部位位于口腔。. In one embodiment, said topical delivery system is used to promote mucosal healing and/or salivary gland regeneration at said wound site, wherein said wound site is located in the oral cavity.
.在一个实施方案中，所述局部给药系统用于抑制肿瘤细胞。. In one embodiment, the topical delivery system is used to inhibit tumor cells.
.在一个实施方案中，所述局部给药系统为敷料的形式。. In one embodiment, the topical delivery system is in the form of a dressing.
.另一方面，本发明还提供了一种用于动态伤口的局部给药系统，其特征在于，所述局部给药系统包括：. On the other hand, the present invention also provides a local drug delivery system for dynamic wounds, characterized in that, the local drug delivery system includes:
(a)鲵皮肤分泌物冻干粉，所述鲵皮肤分泌物冻干粉为20-200目；(a) lyophilized powder of salamander skin secretion, the lyophilized powder of salamander skin secretion is 20-200 mesh;
(b)至少一种药物，所述药物与所述鲵皮肤分泌物冻干粉的干重混合比例为小于等于400％，所述药物的分子量为100-600；(b) at least one drug, the dry weight mixing ratio of the drug and the lyophilized powder of salamander skin secretion is less than or equal to 400%, and the molecular weight of the drug is 100-600;
(c)至少一种溶剂，其量为有效使所述鲵皮肤分泌物冻干粉凝胶化；(c) at least one solvent in an amount effective to gel the lyophilized powder of the salamander skin secretion;
其中，所述至少一种药物和所述至少一种溶剂不属于蛋白变性剂。Wherein, the at least one drug and the at least one solvent are not protein denaturants.
.在一个实施方案中，所述药物包括氨基胍、多柔比星和其药学上可接受的盐中的一种或多种。. In one embodiment, the drug comprises one or more of aminoguanidine, doxorubicin, and pharmaceutically acceptable salts thereof.
.如本文所使用，“局部给药系统”包括生物来源的水凝胶和药物。所述生物来源的水凝胶为由鲵皮肤分泌物冻干粉和溶剂生成的水凝胶，优选为大鲵皮肤分泌物(SSAD)水凝胶。所述药物可以溶解于溶剂中，也可以部分溶解或不溶解。所述药物和所述溶剂不会使蛋白质变性(即不属于蛋白变性剂)。示例性的蛋白变性剂包括强酸、强碱、重金属盐、盐酸胍、尿素、丙酮、TCEP、蛋白酶和一些还原剂(例如半胱氨酸、抗坏血酸、β-巯基乙醇和DTT)等。示例性的药物包括AGE抑制剂和化疗药物。所述药物的分子量可以是100-600。所述局部给药系统通过所述水凝胶的降解来实现对所述药物的控释。由于鲵皮肤分泌物冻干粉遇水成胶，因此所述溶剂可以为任何能够使鲵皮肤分泌物冻干粉稳定成胶的液体。也就是说，临床普遍使用的试剂都可以使鲵皮肤分泌物冻干粉成胶。示例性的溶剂可以为生理盐水等缓冲液、纯水、伤口处的液体等体液、细胞培养基。. As used herein, "topical drug delivery system" includes hydrogels and drugs of biological origin. The hydrogel of biological origin is a hydrogel produced by lyophilized powder of salamander skin secretion and a solvent, preferably giant salamander skin secretion (SSAD) hydrogel. The drug may be dissolved in the solvent, partially dissolved or not dissolved. The drug and the solvent do not denature proteins (ie 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 (such as cysteine, ascorbic acid, β-mercaptoethanol, and DTT), among others. Exemplary drugs include AGE inhibitors and chemotherapy drugs. The molecular weight of the drug may be 100-600. The topical drug delivery system achieves controlled release of the drug through the degradation of the hydrogel. Since the lyophilized powder of salamander skin secretion forms a gel when it meets water, the solvent can be any liquid that can stabilize the lyophilized powder of salamander skin secretion into a gel. That is to say, the reagents commonly used in clinic can make the lyophilized powder of salamander skin secretion into gel. Exemplary solvents may be buffer solutions such as physiological saline, pure water, body fluids such as wound fluid, and cell culture media.
.如本文所使用，“生物来源的”是指源自或取自天然存在的有机体和有机体的部分。换句话说，本发明的局部给药系统的主要成分——生物来源的水凝胶并非是通过基因重组技术获得的。. As used herein, "biologically derived" means derived from or taken from naturally occurring organisms and parts of organisms. In other words, the main component of the topical drug delivery system of the present invention—biologically derived hydrogel is not obtained through genetic recombination technology.
.如本文所使用，“伤口”是指个体皮肤或粘膜中的任何缺损。伤口部位的组织结构的连续性或完整性遭到破坏。伤口可能是由于擦伤、撕脱伤、撕裂伤、穿刺、癌症、糖尿病性溃疡或病变、烧伤、手术或其他损伤造成的。“伤口愈合”是指组织完整性的部分或完全恢复。在一个实施例中，所述伤口部位位于皮肤。在另一个实施例中，所述伤口位于粘膜。“促进伤口愈合”应理解为使皮肤或粘膜从连续性或完整性的中断中恢复或部分恢复。. As used herein, "wound" refers to any defect in the skin or mucous membrane of an individual. The continuity or integrity of the tissue structure at the wound site is disrupted. Wounds may result from abrasions, avulsions, lacerations, punctures, cancer, diabetic ulcers or lesions, burns, surgery, or other injuries. "Wound healing" refers to partial or complete restoration of tissue integrity. In one embodiment, the wound site is on the skin. In another embodiment, the wound is on a mucosa. "Promoting wound healing" is understood as restoring or partially restoring the skin or mucous membranes from a break in continuity or integrity.
.如本文所使用，“动态伤口”是指伤口创面持续受到其他干扰因素影响而一直或时常处于变化状态，以至于较为难以愈合的伤口。所述干扰因素可以是压力(例如，容易受来自咀嚼压力影响的位于口腔内的伤口)、活动拉扯(例如，伤口创面处于容易发生活动、移动的位置，因此容易受到因活动、移动而产生的拉伸力的影响)。所述干扰因素还可以是因为极端环境导致伤口创面持续处于一种不稳定的状态，伤口创面处易发生病理变化(例如，因高血糖、肿瘤等疾病或症状导致伤口创面处容易肿胀、感染、溃烂、流脓、坏死等)。.As used herein, "dynamic wound" refers to a wound whose surface is continuously or constantly changing under the influence of other disturbing factors, so that it is difficult to heal. The interfering factors can be pressure (for example, a wound located in the oral cavity that is easily affected by the pressure from mastication), active pulling (for example, the wound surface is in a position that is prone to activity and movement, and is therefore vulnerable to movement and movement. effect of tension). The interference factor can also be that the wound surface is continuously in an unstable state due to the extreme environment, and the wound surface is prone to pathological changes (for example, due to diseases or symptoms such as hyperglycemia and tumors, the wound surface is prone to swelling, infection, ulceration, pus, necrosis, etc.).
.如本文所使用，“糖尿病性创面”是指由糖尿病(特别是高血糖)引发的且在糖尿病的高血糖等环境下延迟愈合或难以愈合的伤口。. As used herein, "diabetic wound" refers to a wound that is induced by diabetes (particularly hyperglycemia) and whose healing is delayed or difficult to heal under the hyperglycemia and other circumstances of diabetes.
.如本文所使用，“AGE抑制剂”是指抑制AGE(晚期糖基化终末产物)生成和/或活性的物质。示例性的AGE抑制剂包括苯甲酰基噻唑溴化物、吡哆胺、氨基胍。. As used herein, "AGE inhibitor" refers to a substance that inhibits the production and/or activity of AGE (Advanced Glycation End Products). Exemplary AGE inhibitors include benzoylthiazole bromide, pyridoxamine, aminoguanidine.
.如本文所使用，“化疗药物”是指用于治疗肿瘤(或癌症)的药物，例如烷基化剂(例如环磷酰胺)、代谢拮抗剂(例如，5-氟尿嘧啶)、抗癌抗生素(例如，多柔比星)、植物类抗癌剂(例如，紫杉醇)、铂类药物等。. As used herein, "chemotherapy drug" refers to a drug used to treat tumors (or cancer), such as alkylating agents (eg, cyclophosphamide), metabolic antagonists (eg, 5-fluorouracil), anticancer antibiotics ( For example, doxorubicin), plant-based anticancer agents (eg, paclitaxel), platinum-based drugs, and the like.
.如本文所使用，“溶胀比”是指水凝胶的重量因液体吸收而增加的部分，或因溶解而减少的部分。. As used herein, "swell ratio" refers to the portion by which the weight of a hydrogel increases due to liquid absorption, or decreases due to dissolution.
.如本文所使用，“协同作用”是指明显大于每个组分单独施用至伤口所预期效果的结果。在一个具体实施例中，本发明提供的局部给药系统中SSAD水凝胶与所装载的药物AG或DOX具有协同作用。As used herein, "synergy" refers to a result that is significantly greater than the expected effect of each component applied individually to a wound. In a specific embodiment, the SSAD hydrogel in the local drug delivery system provided by the present invention has a synergistic effect with the loaded drug AG or DOX.
.如本文所使用，“再生”是组织或器官受外部作用而部分缺失，在剩余部分的基础上又生长出与缺失部分在形态和/或功能上相同的结构的修复过程。. As used herein, "regeneration" is a repair process in which a tissue or organ is partially lost due to external effects, and a structure identical in shape and/or function to the missing part grows on the basis of the remaining part.
.如本文所使用，“促进”是指所描述对象在既有水平的进一步提高，所述既有水平包括数量水平、表达水平、功能水平、能力水平中的一种或多种。. As used herein, "enhancing" refers to the further improvement of the described object at the existing level, and the existing level includes one or more of the quantitative level, the expression level, the functional level, and the ability level.
附图说明Description of drawings
.为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。. In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Apparently, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort.
.图1为本发明使用的两种动物模型以及装载药物的鲵皮肤分泌物(SSAD)水凝胶促进伤口愈合的可能机制的示意图；Fig. 1 is a schematic diagram of two kinds of animal models used in the present invention and the possible mechanism of salamander skin secretion (SSAD) hydrogel loaded with drugs to promote wound healing;
.图2示出了SSAD的结构特征及其持续的药物释放特性；. Figure 2 shows the structural features of SSAD and its sustained drug release properties;
.图3示出了SSAD诱导的细胞迁移；. Figure 3 shows SSAD-induced cell migration;
.图4示出了腭部的伤口愈合率的评估；. Figure 4 shows the assessment of the wound healing rate of the palate;
.图5示出了术后18天时腭部伤口的免疫荧光染色图像；. Figure 5 shows the immunofluorescence staining image of the palatal wound at 18 days after operation;
.图6示出了SSAD对伤口愈合的治疗机制；. Figure 6 shows the therapeutic mechanism of SSAD on wound healing;
.图7示出了装载DOX的SSAD水凝胶的体内抗癌效率；. Figure 7 shows the in vivo anticancer efficiency of DOX-loaded SSAD hydrogels;
.图8示出了体内微观的抗癌效率；. Figure 8 shows the microscopic anti-cancer efficiency in vivo;
.图9示出了SSAD水凝胶在PBS和唾液中的溶胀比和降解率；. Figure 9 shows the swelling ratio and degradation rate of SSAD hydrogel in PBS and saliva;
.图10示出了与不同浓度的SSAD培养24h的L929细胞和HUVECs的相对细胞活力值；.Figure 10 shows the relative cell viability values of L929 cells and HUVECs cultivated with different concentrations of SSAD for 24h;
.图11示出了与SSAD(0.1mg/mL)培养24h的L929和HUVECs的相对细胞活力值；.Figure 11 shows the relative cell viability values of L929 and HUVECs cultivated with SSAD (0.1mg/mL) for 24h;
.图12示出了与SSAD、IGF-1、SDF-1或SDF-1+IGF-1培养的L929细胞和HUVECs的划痕实验结果图；. Figure 12 shows the results of the scratch experiment of L929 cells and HUVECs cultured with SSAD, IGF-1, SDF-1 or SDF-1+IGF-1;
.图13示出了建立体内尾静脉损伤止血模型来验证不同粒度SSAD粉末的止血特性；.Figure 13 shows the establishment of a tail vein injury hemostatic model in vivo to verify the hemostatic properties of SSAD powders with different particle sizes;
.图14示出了建立体内肝损伤止血模型来验证SSAD的止血能力；. Figure 14 shows the establishment of a hemostatic model of liver injury in vivo to verify the hemostatic ability of SSAD;
.图15示出了体内肝损伤止血模型证明，SSAD可以有效粘附到伤口部位并止血；. Figure 15 shows that the hemostatic model of liver injury in vivo demonstrates that SSAD can effectively adhere to the wound site and stop bleeding;
.图16为正常腭部组织的HE图像(上皮(黄色箭头)，固有层(黑色箭头)，粘膜下层(蓝色箭头)，粘膜腺(●))；.Figure 16 is the HE image of normal palate tissue (epithelium (yellow arrow), lamina propria (black arrow), submucosa (blue arrow), mucosal glands (●));
.图17为正常组织和再生的组织之间的边界的HE图像(正常组织(黑色箭头)，再生的组织(蓝色箭头)，细胞浸润(*))；. FIG. 17 is a HE image of the boundary between normal tissue and regenerated tissue (normal tissue (black arrow), regenerated tissue (blue arrow), cell infiltration (*));
.图18为在术后8天，测量再生的腭部粘膜厚度的代表性HE图像；. FIG. 18 is a representative HE image measuring the thickness of the regenerated palatal mucosa at
.图19示出了对每组的再上皮化定量(黑色箭头表示基线的伤口的距离，红色箭头表示术后18天没有上皮化的距离)；. Figure 19 shows the quantification of re-epithelialization for each group (black arrows indicate the distance of the wound at baseline, red arrows indicate the distance without
.图20为图5a的单独的绿色荧光图像(以评估胶原沉积和炎症细胞浸润)；. Figure 20 is a separate green fluorescence image of Figure 5a (to assess collagen deposition and inflammatory cell infiltration);
.图21为图5a的单独的红色荧光图像(以评估胶原沉积和新血管生成)。. Figure 21 is a single red fluorescence image of Figure 5a (to assess collagen deposition and neovascularization).
具体实施方式Detailed ways
.为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
.需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。.It should be noted that, in this document, the terms "comprising", "comprising" or any other variation thereof are intended to cover a non-exclusive inclusion such that a process, method, article or apparatus comprising a set of elements includes not only those elements , but also includes other elements not expressly listed, or also includes elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the phrase "comprising a ..." does not preclude the presence of additional identical elements in the process, method, article, or apparatus comprising that element.
.如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。. As used in this specification, the term "about", typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 4% of the stated value +/-3%, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, even more typically +/-0.5% of the stated value.
.在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围1～6的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。. In this specification, certain embodiments may be disclosed in a format within a certain range. It should be understood that this description "within a certain range" is merely for convenience and brevity, and should not be construed as an inflexible limitation on the disclosed scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a
.附图详细说明.Detailed description with drawings
.图2：(a)示出了20、60和200目的SSAD粉末的尺寸和相应形成的水凝胶的多孔结构的SEM图像。(b)i)AG和DOX、ii)装载AG的不同孔径(20、60和200目)的SSAD水凝胶和iii)装载DOX的不同孔径(20、60和200目)的SSAD水凝胶的FR-IR特征。(c，d)不同孔径(20、60和200目)的SSAD水凝胶中(c)AG和(d)DOX的累积释放谱。. Figure 2: (a) SEM images showing the size of 20, 60 and 200 mesh SSAD powders and the porous structure of the corresponding formed hydrogels. (b) i) AG and DOX, ii) SSAD hydrogels with different pore sizes (20, 60 and 200 mesh) loaded with AG and iii) SSAD hydrogels with different pore sizes (20, 60 and 200 mesh) loaded with DOX FR-IR characteristics. (c,d) Cumulative release profiles of (c) AG and (d) DOX from SSAD hydrogels with different pore sizes (20, 60, and 200 mesh).
.图3：(a)为有或没有SSAD培养的L929细胞和HUVECs的划痕试验。(b)通过Transwell试验进一步证实，有或没有SSAD处理的L929细胞和HUVECs的迁移能力。(c，d)(c)划痕试验中的相对迁移面积和(d)Transwell试验中从上室迁移到下室的细胞数量的定量分析。*P<0.05，**P<0.01，***P<0.001。. Figure 3: (a) Scratch test of L929 cells and HUVECs cultured with or without SSAD. (b) The migration ability of L929 cells and HUVECs with or without SSAD treatment was further confirmed by Transwell assay. (c,d) Quantification of relative migration area in (c) scratch test and (d) number of cells migrating from upper chamber to lower chamber in Transwell assay. *P<0.05, **P<0.01, ***P<0.001.
.图4：(a)伤口的宏观观察。(b)用公式(3)计算的伤口闭合率。(c)术后18天的伤口部位的HE染色图像。(d)术后8天，不同组别的新生的组织厚度。(e)术后18天，不同组别的再上皮化(re-epithelialization)率。(f)第8天和第18天时伤口部位的Masson's三色染色图像，虚线显示正常组织和再生的组织之间的边界。正常组织(黑色箭头)、再生的组织(蓝色箭头)、再上皮化(黄色箭头)、腺体再生(●)。*P<0.05，**P<0.01，***P<0.001。. Figure 4: (a) Macroscopic observation of the wound. (b) Wound closure rate calculated using formula (3). (c) HE-stained image of the
.图5：(a)血管内皮细胞(CD31，红色)和胶原蛋白I(Col-1，绿色)的免疫荧光染色。(b)肌成纤维细胞(α-SMA，红色)和巨噬细胞(CD68，绿色)的免疫荧光染色。细胞核用4',6-二脒基-2-苯基吲哚(DAPI，蓝色)染色，并且双重染色的组织用黄色荧光识别。(c，d)表示第18天的(c)CD31+细胞和Col-1的百分比和(d)肌成纤维细胞(α-SMA+)和巨噬细胞(CD68+)细胞的百分比的定量的直方图。. Figure 5: (a) Immunofluorescent staining of vascular endothelial cells (CD31, red) and collagen I (Col-1, green). (b) Immunofluorescent staining of myofibroblasts (α-SMA, red) and macrophages (CD68, green). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, blue), and double-stained tissues were identified with yellow fluorescence. (c, d) Histograms representing the quantification of (c) percentages of CD31+ cells and Col-1 and (d) percentages of myofibroblasts (α-SMA+) and macrophages (CD68+) cells at
.图6：(a)SSAD组和对照组之间的转录谱的维恩图。(b)SSAD治疗后显著改变的基因的热图(|log|≥2，P＜0.05)。(c)KEGG通路。(d)20个最显著富集的生物学过程。(e)基于KEGG数据库的鉴定出的差异表达基因的蛋白质-蛋白质相互作用网络的富集分析。(f)第7天时，KSCs(α6+，绿色/CD71-，红色)和增殖细胞(PCNA+，红色)的免疫荧光染色。(g)表示KSCs(α6+/CD71-)和PCNA+细胞的百分比的定量的直方图。. Figure 6: (a) Venn diagram of transcriptional profiles between SSAD and control groups. (b) Heatmap of significantly altered genes after SSAD treatment (|log|≥2, P<0.05). (c) KEGG pathway. (d) The 20 most significantly enriched biological processes. (e) Enrichment analysis of protein-protein interaction networks of identified differentially expressed genes based on KEGG database. (f) Immunofluorescent staining of KSCs (α6+, green/CD71-, red) and proliferating cells (PCNA+, red) at
.图7：(a)4种不同的处理方式下，对荷瘤伤口的宏观观察。(b，c)不同处理后的18天，切除的肿瘤的(b)照片和(c)重量。(d)不同处理后，不同组别的肿瘤的生长曲线。(e-g)不同处理的组别的小鼠的(e)肿瘤复发曲线、(f)体重曲线和(g)存活曲线(n＝9)。*P<0.05，**P<0.01，***P<0.001。.Figure 7: (a) Macroscopic observation of tumor-bearing wounds under 4 different treatment methods. (b, c) (b) photographs and (c) weights of resected
.图8：(a)为不同组别的肿瘤组织的HE染色、TUNEL染色和Ki67染色(红色：凋亡细胞；绿色：Ki67+细胞；蓝色：细胞核)图像。(b，c)(b)Ki67的表达和(c)TUNEL检测结果(每个视野的细胞数)的定量。(d)主要器官的HE染色的代表性组织学图像。*P<0.05，**P<0.01，***P<0.001。. Figure 8: (a) HE staining, TUNEL staining and Ki67 staining (red: apoptotic cells; green: Ki67+ cells; blue: nuclei) images of tumor tissues in different groups. (b,c) (b) Ki67 expression and (c) quantification of TUNEL assay results (number of cells per field of view). (d) Representative histological images of HE staining of major organs. *P<0.05, **P<0.01, ***P<0.001.
.图13：(a-c)(a)20目SSAD、(b)60目SSAD和(c)200目SSAD的术后出血。(d，e)表示不同粒度的SSAD组的(d)出血时间或(e)术后失血量的直方图。. FIG. 13 : Postoperative bleeding of (a-c) (a) 20 mesh SSAD, (b) 60 mesh SSAD and (c) 200 mesh SSAD. (d, e) Histograms representing (d) bleeding time or (e) postoperative blood loss in the SSAD group with different granularity.
.图14：(a)皮肤消毒。(b)腹腔的切除。(c)肝脏的暴露。(d)(c)的放大图。(e)用一次性活检穿孔器造成的肝脏损伤(直径＝5mm)。. Figure 14: (a) Skin disinfection. (b) Resection of the abdominal cavity. (c) Liver exposure. (d) Enlarged view of (c). (e) Liver injury (diameter = 5 mm) with a disposable biopsy punch.
.图15：(a，b)(a)SSAD或(b)对照组的术后出血。(c，d)表示(c)出血时间和(d)术后失血量的直方图。. FIG. 15 : (a,b) Postoperative bleeding in (a) SSAD or (b) control group. (c,d) Histograms representing (c) bleeding time and (d) postoperative blood loss.
.实施例一：材料与方法.Embodiment one: materials and methods
.1.1.SSAD的制备和特征.1.1. Preparation and characterization of SSAD
.1.1.1.SSAD的制备、SEM和FT-IR测量.1.1.1. Preparation, SEM and FT-IR measurement of SSAD
.如前所述(参考文献17)，收集中国大鲵的背部粘液以获得鲵皮肤分泌物(还可以理解为“鲵粘液提取物”或“鲵皮肤粘液提取物”)。本发明实施例中选择中国大鲵皮肤分泌物(SSAD)的原因在于，其能够代表鲵这种两栖动物受刺激所分泌的粘液。且在本发明中，鲵皮肤分泌物为冻干粉的粉末的形式。根据实际情况可选用其他种类的鲵。简言之，中国大鲵在机械刺激下(轻轻刮取其皮肤)开始分泌粘液。将粘液收集进干净的管子后，用灭菌的PBS洗涤、震荡并离心。接下来，将粘液冷冻干燥24h，用冷冻球磨机在0℃下以不同次数研磨成粉末，并最后用相应的筛目尺寸(20、60和200目)分成不同的粒度(particle sizes)。. The dorsal mucus of Chinese giant salamanders was collected to obtain salamander skin secretions (also known as "salamander mucus extract" or "salamander skin mucus extract") as previously described (ref. 17). The reason why Chinese giant salamander skin secretion (SSAD) is selected in the embodiment of the present invention is that it can represent the mucus secreted by the stimulated amphibian of the Chinese giant salamander. And in the present invention, the salamander skin secretion is in the form of freeze-dried powder. Other types of salamanders can be used according to the actual situation. Briefly, Chinese giant salamanders began to secrete mucus when mechanically stimulated (by gently scraping their skin). After collecting mucus into clean tubes, wash with sterile PBS, vortex and centrifuge. Next, the mucus was freeze-dried for 24 h, ground into powder with a freezer ball mill at 0°C at different times, and finally divided into different particle sizes with corresponding mesh sizes (20, 60, and 200 mesh).
.需要特别说明的是，本发明实验选用20目、60目、200目的鲵皮肤分泌物，是为了验证不同筛目尺寸的鲵皮肤分泌物冻干粉(简称“粉末”)形成的水凝胶的药物释放性能。当需要较快释放药物时，可以选择筛目较小的粉末(例如20目)；而需要较慢释放药物时，可以选择筛目较小的粉末(例如200目)。因此，可以根据实际的药物和降解速率的需要，选择不同筛目的粉末(甚至可以选择14-300目的粉末)以实现对药物的控释。.It should be noted that the experiment of the present invention selects salamander skin secretions of 20 mesh, 60 mesh, and 200 mesh in order to verify the hydrogel formed by the lyophilized powder ("powder") of salamander skin secretions of different mesh sizes. drug release properties. When faster drug release is required, a powder with a smaller mesh size (for example, 20 mesh) can be selected; and when a slower drug release is required, a powder with a smaller mesh size (for example, 200 mesh) can be selected. Therefore, powders of different mesh sizes (even 14-300 mesh powders can be selected) can be selected according to the needs of actual drugs and degradation rates to achieve controlled release of drugs.
.氨基胍是一种AGE抑制剂，具有亲核性肼官能团-NH2NH2和α-二羰基导向的胍官能团-NH-C(＝NH)NH2；而多柔比星(又称阿霉素)是一种蒽环类抗生素，可以抑制DNA和RNA的合成，进而实现抗肿瘤的功效。上述这两种药物的分子量、结构、用途各不相同，且都没有破坏鲵皮肤分泌物水凝胶的多孔结构。也就是说，鲵皮肤分泌物水凝胶(例如，SSAD水凝胶)实际能够装载的药物范围较广，只要不会使SSAD水凝胶中的蛋白质变性即可。使蛋白质变性的方法是本领域技术人员已知的，例如化学方法(例如蛋白变性剂，包括强酸、强碱、重金属盐、盐酸胍、尿素、丙酮、TCEP、蛋白酶和一些还原剂(例如半胱氨酸、抗坏血酸、β-巯基乙醇和DTT))和物理方法(加热(高温)、紫外线及X射线照射、超声波、剧烈振荡或搅拌等)。本发明提供的局部给药系统适用于65℃及以下的温度。.Aminoguanidine is a kind of AGE inhibitor, has nucleophilic hydrazine functional group-NH2 NH2 and guanidine functional group-NH-C (=NH) NH2 of α-dicarbonyl orientation; And doxorubicin (also known as A Mycin) is an anthracycline antibiotic that can inhibit the synthesis of DNA and RNA, thereby achieving antitumor effects. The above two drugs have different molecular weights, structures, and uses, and none of them can destroy the porous structure of the salamander skin secretion hydrogel. That is to say, the salamander skin secretion hydrogel (for example, SSAD hydrogel) can actually load a wide range of drugs, as long as it does not denature the protein in the SSAD hydrogel. Methods to denature proteins are known to those skilled in the art, such as chemical methods (such as protein denaturants, including strong acids, strong bases, heavy metal salts, guanidine hydrochloride, urea, acetone, TCEP, proteases and some reducing agents (such as cysteine Amino acid, ascorbic acid, β-mercaptoethanol and DTT)) and physical methods (heating (high temperature), ultraviolet and X-ray irradiation, ultrasonic waves, vigorous shaking or stirring, etc.). The topical drug delivery system provided by the present invention is suitable for the temperature of 65°C and below.
.因此，SSAD水凝胶能够装载的药物还可以包括氨基胍和多柔比星及其药学上可接受的盐、AGE抑制剂(例如苯酰基噻唑溴化物、吡哆胺)、抗生素(例如青霉素、链霉素、多西环素)、化疗药物(例如紫杉醇、高三尖杉酯碱、环磷酰胺、奈达铂)。SSAD水凝胶还可以装载冻干粉(例如蛋白冻干粉、多肽冻干粉)、表皮生长因子和生物活性物质。.Therefore, the drugs that SSAD hydrogel can load can also include aminoguanidine and doxorubicin and pharmaceutically acceptable salts thereof, AGE inhibitors (such as benzoyl thiazole bromide, pyridoxamine), antibiotics (such as penicillin , streptomycin, doxycycline), chemotherapy drugs (eg paclitaxel, homoharringtonine, cyclophosphamide, nedaplatin). SSAD hydrogel can also be loaded with lyophilized powder (such as protein lyophilized powder, polypeptide lyophilized powder), epidermal growth factor and biologically active substances.
.同理，使鲵皮肤分泌物冻干粉凝胶化的溶剂也应不会使蛋白质变性，例如PBS(磷酸盐缓冲液)、蒸馏水、去离子水、生理盐水(NaCl缓冲液)、Tris缓冲液(TBS)、柠檬酸盐缓冲液、氯己定水溶液、人类全血、含有水分的人类血液提取物。其中，含有水分的人类血液提取物包括血浆、血清、血细胞、富血小板血浆(PRP)和富血小板血浆纤维蛋白(PRF)中的一种或多种。.Similarly, the solvent used to gel the lyophilized powder of salamander skin secretion should not denature the protein, such as PBS (phosphate buffer saline), distilled water, deionized water, normal saline (NaCl buffer), Tris buffer solution (TBS), citrate buffer, chlorhexidine aqueous solution, human whole blood, human blood extract containing water. Wherein, the human blood extract containing water includes one or more of plasma, serum, blood cells, platelet-rich plasma (PRP) and platelet-rich plasma fibrin (PRF).
.在一些实施方案中，SSAD水凝胶通过非共价相互作用的物理包封或吸附来装载药物。在一些实施方案中，药物通过羟基、羧基、氨基中的至少一种与SSAD水凝胶连接。. In some embodiments, SSAD hydrogels are loaded with drugs by physical encapsulation or adsorption through non-covalent interactions. In some embodiments, the drug is linked to the SSAD hydrogel through at least one of hydroxyl, carboxyl, and amino groups.
.通过SEM和FT-IR检验SSAD粉末和具有不同孔径的载药(AG或DOX)水凝胶。在支架上安装了SSAD粉末和不同尺寸(20、60和200目)的冻干SSAD水凝胶。在金溅射镀膜(goldsputter-coating)后，所有样本在10kV和5kV下、用SEM(SU8010，ZEISS MERLIN Compact，日本)成像。使用FT-IR光谱仪(Nicolet 670，美国)在透射模式下记录AG和DOX以及载药的SSAD水凝胶的FT-IR光谱(4000-400cm-1)，且光谱数据以吸收度单位记录。. Examination of SSAD powder and drug-loaded (AG or DOX) hydrogels with different pore sizes by SEM and FT-IR. SSAD powder and lyophilized SSAD hydrogels of different sizes (20, 60 and 200 mesh) were mounted on the racks. After gold sputter-coating, all samples were imaged with SEM (SU8010, ZEISS MERLIN Compact, Japan) at 10 kV and 5 kV. FT-IR spectra (4000-400 cm−1 ) of AG and DOX and drug-loaded SSAD hydrogels were recorded in transmission mode using an FT-IR spectrometer (Nicolet 670, USA), and spectral data were recorded in absorbance units.
.1.1.2.SSAD水凝胶溶胀和降解实验.1.1.2. SSAD hydrogel swelling and degradation experiments
.在PBS和人类唾液中评估了不同粒度(20、60和200目)的SSAD水凝胶的溶胀比和降解率。简言之，用PBS胶化100mg SSAD。之后，将水凝胶浸泡在PBS或人类唾液(5mL)中。对于SSAD水凝胶溶胀实验，在预定的时间点，称量SSAD水凝胶的质量并将相同体积的新鲜PBS或人类唾液加回到试管。对于SSAD水凝胶降解实验，在预定的时间点，收集SSAD水凝胶并将其冷冻干燥24h。溶胀比根据公式(1)计算：. The swelling ratio and degradation rate of SSAD hydrogels with different particle sizes (20, 60 and 200 mesh) were evaluated in PBS and human saliva. Briefly, 100 mg of SSAD was gelled with PBS. Afterwards, the hydrogel was soaked in PBS or human saliva (5 mL). For the SSAD hydrogel swelling experiments, at predetermined time points, the mass of the SSAD hydrogel was weighed and the same volume of fresh PBS or human saliva was added back to the test tube. For the SSAD hydrogel degradation experiments, at predetermined time points, SSAD hydrogels were collected and freeze-dried for 24 h. The swelling ratio is calculated according to formula (1):
.
.式中，Wt代表不同时间间隔，在PBS或人类唾液中溶胀后的SSAD水凝胶的重量，Wb代表SSAD水凝胶在基线的重量。In the formula, Wt represents the weight of SSAD hydrogel after swelling in PBS or human saliva at different time intervals, and Wb represents the weight of SSAD hydrogel at baseline.
.降解率根据公式(2)计算：.Degradation rate is calculated according to formula (2):
.
.式中，Wt代表不同时间间隔，在PBS或人类唾液中溶胀后的SSAD水凝胶冻干的重量，且Wb代表SSAD粉末在基线的重量。In the formula, Wt represents the lyophilized weight of SSAD hydrogel after swelling in PBS or human saliva at different time intervals, and Wb represents the weight of SSAD powder at baseline.
.1.1.3.体外的药物控释.1.1.3. Controlled drug release in vitro
.使用AG或DOX评估了具有不同粒度(20、60和200目)的SSAD水凝胶的释放动力学。简言之，50mg AG或10mg DOX分别与500mg SSAD或1000mg SSAD(20、60或200目)混合，以在PBS中形成装载药物的SSAD水凝胶。之后，将装载药物的水凝胶浸泡在PBS(5mL，pH 7.4)中。在预定的时间点，收集样本并在230nm或480nm的波长下检测，以确定AG或DOX的浓度。同时，将相同体积的PBS加回到每个试管，以保持恒定的体积。. The release kinetics of SSAD hydrogels with different particle sizes (20, 60 and 200 mesh) were evaluated using AG or DOX. Briefly, 50 mg AG or 10 mg DOX were mixed with 500 mg SSAD or 1000 mg SSAD (20, 60 or 200 mesh), respectively, to form drug-loaded SSAD hydrogels in PBS. Afterwards, the drug-loaded hydrogel was soaked in PBS (5 mL, pH 7.4). At predetermined time points, samples were collected and detected at a wavelength of 230 nm or 480 nm to determine the concentration of AG or DOX. At the same time, add the same volume of PBS back to each tube to maintain a constant volume.
.1.2.细胞增殖和迁移试验.1.2. Cell proliferation and migration assay
.1.2.1.CCK-8检测.1.2.1. CCK-8 Detection
.将200μL培养基中全部5,000个细胞接种于96孔板的每个孔。在培养箱(37℃，5％CO2)中培养24h后，用200μL含SSAD的水凝胶代替培养基12h和24h。在所示时间点，向每孔加入100μL新鲜Dulbecco's modified eagle培养基(DMEM)和10μL CCK8溶液，在37℃下培养2小时，并用酶标仪(EnSpire，PerkinElmer，Singapore)检测在450nm的吸光度。. Seed a total of 5,000 cells in 200 μL of medium into each well of a 96-well plate. After culturing in an incubator (37° C., 5% CO2 ) for 24 h, the culture medium was replaced with 200 μL of SSAD-containing hydrogel for 12 h and 24 h. At the indicated time points, 100 μL of fresh Dulbecco's modified eagle medium (DMEM) and 10 μL of CCK8 solution were added to each well, incubated at 37°C for 2 hours, and the absorbance at 450 nm was detected with a microplate reader (EnSpire, PerkinElmer, Singapore).
.1.2.2.划痕伤口愈合试验.1.2.2. Scratch wound healing test
.对于划痕试验，在6孔板的每个孔中接种6×105个细胞。并且在它们达到汇合(confluence)后，用200μL的吸头将其划伤，接着用PBS洗涤3次，再用含有SSAD的DMEM培养24h。在0和24h时在显微镜下对细胞拍照。. For the scratch assay, seed 6 x105 cells in each well of a 6-well plate. And after they reached confluence, they were scratched with a 200 μL tip, washed three times with PBS, and then cultured with DMEM containing SSAD for 24 h. Cells were photographed under a microscope at 0 and 24 h.
.1.2.3.Transwell迁移试验.1.2.3. Transwell migration test
.基于划痕伤口愈合的结果，随后进行了Transwell迁移试验。简言之，将100μL含有2×105个细胞(L929或HUVECs)的DMEM接种于insert小室的上室，且下室装入800μL补充有SSAD的DMEM。培养24h后，取出insert小室并将其清洗，并且用0.1％结晶紫对已经迁移到膜的下侧的细胞进行染色并在显微镜下计数，如前所述(参考文献50)。. Based on the results of scratch wound healing, a Transwell migration test was subsequently performed. Briefly, 100 μL of DMEM containing 2×105 cells (L929 or HUVECs) was seeded in the upper chamber of the insert chamber, and 800 μL of DMEM supplemented with SSAD was filled in the lower chamber. After 24 h of incubation, the insert chambers 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 (ref. 50).
.1.3.动物研究.1.3. Animal studies
.1.3.1.高血糖症下的硬腭粘膜伤口愈合模型.1.3.1. Hard palate mucosal wound healing model under hyperglycemia
.动物购自重庆医科大学实验动物中心，并在无特定病原体(SPF)条件下饲养于重庆医科大学附属口腔医院的动物实验中心。.The animals were purchased from the Experimental Animal Center of Chongqing Medical University, and were raised in the Animal Experiment Center of the Affiliated Stomatological Hospital of Chongqing Medical University under specific pathogen-free (SPF) conditions.
.本发明使用了40只(8-12周)、平均体重为200-220g的雄性Sprague-Dawley(SD)大鼠，并通过腹腔注射链脲佐菌素(streptozotocin，STZ，60mg/kg)诱导2型糖尿病。在注射STZ后1周，只有血糖水平高于≥250mg/dL的大鼠被纳入后续研究。硬腭粘膜损伤的诱导方法如First及其同事所述(参考文献51)。简言之，所有的动物都用1％戊巴比妥钠(30mg/kg)腹腔注射麻醉。然后，用开睑器(blepharostat)打开口腔，并用碘酒和75％乙醇对腭部粘膜消毒。接着，用一次性活检穿孔器(直径＝3mm)在大鼠腭部(位于中间)形成全层硬腭粘膜缺损。所有动物被随机分为以下四组，并根据其组别(n＝10)处理(这些处理每天重复进行)：.The present invention has used 40 (8-12 weeks), the male Sprague-Dawley (SD) rat of average body weight is 200-220g, and induces by intraperitoneal injection streptozotocin (streptozotocin, STZ, 60mg/kg) Type 2 diabetes. One week after STZ injection, only rats with blood glucose levels above ≥250 mg/dL were included in the follow-up study. Hard palate mucosal lesions were induced as described by First and colleagues (ref. 51). Briefly, all animals were anesthetized with 1% sodium pentobarbital (30 mg/kg) intraperitoneally. Then, the oral cavity was opened with a blepharostat, and the palatal mucosa was disinfected with tincture of iodine and 75% ethanol. Next, a full-thickness hard palate mucosal defect was created in the rat palate (located in the middle) 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):
-对照组：空白对照，未使用任何处理- Control group: blank control, no treatment was used
-AG组：AG(0.4mg/部位)-AG group: AG (0.4mg/site)
-SSAD组：SSAD水凝胶(每个部位，5mg SSAD粉末)-SSAD group: SSAD hydrogel (per site, 5 mg SSAD powder)
-SSAD+AG组：装载AG的SSAD水凝胶(每个部位，5mg SSAD粉末+0.4mg AG)-SSAD+AG group: SSAD hydrogel loaded with AG (per site, 5 mg SSAD powder + 0.4 mg AG)
.鲵皮肤分泌物冻干粉的剂量与预期给药的伤口大小相关。药物与鲵皮肤分泌物冻干粉的干重比为小于等于4:1，优选为1:1。如果装载的药物过多(即鲵皮肤分泌物冻干粉过少)，会影响局部给药系统中水凝胶的形成及其粘性(即难以成胶且粘性较弱)。这会影响局部给药系统与伤口的粘合程度，进而无法实现覆盖创面的敷料作用。. The dose of lyophilized powder of salamander skin secretion is related to the size of the expected wound. The dry weight ratio of the drug to the lyophilized salamander skin secretion powder is less than or equal to 4:1, preferably 1:1. If the loading of drugs is too much (that is, the lyophilized powder of salamander skin secretion is too little), it will affect the formation and viscosity of the hydrogel in the topical drug delivery system (that is, it is difficult to form a gel and the viscosity is weak). This will affect the degree of adhesion of the topical drug delivery system to the wound, thereby making it impossible to achieve the dressing effect of covering the wound.
.本发明使用的局部AG(2mg/kg)的剂量是对于全身给药来说，是微不足道的——只是一些报道中用于促进腭部伤口愈合的全身给药AG(100mg/kg)的1/50。The dose of topical AG (2mg/kg) used in the present invention is negligible for systemic administration - only 1% of systemically administered AG (100mg/kg) in some reports for promoting palatal wound healing /50.
.在所示时间点用数码相机(Nikon，JY67ON，日本)拍摄伤口照片，并将数码照片传输到计算机，以用ImageJ软件(National Institutes ofHealth，NIH，美国)分析腭部切口的闭合。伤口闭合率根据公式(3)计算：Wound photographs were taken with a digital camera (Nikon, JY67ON, Japan) at the indicated time points and transferred to a computer for analysis of palatal incision closure with ImageJ software (National Institutes of Health, NIH, USA). The wound closure rate was calculated according to formula (3):
.
.式中，S初始是初始的伤口尺寸，S当前是当前的伤口尺寸。每个伤口测量3次，并记录平均伤口尺寸。在术后8天和18天，每组5只大鼠被安乐死以得到硬腭进行组织学分析。. In the formula, Sinitial is the initial wound size, Scurrent is the current wound size. Each wound was measured 3 times, and the average wound size was recorded. At
.样本在4％多聚甲醛中固定24h，并在10％乙二胺四乙酸(EDTA，pH 8.0)中脱钙2-3个月。之后，用不同浓度的乙醇(60-100％)对其脱水。随后，将样本用石蜡包埋并切成5-μm的切片。对于每个样本，制备了超过10张载玻片，并在每个样本的伤口区域在垂直于上腭中线取样。然后，选择伤口相对较宽的切片进行组织学分析并用HE(Solarbio，中国)和Masson's三色法(Solarbio)对其染色。. The samples were fixed in 4% paraformaldehyde for 24h and decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 8.0) for 2-3 months. Afterwards, it was dehydrated with different concentrations of ethanol (60-100%). Subsequently, samples were embedded in paraffin and cut into 5-μm sections. For each specimen, more than 10 slides were prepared and samples were taken perpendicular to the midline of the palate in the wound area of each specimen. Then, relatively wide sections of the wound were selected for histological analysis and stained with HE (Solarbio, China) and Masson's trichrome method (Solarbio).
.术后18天检测再上皮化率，根据公式(4)计算：.The re-epithelialization rate was detected 18 days after the operation, and calculated according to the formula (4):
.
.式中，Db代表在基线的粘膜缺损的距离(3mm)，Dn代表术后18天时没有上皮化的距离(图19)。In the formula,Db represents the distance of the mucosal defect at baseline (3mm), andDn represents the distance without epithelialization at 18 days after surgery ( FIG. 19 ).
.此外，进行了免疫荧光染色。将切片用3％牛血清白蛋白(BSA，A8020，Solarbio)封闭后，用一抗(Abcam，美国)进行免疫染色，4℃过夜。然后用相应的二抗(Abcam)室温孵育1h，用DAPI(Goodbio科技有限公司，中国)复染细胞核。所有图像都是用倒置的荧光显微镜(Nikon，Nikon Eclipse Ti-SR，日本)获得的。.In addition, immunofluorescent staining was performed. The sections were blocked with 3% bovine serum albumin (BSA, A8020, Solarbio), and then immunostained with primary antibody (Abcam, USA) at 4°C overnight. Then, the corresponding secondary antibody (Abcam) was incubated at room temperature for 1 h, and the nuclei were counterstained with DAPI (Goodbio Technology Co., Ltd., China). All images were acquired with an inverted fluorescence microscope (Nikon, Nikon Eclipse Ti-SR, Japan).
.为了进一步阐明SSAD促进伤口愈合的机制，使用PCNA染色评估了修复的组织的细胞增殖，以及进行了转录组学分析。建立了腭部粘膜缺损模型的SD大鼠(直径＝3mm)被随机分为SSAD组(接受SSAD敷料，n＝16)和对照组(无处理，n＝16)：在第3天和第7天，每组中一半的大鼠(n＝8)被处死并腭部粘膜被收集用于转录组分析。RNA的提取、纯化、逆转录、文库构建和测序均于深圳BGI有限公司(深圳，中国)进行。. To further elucidate the mechanism by which SSAD promotes wound healing, cell proliferation in repaired tissues was assessed using PCNA staining, as well as transcriptomic analysis. SD rats (diameter=3mm) with established palatal mucosal defect model were randomly divided into SSAD group (receiving SSAD dressing, n=16) and control group (no treatment, n=16): on the 3rd day and 7th day On
.完成测序后，根据得到的数据库进行生物信息学分析，并且用RSEM(v1.2.12)计算基因的表达水平。使用PossionDis、以错误发现率(FDR)≤0.001且|Log2Ratio|≥1进行差异表达分析。使用维恩图来检测两组中共表达的基因。为了深入了解表型的变化，通过基于超几何检验(https://en.wikipedia.org/wiki/hypergeometric_distribution)的Phyper，对两组中共表达的基因进行GO和KEGG富集分析(http://www.geneontology.org/和https://www.kegg.jp/)。通过Bonferroni校正，对术语和通路的显著性水平以Q值≤0.05进行校正。此外，共表达基因的PPI网络分析也通过基因/蛋白相互作用检索搜查工具算法(Search Tool for the Retrieval of Interacting Genes/Proteins algorithm)进行。. After the sequencing is completed, bioinformatics analysis is performed according to the obtained database, and the expression level of the gene is calculated with RSEM (v1.2.12). Differential expression analysis was performed using PossionDis with false discovery rate (FDR)≤0.001 and |Log2Ratio|≥1. A Venn diagram was used to detect co-expressed genes in the two groups. To gain insight into phenotypic changes, GO and KEGG enrichment analysis (http:// www.geneontology.org/ and https://www.kegg.jp/). Significance levels for terms and pathways were corrected for Q-values ≤ 0.05 by Bonferroni correction. In addition, the PPI network analysis of co-expressed genes was also performed by the Search Tool for the Retrieval of Interacting Genes/Proteins algorithm (Search Tool for the Retrieval of Interacting Genes/Proteins algorithm).
.1.3.2.抗肿瘤功效和伤口愈合模型.1.3.2. Antitumor Efficacy and Wound Healing Model
.本发明使用了36只雌性C57BL/6小鼠(8-10周)。除去注射部位的毛后，将1×106个B16F10细胞注射到每只C57BL/6小鼠的背部，形成肿瘤。当肿瘤达到约50mm3时，将荷瘤C57BL6小鼠随机分组如下(n＝9)：. Thirty-six female C57BL/6 mice (8-10 weeks) were used in the present invention. After removing the hair from the injection site, 1×106 B16F10 cells were injected into the back of each C57BL/6 mouse to form tumors. When the tumor reached about50 mm, the tumor-bearing C57BL6 mice were randomly divided into groups as follows (n=9):
-对照组：空白对照，未使用任何处理- Control group: blank control, no treatment was used
-DOX组：DOX(每个部位，4mg/kg)-DOX group: DOX (each part, 4mg/kg)
-SSAD组：SSAD水凝胶(每个部位，8mg SSAD粉末)-SSAD group: SSAD hydrogel (per site, 8mg SSAD powder)
-SSAD+DOX组：载DOX的SSAD水凝胶(每个部位，8mg SSAD粉末+4mg/kg DOX)-SSAD+DOX group: DOX-loaded SSAD hydrogel (per site, 8mg SSAD powder + 4mg/kg DOX)
.然后，在每个肿瘤部位，随后建立一个10mm圆形全层皮肤缺损，并使用各组相应的材料来覆盖这个由肿瘤切除造成的伤口。鉴于本发明的荷瘤伤口在相对干燥的环境下会出现结痂，这些荷瘤伤口只在第0天接受合理剂量的药物处理以避免刺激伤口。需要强调的是，鲵皮肤分泌物和药物的用量原理如上所述。处理后，每2天记录小鼠的体重、肿瘤尺寸和存活率。肿瘤的大小根据公式(5)计算：. Then, at each tumor site, a 10mm circular full-thickness skin defect was subsequently created, and each set of corresponding materials was used to cover this wound caused by tumor resection. In view of the fact that the tumor-bearing wounds of the present invention will form scabs in a relatively dry environment, these tumor-bearing wounds are only treated with a reasonable dose of drugs on
.
.式中，L代表肿瘤的长度，W代表肿瘤的宽度。. In the formula, L represents the length of the tumor, and W represents the width of the tumor.
.在治疗后18天，小鼠被处死，并且肿瘤被收集、称重并拍照。经过一系列常规处理(组织固定、脱水、包埋、切片)后，选择4组的组织切片并用HE染色来组织学分析。使用Ki67免疫组化法来评估肿瘤细胞的增殖情况。实验人员还进行了TUNEL检测以检测任何凋亡的肿瘤细胞。. 18 days after treatment, mice were sacrificed and tumors were harvested, weighed and photographed. After a series of routine treatments (tissue fixation, dehydration, embedding, sectioning), tissue sections from 4 groups were selected and stained with HE for histological analysis. Tumor cell proliferation was assessed using Ki67 immunohistochemistry. The experimenters also performed a TUNEL assay to detect any apoptotic tumor cells.
.1.5.统计分析.1.5. Statistical analysis
.所有数据均以平均值±平均值的标准误差表示。统计分析用GraphPad Prism(GraphPad软件，美国)进行。统计学显著是通过使用单因素方差分析(ANOVA)或非配对t检验来评估的。P值至少<0.05为显著。. All data are presented as mean ± standard error of the mean. Statistical analysis was performed with GraphPad Prism (GraphPad software, USA). Statistical significance was assessed using one-way analysis of variance (ANOVA) or unpaired t-test. A P value of at least <0.05 was considered significant.
.实施例二：SSAD的结构特征和药物释放特性.Example 2: Structural features and drug release properties of SSAD
.如图2a所示，很容易获得不同筛目尺寸的SSAD粉末，并且这些粉末导致具有不同多孔特性的水凝胶。形成的水凝胶意味着，SSAD粒度和水合过程中的相关变化直接影响了所形成的水凝胶的孔径尺寸。通过将SSAD颗粒与水混合后，它们会通过氢键和SSAD蛋白的氨基酸残基之间的S-S键水合并溶胀，以形成具有多孔结构的水凝胶。水凝胶表面上的孔为圆形并且相互连接(interconnected)，这可能是由两种因素的结果：微孔从SSAD粉末的水化形成，而大孔由SSAD的粒度决定。这一观察证实，越密集的筛目与越小的孔有关，并显示相应的水凝胶网络可能越稳定。. As shown in Fig. 2a, SSAD powders with different mesh sizes are readily obtained, and these powders lead to hydrogels with different porous properties. The formed hydrogels imply that the SSAD particle size and related changes in the hydration process directly affect the pore size of the formed hydrogels. After mixing SSAD particles with water, they hydrate and swell through hydrogen bonds and S-S bonds between amino acid residues of SSAD proteins to form a hydrogel with a porous structure. The pores on the hydrogel surface are circular and interconnected, which may be the result of two factors: the micropores form from the hydration of the SSAD powder, while the macropores are determined by the particle size of the SSAD. This observation confirms that denser meshes are associated with smaller pores and suggests that the corresponding hydrogel networks may be more stable.
.傅里叶变换红外(FT-IR)光谱进一步表明了AG或DOX分子在SSAD中的成功装载，如图2b所示。对于纯的AG或DOX分子，可以在3440cm-1和3326cm-1分别观察到可归因于主烷基链的伸缩振动的吸收带(图2b-i)。在SSAD装载药物之前，N-H的伸缩振动峰在3435cm-1出现(图2b-ii和2b-iii)。然而，在与AG或DOX整合后，出现了2个峰(3274cm-1或3500cm-1)，分别如图2b-ii和图2b-iii所示。鉴于药物分子中没有活性化学基团，它们在SSAD水凝胶中的装载主要可以归因于通过多种非共价相互作用的物理包封(physical entrapment)或吸附。. Fourier transform infrared (FT-IR) spectroscopy further indicated the successful loading of AG or DOX molecules in SSAD, as shown in Fig. 2b. For pure AG or DOX molecules, absorption bands attributable to the stretching vibrations of the main alkyl chains can be observed at 3440 cm−1 and 3326 cm−1 , respectively (Fig. 2b–i). Before SSAD was loaded with drugs, the stretching vibration peak of NH appeared at 3435 cm−1 (Fig. 2b-ii and 2b-iii). However, after integration with AG or DOX, 2 peaks (3274 cm−1 or 3500 cm−1 ) appeared, as shown in Figure 2b-ii and Figure 2b-iii, respectively. Given that there are no active chemical groups in drug molecules, their loading in SSAD hydrogels can be mainly attributed to physical entrapment or adsorption through various non-covalent interactions.
.对由不同粒度的粉末(20、60和200目)制得的SSAD水凝胶的药物释放特性进行了评估。AG(Mw：111.55)和DOX(Mw：543.52)被选为模型药物来装载至SSAD水凝胶。在评估期间(24h)。从20、60和200目的SSAD水凝胶中累积释放的AG分别为64％、56％和50％(图2c)。从20、60和200目的SSAD水凝胶中累积释放的DOX分别为73％、70％和51％(图2d)。随着SSAD粉末的粒度的增加，相应的SSAD水凝胶的药物释放率也增加。此外，为了分析SSAD水凝胶的体外溶胀和降解行为，将其浸入磷酸盐缓冲盐溶液(PBS)或人类唾液中，37℃，24h。在不同的时间间隔，测量溶胀比和降解率(图9)。SSAD水凝胶在PBS和唾液中都会逐渐降解。在早期的8小时，20目SSAD水凝胶的吸水的重量重于降解的重量。8小时后，所有的水凝胶(20、60、200目)逐渐降解，并在36小时后，达到了平衡状态(图9a-b)。此外，还评估了SSAD水凝胶的生物降解。结果(图9c-d)显示，随着SSAD粒度的增大，SSAD水凝胶的降解率增大，因为SSAD的孔径越大，降解速率越快。. The drug release properties of SSAD hydrogels prepared from powders of different particle sizes (20, 60 and 200 mesh) were evaluated. AG (Mw : 111.55) and DOX (Mw : 543.52) were selected as model drugs to be loaded into SSAD hydrogels. During the evaluation period (24h). The cumulative release of AG from 20, 60 and 200 mesh SSAD hydrogels was 64%, 56% and 50%, respectively (Fig. 2c). The cumulative release of DOX from 20, 60 and 200 mesh SSAD hydrogels was 73%, 70% and 51%, respectively (Fig. 2d). As the particle size of SSAD powder increased, the drug release rate of the corresponding SSAD hydrogel also increased. Furthermore, to analyze the in vitro swelling and degradation behavior of SSAD hydrogels, they were immersed in phosphate-buffered saline (PBS) or human saliva at 37 °C for 24 h. At different time intervals, the swelling ratio and degradation rate were measured (Fig. 9). SSAD hydrogels gradually degrade in both PBS and saliva. In the early 8 hours, the 20-mesh SSAD hydrogel absorbed more weight than degraded. After 8 hours, all hydrogels (20, 60, 200 mesh) gradually degraded, and after 36 hours, reached an equilibrium state (Fig. 9a-b). In addition, the biodegradation of SSAD hydrogels was also evaluated. The results (Fig. 9c–d) showed that with the increase of SSAD particle size, the degradation rate of SSAD hydrogel increased, because the larger the pore size of SSAD, the faster the degradation rate.
.此外，相同粒度的SSAD水凝胶似乎在唾液中更稳定，与在PBS中相比，其溶胀率更平缓。这可能是因为某些物质(例如，唾液含有的蛋白质)导致相对饱和的(saturated)溶胀环境。因此，由于其的稳定性，SSAD水凝胶成为一种潜在的新型敷料，用于口腔的伤口及其他相关伤口的微环境。.Furthermore, SSAD hydrogels of the same particle size appeared to be more stable in saliva, with a gentler swelling rate than in PBS. This may be because certain substances (eg, proteins contained in saliva) result in a relatively saturated swelling environment. Therefore, due to its stability, SSAD hydrogel becomes a potential novel dressing for wounds in the oral cavity and other related wound microenvironments.
.实施例三：体外细胞学研究.Example 3: In vitro cytology research
.SSAD诱导的细胞增殖：.SSAD-induced cell proliferation:
.用细胞计数试剂盒-8(CCK-8)试验，评估SSAD与L929成纤维细胞和人脐静脉内皮细胞(HUVECs)的生物相容性，这两种细胞在皮肤和粘膜再生中发挥重要作用。在本发明的初步实验中(图10)，测试了一系列浓度(从5到0.005mg/mL)的SSAD对L929细胞和HUVECs的细胞毒性。在这些不同的浓度中，0.1mg/mL SSAD似乎显著促进细胞增殖，并被选择用于后续的细胞实验。CCK8试验结果表明，在暴露于SSAD条件培养基(0.1mg/mL)24h后，L929细胞和HUVECs仍然显示出相对高的细胞活力值(分别为对照培养基的105％和110％)，这表明SSAD表现出良好的生物相容性(图11)。.Using the Cell Counting Kit-8 (CCK-8) assay to assess the biocompatibility of SSAD with L929 fibroblasts and human umbilical vein endothelial cells (HUVECs), which play important roles in skin and mucosal regeneration . In the preliminary experiments of the present invention (Figure 10), a range of concentrations (from 5 to 0.005 mg/mL) of SSAD were tested for cytotoxicity on L929 cells and HUVECs. Among these different concentrations, 0.1 mg/mL SSAD appeared to significantly promote cell proliferation and was selected for subsequent cell experiments. The results of the CCK8 test showed that after 24 h exposure to SSAD-conditioned medium (0.1 mg/mL), L929 cells and HUVECs still showed relatively high cell viability values (105% and 110% of the control medium, respectively), which indicated that SSAD exhibited good biocompatibility (Fig. 11).
.SSAD诱导的细胞迁移：.SSAD-induced cell migration:
.用划痕(图3a)和Transwell迁移(图3b)试验，来体外验证SSAD对L929细胞和HUVECs迁移的影响。与对照组相比，SSAD条件培养基可以显著促进L929细胞(对照组的1.5倍)和HUVECs(对照组的1.7倍)的侧向(lateral)迁移(图3c)。与对照组相比，SSAD条件培养基还显著促进L929细胞(对照组的2.1倍)和HUVECs(对照组的3.0倍)的跨膜迁移(图3d)。由于细胞迁移是伤口愈合的关键步骤，上述结果表明，SSAD可以极大地增加血管内皮细胞(HUVECs)和成纤维细胞(L929)的迁移。.Using scratch (Fig. 3a) and Transwell migration (Fig. 3b) assays to verify the effect of SSAD on the migration of L929 cells and HUVECs in vitro. Compared with the control group, SSAD-conditioned medium could significantly promote the lateral migration of L929 cells (1.5 times of the control group) and HUVECs (1.7 times of the control group) (Fig. 3c). Compared with the control group, SSAD-conditioned medium also significantly promoted the transmembrane migration of L929 cells (2.1 times that of the control group) and HUVECs (3.0 times that of the control group) (Fig. 3d). Since cell migration is a critical step in wound healing, the above results suggest that SSAD can greatly increase the migration of vascular endothelial cells (HUVECs) and fibroblasts (L929).
.众所周知，一些生长因子的表达可能在伤口愈合的过程中有益。生物检定法(bioassay)结果显示，SSAD含有大量的生长因子，如血管内皮生长因子(VEGF)、胰岛素样生长因子1(IGF-1)、基质细胞衍生因子1(SDF-1)等(表1)。根据SSAD所含各生长因子的含量，本发明实验人员选择了SSAD所含最多的两种生长因子，即SDF-1和IGF-1。与单独给药SDF-1或IGF-1或者同时给药SDF-1和IGF-1时相比，SSAD在促进细胞行为方面表现得更好(图12)，其表明这是SSAD在促进细胞增殖和迁移的综合结果。It is known that the expression of some growth factors may be beneficial in the process of wound healing. Bioassay results showed that SSAD contained a large number of growth factors, such as vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), stromal cell-derived factor 1 (SDF-1), etc. (Table 1 ). According to the content of each growth factor contained in SSAD, the experimenters of the present invention selected the two growth factors contained most in SSAD, ie SDF-1 and IGF-1. Compared with the administration of SDF-1 or IGF-1 alone or the simultaneous administration of SDF-1 and IGF-1, SSAD performed better in promoting cell behavior (Figure 12), which indicated that it was SSAD that promoted cell proliferation and migration results.
.表1 SSAD的生物检定法结果.Table 1 SSAD bioassay results
.实施例四：体内动物研究之硬腭粘膜损伤模型.Example 4: Hard palate mucosal injury model for in vivo animal research
.接受FGG的患者遭受与腭部粘膜缺损有关的并发症，如疼痛、感染和进食时的不适，尤其是糖尿病患者。虽然近年来，糖尿病溃疡受到了很多关注，但关于糖尿病患者口内伤口愈合的研究却很少。据发明人所知，本发明技术方案首次采用了装载AG来局部使用，以促进高血糖症的存在下的腭部伤口愈合。由于口腔的潮湿和不稳定的环境，SSAD水凝胶不可能长时间、稳定地留在原处。因此，AG的相对快速释放可能是体内应用的更好选择，这可以最大程度地提高生物利用度。此外，随着粒度的增加，相同重量的SSAD粉末需要更多的液体进行凝胶化，这表明具有较大粒度的SSAD粉末可能具有更好的止血效果(图13)。所有的这些数据构成了选择20目的SSAD来治疗腭部粘膜损伤模型的理由。.Patients receiving FGG suffer from complications related to palatal mucosal defects such as pain, infection, and discomfort while eating, especially in diabetic patients. Although diabetic ulcers have received a lot of attention in recent years, little research has been done on intraoral wound healing in diabetic patients. As far as the inventors know, the technical solution of the present invention is the first to load AG for local application to promote palatal wound healing in the presence of hyperglycemia. Due to the moist and unstable environment of the oral cavity, it is impossible for the SSAD hydrogel to stay in place stably for a long time. Therefore, the relatively fast release of AG may be a better choice for in vivo applications, which can maximize bioavailability. In addition, as the particle size increased, the same weight of SSAD powder required more liquid for gelation, suggesting that SSAD powder with larger particle size might have better hemostatic effect (Fig. 13). All of these data constitute the reason for choosing 20 mesh SSAD to treat palatal mucosal injury model.
.在本发明的整个实验期间，没有观察到异常的生理症状。与其他两组相比，用SSAD和装载AG的SSAD(SSAD+AG)处理的小鼠显示出较少的出血(图4a)。此外，经典的肝脏损伤-止血模型(图14)表明，SSAD可以有效地粘附到伤口部位，密封并止血(图15)。如图4b和表2所示，在术后4天，SSAD+AG组(61.2±4.3％)、AG组(55.1±9.8％)和SSAD组(56.8±13.1％)的动物伤口闭合率显著快于对照组(37.7±9.9％)，其中SSAD+AG组愈合最快。在术后8天，SSAD+AG组的动物的伤口闭合率(78.3±5.1％)仍显著高于对照组(53.7±15.3％)。在术后18天，SSAD+AG组的伤口几乎完全愈合，平均伤口闭合率为97.4±3.3％，其也高于对照组(88.3±9.8％)、SSAD组(92.5±7.1％)和AG组(92.5±9.0％)，且SSAD+AG组与对照组之间的差异显著(P＜0.05)。因此，SSAD+AG在相对较短的时间内极大地加快口腔粘膜缺损的愈合。. During the entire experimental period of the present invention, no abnormal physiological symptoms were observed. Mice treated with SSAD and AG-loaded SSAD (SSAD+AG) showed less bleeding compared to the other two groups (Fig. 4a). Furthermore, the classic liver injury-hemostasis model (Figure 14) demonstrated that SSAD can effectively adhere to the wound site, seal and stop bleeding (Figure 15). As shown in Figure 4b and Table 2, at
.表2按公式(3)计算的各组的伤口闭合率.Wound closure rate of each group calculated by formula (3) in table 2
.本发明中，全层(full-thickness)硬腭粘膜缺损的直径为3mm，发明人还试图将伤口生成于硬腭的中间，这对每只大鼠都是恒定的。据此，可以粗略计算出伤口相对于上颌的位置。更重要的是，正常的口腔组织由上皮、固有层和粘膜下层组成，其中有小唾液腺(●)存在(图16)。此外，在愈合期间，再生的腭部粘膜的厚度显著高于正常组织，并且再生的组织中的胶原蛋白排列相对无序，细胞浸润较多(图17)。因此，进行苏木精-伊红(HE)染色以观察治疗组的粘膜厚度、上皮完整性和腺体再生，其中黑色虚线表示正常组织(黑色箭头)和再生的组织(蓝色箭头)的边界(图4c)。尽管在术后18天，4组之间的再生的腭部粘膜厚度没有统计学上的显著差异，但在术后8天，SSAD+AG组的再生的腭部粘膜厚度(847.4±17.8μm)显著高于SSAD组(285.4±5.1μm)、AG组(299.6±11.4μm)和对照组(248.8±29.8μm)(图18和图4d，P＜0.001)，这可以归因于伤口愈合期间的组织重塑。具体地，在早期恢复阶段，大量的细胞外基质(ECM)由在伤口边缘、经迁移和增殖的角质细胞和成纤维细胞分泌，并然后，肉芽组织变得成熟以形成疤痕，其特征是持续的胶原合成和胶原分解代谢。In the present invention, the diameter of the full-thickness hard palate mucosal defect was 3 mm, and the inventors also tried to generate the wound in the middle of the hard palate, which was constant for each rat. From this, the position of the wound relative to the maxilla can be roughly calculated. More importantly, normal oral tissues consist of epithelium, lamina propria, and submucosa, in which minor salivary glands (●) are present (Fig. 16). In addition, during the healing period, the thickness of the regenerated palatal mucosa was significantly higher than that of normal tissue, and the arrangement of collagen in the regenerated tissue was relatively disordered, and the cell infiltration was more infiltrated (Fig. 17). Therefore, hematoxylin-eosin (HE) staining was performed to observe mucosal thickness, epithelial integrity, and gland regeneration in the treatment group, where the black dotted line indicates the boundary between normal tissue (black arrow) and regenerated tissue (blue arrow) (Fig. 4c). Although there was no statistically significant difference in the regenerated palatal mucosal thickness among the 4 groups at 18 days postoperatively, at 8 days postoperatively, the regenerated palatal mucosal thickness in the SSAD+AG group was (847.4±17.8 μm) significantly higher than SSAD group (285.4±5.1μm), AG group (299.6±11.4μm) and control group (248.8±29.8μm) (Figure 18 and Figure 4d, P<0.001), which can be attributed to the Organizational remodeling. Specifically, in the early recovery phase, a large amount of extracellular matrix (ECM) is secreted by migrating and proliferating keratinocytes and fibroblasts at the wound edge, and then, granulation tissue becomes mature to form a scar characterized by persistent Collagen synthesis and collagen catabolism.
.上皮钉突(黄色箭头，图4c)是粘膜愈合的象征。根据图19中显示的标准，计算了各组的再上皮化率，SSAD+AG组的再上皮化率(99.5±0.5％)显著高于SSAD组(93.7±3.3％)、AG组(91.4±0.8％)和对照组(72.3±1.9％)(图4e)。更有趣的是，以前几乎没有唾液可以在全层粘膜缺损区域再生。在本发明中，在SSAD+AG组观察到一些唾液腺(●)，而在AG、SSAD和对照组中没有。附属器(appendage)再生是无疤痕愈合的关键指标之一。. Epithelial spikes (yellow arrows, Fig. 4c) are a sign of mucosal healing. According to the standard shown in Figure 19, the re-epithelialization rate of each group was calculated, and the re-epithelialization rate of the SSAD+AG group (99.5±0.5%) was significantly higher than that of the SSAD group (93.7±3.3%) and the AG group (91.4±0.5%). 0.8%) and the control group (72.3±1.9%) (Fig. 4e). More interestingly, little saliva was previously available to regenerate in areas of full-thickness mucosal defects. In the present invention, some salivary glands (•) were observed in the SSAD+AG group, but not in the AG, SSAD and control groups. Appendage regeneration is one of the key indicators of scar-free healing.
.胶原蛋白是固有层的主要成分，并且胶原蛋白的合成与降解平衡在伤口再生中起重要作用。进行Masson’s三色染色来观察胶原蛋白的沉积(图4f)。在术后8天(即伤口愈合的相对早期阶段)，SSAD+AG组中激增的胶原纤维密集排布，呈粗大的束状形态。在术后18天，SSAD+AG和SSAD组的再生的胶原逐渐重建至正常水平，纤维基质排列更加密集(图4f，40×)；然而AG和对照组的胶原纤维仍处于相对无序的排列状态(图4f)。上述数据显示，SSAD+AG组的基质胶原沉积在伤口愈合的相对后期逐渐重建至正常水平，表明装载AG的SSAD可以通过加速胶原的合成和成熟，显著促进高血糖症下的组织再生。上述联合作用优于单独施用SSAD或AG且远优于空白对照组。因此，装载AG的SSAD可以视为促进糖尿病的伤口愈合的有效方法。. Collagen is the main component of the lamina propria, and the balance of synthesis and degradation of collagen plays an important role in wound regeneration. Masson's trichrome staining was performed to observe collagen deposition (Fig. 4f). At postoperative day 8 (i.e., a relatively early stage of wound healing), the proliferated collagen fibers in the SSAD+AG group were densely arranged in a thick bundle-like morphology. On
.还进行组织再生有关的相关生物标记物的免疫荧光染色，来比较4组之间的再生效果。CD31(红色)用于对血管内皮细胞染色，其证明新血管生成；Col-1(绿色)用于对胶原蛋白染色，其是ECM沉积的重要标记物(图5a)。α-平滑肌肌动蛋白(α-SMA，红色)用于对肌成纤维细胞染色，其是成熟血管的收缩的原因之一，而巨噬细胞标记物CD68(绿色)用于评估炎症水平(图5b)。.Immunofluorescent staining of relevant biological markers related to tissue regeneration was also carried out to compare the regeneration effect among the 4 groups. CD31 (red) was used to stain vascular endothelial cells, which demonstrates neovascularization; Col-1 (green) was used to stain collagen, an important marker of ECM deposition (Fig. 5a). α-Smooth muscle actin (α-SMA, red) was used to stain myofibroblasts, which are responsible for the constriction of mature blood vessels, while the macrophage marker CD68 (green) was used to assess the level of inflammation (Fig. 5b).
.在术后18天，SSAD+AG治疗组的每个显微镜视野中CD31+和Col-1+细胞的表达密度(38.6±6.5％，或33.0±4.2％)显著高于对照组(1.2±0.5％，或3.4±1.2％)、SSAD组(16.1±4.0％，或11.8±4.3％)和AG组(8.9±2.6％，或7.4±2.1％)(图5c)。SSAD+AG治疗组的每个显微镜视野中α-SMA+细胞的表达密度(2.1±0.8％)显著高于对照组(0±0％)、SSAD组(0.5±0.4％)和AG组(0.3±0.3％)(图5d)。SSAD+AG治疗组的每个显微镜视野中CD68+细胞的表达密度(0.6±0.2％)明显低于对照组(11.5±2％)、SSAD组(1.3±0.3％)和AG组(1.3±0.3％)(图5d)。提供了图5a的单独的荧光图像来评估胶原沉积和新血管生成(图20和图21)。这些结果进一步表明，装载AG的SSAD可以提高SSAD在促进血管形成、增强ECM重塑和减少炎症中的表现。.18 days after operation, the expression density of CD31+ and Col-1+ cells in each microscope field in the SSAD+AG treatment group (38.6±6.5%, or 33.0±4.2%) was significantly higher than that in the 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. 5c). The expression density of α-SMA+ cells in each microscope field in the SSAD+AG treatment group (2.1±0.8%) was significantly higher than that in the control group (0±0%), the SSAD group (0.5±0.4%) and the AG group (0.3±0.0%). 0.3%) (Fig. 5d). The expression density of CD68+ cells in each microscope field in the SSAD+AG treatment group (0.6±0.2%) was significantly lower than that in the control group (11.5±2%), the SSAD group (1.3±0.3%) and the AG group (1.3±0.3%) ) (Fig. 5d). Separate fluorescent images of Figure 5a are provided to assess collagen deposition and neovascularization (Figure 20 and Figure 21). These results further suggest that AG-loaded SSADs can enhance the performance of SSADs in promoting angiogenesis, enhancing ECM remodeling, and reducing inflammation.
.总之，通过以可控的方式释放AG并促进L929细胞和HUVEC的增殖和迁移，装载AG的SSAD被认为通过在增生期(proliferative phase)的角质细胞之间的相互作用，来促进肉芽组织的形成和新血管生成。.In conclusion, by releasing AG in a controllable manner and promoting the proliferation and migration of L929 cells and HUVEC, AG-loaded SSAD is thought to promote the formation of granulation tissue through the interaction between keratinocytes in the proliferative phase. formation and neovascularization.
.为了进一步阐明SSAD在促进伤口愈合的基本机制，对再生的口腔粘膜进行了转录组学分析。维恩图(图6a)表明，两组中有994个差异表达基因(DEGs)共表达。SSAD处理后的DEGs的热图如图6b所示。分析了与共表达的DEGs相应的蛋白质-蛋白质相互作用(PPI)网络，并且每个节点的颜色强度(从蓝色到红色)和尺寸明确地代表了它们与其他蛋白质分子的联系程度。如图6c所示，PPI分析揭示，主要的相关信号分子是与胶原蛋白相关的蛋白质，如Col1a1、Col3a1和Col6a1。上调的DEGs的京都基因与基因组百科全书(KEGG)通路富集分析(图6d)显示，这些基因在胶原蛋白的消化和吸收、ECM-受体相互作用和细胞粘附分子(CAMs)中显著富集，表明SSAD可以加速细胞粘附、胶原蛋白沉积和ECM产生。此外，尽管并不显著，一些基因还在其他信号通路富集，如调节干细胞多能性的信号通路，这提示SSAD还可能通过募集干细胞来促进伤口愈合。基于KEGG数据库进行功能富集分析，并且前20个基因本体论(GO)术语如图6e所示，其中显著富集的生物学过程是与细胞-基质粘附有关的ECM形成、细胞粘附和迁移、ECM机化(organization)、胶原纤维机化、多细胞生物体发育和细胞-基质粘附的正调控。上述所有的转录组结果，都通过相应抗体的免疫荧光染色得到了验证。图5的结果显示，SSAD治疗后，在SSAD相关组别中Col-1、CD31和α-SMA的阳性染色率很高，这与转录组数据一致。上述结果表明，通过SSAD处理，与伤口愈合相关的通路和生物学过程被激活，如ECM-受体相互作用、细胞-基质粘附和ECM机化。. To further elucidate the basic mechanism of SSAD in promoting wound healing, transcriptomic analysis of the regenerated oral mucosa was performed. The Venn diagram (Fig. 6a) showed that 994 differentially expressed genes (DEGs) were co-expressed in the two groups. The heatmap of DEGs after SSAD treatment is shown in Fig. 6b. The protein-protein interaction (PPI) network corresponding to the coexpressed DEGs was analyzed, and the color intensity (from blue to red) and size of each node clearly represented their degree of connection with other protein molecules. As shown in Figure 6c, PPI analysis revealed that the major associated signaling molecules were collagen-associated proteins such as Col1a1, Col3a1 and Col6a1. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the upregulated DEGs (Fig. 6d) revealed that these genes were significantly enriched in collagen digestion and uptake, ECM-receptor interactions, and cell adhesion molecules (CAMs) set, indicating that SSAD can accelerate cell adhesion, collagen deposition, and ECM production. In addition, although not significantly, some genes were also enriched in other signaling pathways, such as those regulating stem cell pluripotency, suggesting that SSAD may also promote wound healing by recruiting stem cells. Functional enrichment analysis was performed based on the KEGG database, and the top 20 Gene Ontology (GO) terms are shown in Figure 6e, in which the significantly enriched biological processes were ECM formation, cell adhesion and Positive regulation of migration, ECM organization, collagen fiber organization, development of multicellular organisms, and cell-matrix adhesion. All transcriptome results described above were verified by immunofluorescent staining with the corresponding antibodies. The results in Figure 5 showed that after SSAD treatment, the positive staining rates of Col-1, CD31, and α-SMA were high in SSAD-related groups, which was consistent with the transcriptome data. The above results indicated that pathways and biological processes related to wound healing were activated by SSAD treatment, such as ECM-receptor interaction, cell-matrix adhesion, and ECM organization.
.伤口愈合一般可分为4个阶段：止血、炎症、细胞增殖和组织重塑。干细胞的激活和向伤口区域的有效募集是促进伤口再上皮化的关键。角质细胞干细胞(KSCs)归巢到伤口部位在伤口愈合中起重要作用并可以维持和修复上皮组织和保持组织的增殖潜力，由于其有分化成各种功能性细胞的能力。生物检定法结果显示，SSAD含有大量的生长因子，如VEGF、IGF-1、SDF-1等(表1)。大多数生长因子被认为可以促进细胞增殖和迁移，从而促进伤口愈合。特别地，SDF-1可以募集干细胞至伤口部位；在伤口部位，干细胞会分化成内皮细胞和成纤维细胞，通过细胞活化/增殖或间质转型(mesenchymal transition)变成肌成纤维细胞，肌成纤维细胞在软组织再生中发挥关键作用。另外，IGF-1的表达还在介导伤口愈合中起重要作用。在本发明中，为了进一步验证SSAD是否可以募集内源性干细胞并激活细胞粘附和迁移通路，实验人员通过免疫荧光法评估了再生的组织中的干细胞募集和细胞增殖，如上述转录组分析表明。.Wound healing can generally be divided into 4 stages: hemostasis, inflammation, cell proliferation and tissue remodeling. Activation and effective recruitment of stem cells to the wound area are key to promote wound re-epithelialization. Keratinocyte stem cells (KSCs) homing to wound sites play an important role in wound healing and can maintain and repair epithelial tissue and maintain tissue proliferative potential due to their ability to differentiate into various functional cells. The results of biological assays showed that SSAD contained a large number 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 the wound site; at the wound site, the stem cells will differentiate into endothelial cells and fibroblasts, and become myofibroblasts through cell activation/proliferation or mesenchymal transition. Fibroblasts play a key role in soft tissue regeneration. In addition, the expression of IGF-1 also plays an important role in mediating wound healing. In the present invention, in order to further verify whether SSAD can recruit endogenous stem cells and activate cell adhesion and migration pathways, experimenters evaluated stem cell recruitment and cell proliferation in regenerated tissues by immunofluorescence, as indicated by the above-mentioned transcriptome analysis .
.对SSAD治疗组和空白对照组进行KSCs(α6+/CD71-)免疫荧光染色和增殖细胞核抗原(PCNA)染色。结果(图6f和6g)显示，在术后7天，SSAD治疗组比对照组有显著更多的KSC被募集到伤口部位(P<0.001)。在同一时间点，SSAD治疗组的PCNA+角质细胞的数量也显著多于对照组。. KSCs (α6+/CD71-) immunofluorescence staining and proliferating cell nuclear antigen (PCNA) staining were performed on the SSAD treatment group and the blank control group. The results (Figures 6f and 6g) showed that at 7 days after surgery, significantly more KSCs were recruited to the wound site in the SSAD-treated group than in the control group (P<0.001). At the same time point, the number of PCNA+ keratinocytes in the SSAD-treated group was also significantly higher than that in the control group.
.基于上述结果，可得出结论：SSAD可以促进KSC募集到伤口部位，并保持一个更大的、有加速伤口愈合功能的再生的上皮角质细胞池。此外，SSAD可以促进细胞粘附以及ECM和胶原蛋白的沉积，使其成为有前景的用于软组织再生的敷料。.Based on the above results, it can be concluded that SSAD can promote the recruitment of KSCs to the wound site and maintain a larger pool of regenerating epithelial keratinocytes with the function of accelerating wound healing. In addition, SSAD can promote cell adhesion and deposition of ECM and collagen, making it a promising dressing for soft tissue regeneration.
.实施例五：体内动物研究之荷瘤伤口愈合模型.Example 5: Tumor-bearing wound healing model for in vivo animal research
.受上述实验中体外载药和控释的优秀表现的鼓励，进一步提出了协同联合局部化疗与组织再生，以治疗皮肤肿瘤的设想。对于荷瘤伤口愈合模型，考虑到局部施用DOX的强刺激性，在DOX的总剂量相同的条件下，相对较低的DOX的释放速率可能有利于荷黑色素瘤伤口愈合。由上，最终选择了可以更加稳定和持续地释放药物的200目SSAD水凝胶。恶性黑色素瘤的发病率在许多地区上升，且黑色素瘤可以发生于皮肤或粘膜。其中，皮肤恶性黑色素瘤在高加索人中最常见，而粘膜恶性黑色素瘤在黄种人(如日本人)中最常见。口腔内的恶性黑色素瘤大多发生在上牙龈和硬腭。因此，黑色素瘤被选作肿瘤模型。Encouraged by the excellent performance of in vitro drug loading and controlled release in the above experiments, the idea of synergistically combining local chemotherapy and tissue regeneration to treat skin tumors was further proposed. For the tumor-bearing wound healing model, considering the strong irritation of local application of DOX, under the same total dose of DOX, a relatively low release rate of DOX may be beneficial to the healing of melanoma-bearing wounds. Based on the above, the 200-mesh SSAD hydrogel that can release drugs more stably and continuously was finally selected. The incidence of malignant melanoma is increasing in many regions, and melanoma can arise in the skin or mucous membranes. Among them, cutaneous melanoma is most common in Caucasians, while mucosal melanoma is most common in people of yellow race (such as Japanese). Malignant melanomas in the oral cavity mostly occur on the upper gums and hard palate. Therefore, melanoma was chosen as a tumor model.
.将B16F10荷瘤小鼠随机分为4组：对照组、SSAD组、DOX组和SSAD+DOX组。图7a显示了在所示时间点的荷瘤伤口愈合的照片。在各种处理的18天后，切除的肿瘤的照片(图7b)可以进一步直观地说明各组的治疗效果。.The B16F10 tumor-bearing mice were randomly divided into 4 groups: control group, SSAD group, DOX group and SSAD+DOX group. Figure 7a shows photographs of tumor-bearing wound healing at the indicated time points. After 18 days of each treatment, photographs of resected tumors (Fig. 7b) can further visually illustrate the treatment effect of each group.
.还对各组切除的肿瘤进行了称重(图7c)和测量尺寸(图7d)。对照组的平均肿瘤尺寸迅速增加，并在18天时超过了4,000mm3和2.8g，有着最高的肿瘤复发率(多达67％)(图7e)。有趣的是，SSAD水凝胶组的肿瘤体积增加缓慢且平均肿瘤尺寸(1,500mm3，1.8g)小于对照组，肿瘤复发率为44％，这证明SSAD本身已经在一定程度上表现出抗肿瘤活性。在DOX组中也观察到比对照组稍微更明显的肿瘤抑制(1000mm3，0.78g)，复发率为22％。然而，令人注目地，在评估期间，SSAD+DOX组的小鼠没有出现肿瘤复发(图7e)。同时，在所有4组中，没有发现明显的体重异常(图7f)。在整个治疗期间，对照组、DOX组和SSAD组的荷瘤小鼠的存活率分别为78％、89％和100％；然而，SSAD+DOX治疗组的小鼠不仅存活率为100％，而且还保持无瘤状态(图7g)。The resected tumors in each group were also weighed (Fig. 7c) and measured in size (Fig. 7d). The average tumor size of the control group increased rapidly and exceeded 4,000 mm3 and 2.8 g at 18 days, with the highest rate of tumor recurrence (up to 67%) (Fig. 7e). Interestingly, the tumor volume of the SSAD hydrogel group increased slowly and the average tumor size (1,500 mm3 , 1.8 g) was smaller than that of the control group, and the tumor recurrence rate was 44%, which proves that SSAD itself has shown anti-tumor effects to a certain extent active. Slightly more pronounced tumor suppression was also observed in the DOX group than in the control group (1000 mm3 , 0.78 g), with a recurrence rate of 22%. Strikingly, however, mice in the SSAD+DOX group did not exhibit tumor recurrence during the evaluation period (Fig. 7e). Meanwhile, in all 4 groups, no obvious body weight abnormalities were found (Fig. 7f). During the entire treatment period, the survival rates of tumor-bearing mice in the control group, DOX group and SSAD group were 78%, 89% and 100%, respectively; however, the mice in the SSAD+DOX treatment group not only survived 100%, but also Tumor-free status was also maintained (Fig. 7g).
.这些令人鼓舞的结果表明：在SSAD+DOX组中，肿瘤细胞可以在早期被SSAD水凝胶释放的DOX有效消除，而且SSAD治愈全层皮肤缺损的能力并没有显著受到相对较短的DOX治疗的影响。虽然在装载DOX的SSAD附近的正常皮肤细胞也可能被释放的DOX清除，但SSAD可以诱导相对健康的组织的多种类型的细胞迁移到伤口部位，在伤口部位它们可以参与到随后的伤口愈合过程。这种协同作用在随后的组织愈合过程中发挥了重要作用。更重要的是，SSAD含有大量的、在伤口愈合过程中募集干细胞并激活伤口愈合相关的信号传导途径和生物学过程的生长因子，从而促进新血管生成、ECM产生以及胶原蛋白的沉积。同时，SSAD本身温和的抑制肿瘤作用，可能归因于所募集的干细胞的免疫调节。此外，由200目的细颗粒组成的SSAD水凝胶可以限制营养物质与代谢物的交换。虽然这种限制可能非常弱且对正常组织没有明显的影响，但可能会更好地抑制快速生长过程中需要更多营养和氧气并产生更多废物的实体肿瘤。.These encouraging results indicated that in the SSAD+DOX group, tumor cells could be effectively eliminated by DOX released from SSAD hydrogel at an early stage, and that the ability of SSAD to heal full-thickness skin defects was not significantly affected by the relatively short DOX Effect of treatment. Although normal skin cells in the vicinity of a DOX-loaded SSAD may also be cleared by the released DOX, the SSAD induces the migration of multiple cell types from relatively healthy tissues to the wound site, where they can 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 wound healing and activate wound healing-related signaling pathways and biological processes, thereby promoting neovascularization, ECM production, and collagen deposition. Meanwhile, the mild tumor suppressive effect of SSAD itself may be attributed to the immune regulation of the recruited stem cells. In addition, the SSAD hydrogel composed of 200-mesh fine particles can limit the exchange of nutrients and metabolites. While this confinement may be very weak and have no appreciable effect on normal tissues, it may better suppress solid tumors that require more nutrients and oxygen and produce more waste products during rapid growth.
.最后，对第18天的肿瘤组织进行了HE、末端脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)和Ki67染色(图8a)。HE图像揭示，SSAD+DOX组诱导出最多的肿瘤细胞破坏。如TUNEL染色所示，与其他三组相比，SSAD+DOX组的凋亡荧光表达最高。此外，几乎看不见SSAD+DOX组的Ki67+肿瘤细胞表达，这表明装载DOX的SSAD伤口敷料显著降低了肿瘤细胞的增殖。凋亡细胞和Ki67+肿瘤细胞的定量分析如图8b和8c所示。总体上，很明显，与其他组相比，SSAD+DOX对黑色素瘤的体内抑制效果最好。此外，在本发明中不同的处理后，主要器官没有明显的组织病理学变化(图8d)，这表明SSAD可以作为局部药物递送的安全载体。Finally, tumor tissues at
.总结：.Summarize:
.近年来，局部药物递送受到越来越多的关注。然而，在某些场景下(例如口腔中或肿瘤切除后的创面)，局部药物递送的治疗效果仍然受限。本发明提供的局部给药系统被证明是一种有效的用于各种药物(例如AG和DOX)的局部药物持续释放系统。.Local drug delivery has received increasing attention in recent years. However, the therapeutic effect of local drug delivery is still limited in some scenarios (such as in the oral cavity or on wounds after tumor resection). The topical drug delivery system provided by the present invention proved to be an effective local drug sustained release system for various drugs such as AG and DOX.
.本发明涉及的装载氨基胍或多柔比星的局部给药系统，展示了可控的药物释放和促进愈合的性能，并分别在糖尿病性创面(糖尿病大鼠腭部粘膜缺损模型)或肿瘤切除后的创面(C57BL/6小鼠背部黑色素瘤模型)中得到了验证，如图1所示。上述两种伤口都为动态伤口，其中口内糖尿病性创面是处于糖尿病的高血糖下的湿态伤口，其受到咀嚼等压力和细菌的干扰；背部皮肤肿瘤切除后的创面是处于肿瘤环境下的干态伤口，其受到拉伸并有再次发展成肿瘤的风险。即便处在易遭受干扰且多变的环境中，本发明提供的局部给药系统却能够长时间稳定粘附在动态伤口上并保持良好的贴合，不会因受动态伤口所处的环境的影响而丧失粘性和完整性；这展现了本发明提供的局部给药系统的优秀的粘性(干湿粘结)与适应性(柔性粘结)，并保证了稳定且可控的药物释放。即本发明的局部递送系统发挥药物局部递送和促进伤口愈合的双重功能，以促进上述处于难以愈合状态的动态伤口的愈合。另一方面，在本发明的局部递送系统中，鲵皮肤分泌物水凝胶和所装载的药物在促进伤口愈合(特别是动态伤口的愈合)中起到协同作用，在药物局部递送的同时，促进伤口愈合。其促进伤口愈合的作用表现在促进细胞迁移和增殖、加速ECM沉积(上调与细胞粘附和细胞外基质沉积有关的通路)和募集内源性干细胞到伤口部位等方面。.The local drug delivery system loaded with aminoguanidine or doxorubicin related to the present invention has demonstrated controllable drug release and healing-promoting properties, and is effective in diabetic wounds (diabetic rat palatal mucosa defect model) or tumors respectively. It was verified in the resected wound (C57BL/6 mouse dorsal melanoma model), as shown in Figure 1. The above two kinds of wounds are dynamic wounds, and the diabetic wound in the mouth is a wet wound under the hyperglycemia of diabetes, which is disturbed by pressure such as mastication and bacteria; the wound after resection of the back skin tumor is a dry wound under the tumor environment. A wound that is stretched and at risk of developing a tumor again. Even in a disturbed and changeable environment, the local drug delivery system provided by the present invention can stably adhere to the dynamic wound for a long time and maintain a good fit without being affected by the environment where the dynamic wound is located. This shows the excellent viscosity (dry and wet bonding) and adaptability (flexible bonding) of the topical drug delivery system provided by the present invention, and ensures stable and controllable drug release. That is, the local delivery system of the present invention exerts the dual functions of local drug delivery and wound healing promotion, so as to promote the healing of the aforementioned dynamic wounds that are difficult to heal. On the other hand, in the local delivery system of the present invention, the salamander skin secretion hydrogel and the loaded drug play a synergistic role in promoting wound healing (especially dynamic wound healing), while the drug is delivered locally, Promotes wound healing. Its role in promoting wound healing is manifested in promoting cell migration and proliferation, accelerating ECM deposition (upregulating pathways related to cell adhesion and extracellular matrix deposition), and recruiting endogenous stem cells to the wound site.
.可以根据实际需求(例如，对药物释放速率的要求、伤口的类型)，通过控制相应的鲵皮肤分泌物冻干粉粒度来制备具有针对性的孔隙结构的装载药物的水凝胶。.A drug-loaded hydrogel with a targeted pore structure can be prepared by controlling the particle size of the corresponding lyophilized powder of salamander skin secretions according to actual needs (eg, requirements for drug release rate, type of wound).
.上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。.The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation, the above-mentioned specific implementation is only illustrative, rather than limiting, those of ordinary skill in the art Under the enlightenment of the present invention, many forms can also be made without departing from the gist of the present invention and the protection scope of the claims, and these all belong to the protection of the present invention.
.参考文献.references
1.Ongnok,B.；Chattipakorn,N.；Chattipakorn,S.C.,Doxorubicin andcisplatin induced cognitive impairment:Thepossiblemechanismsandinterventions.ExpNeurol2020,324,113118.1. Ongnok, B.; Chattipakorn, N.; Chattipakorn, S.C., Doxorubicin and cisplatin induced cognitive impairment: The possible mechanisms and interventions. ExpNeurol2020, 324, 113118.
2.Oliva,N.；Conde,J.；Wang,K.；Artzi,N.,Designing Hydrogels for On-Demand Therapy.Acc ChemRes 2017,50(4),669-679.2. Oliva, N.; Conde, J.; Wang, K.; Artzi, N., Designing Hydrogels for On-Demand Therapy. Acc ChemRes 2017, 50(4), 669-679.
3.Katz,J.；Bhattacharyya,I.；Farkhondeh-Kish,F.；Perez,F.M.；Caudle,R.M.；Heft,M.W.,Expression of the receptor of advanced glycation end products ingingival tissues of type 2diabetes patients with chronic periodontal disease:a study utilizing immunohistochemistry and RT-PCR.JClinPeriodontol2005,32(1),40-4.3. Katz, J.; Bhattacharyya, I.; Farkhondeh-Kish, F.; Perez, F.M.; : a study utilizing immunohistochemistry and RT-PCR. JClinPeriodontol2005, 32(1), 40-4.
4.Li,Y.；Chang,Y.；Ye,N.；Dai,D.；Chen,Y.；Zhang,N.；Sun,G.；Sun,Y.,AdvancedGlycation End Products Inhibit the Proliferation of Human Umbilical VeinEndothelial Cells by Inhibiting Cathepsin D.IntJMol Sci 2017,18(2).4. Li, Y.; Chang, Y.; Ye, N.; Dai, D.; Chen, Y.; Zhang, N.; Sun, G.; Sun, Y., Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.IntJMol Sci 2017,18(2).
5.Okonkwo,U.A.；DiPietro,L.A.,Diabetes and Wound Angiogenesis.Int JMolSci 2017,18(7).5. Okonkwo, U.A.; DiPietro, L.A., Diabetes and Wound Angiogenesis. Int JMolSci 2017, 18(7).
6.Bao,Y.；Chen,H.；Cai,Z.；Zheng,J.；Zou,J.；Shi,Y.；Jiang,L.,Advancedglycation end products inhibit neural stem cell differentiation viaupregualtion ofHDAC3 expression.Brain Res Bull 2020,159,1-8.6. Bao, Y.; Chen, H.; Cai, Z.; Zheng, J.; Zou, J.; Shi, Y.; Jiang, L., Advanced glycation end products inhibit neural stem cell differentiation via upregualtion of HDAC3 expression. Brain Res Bull 2020,159,1-8.
7.Li,Y.；Wang,L.；Zhang,M.；Huang,K.；Yao,Z.；Rao,P.；Cai,X.；Xiao,J.,Advanced glycation end products inhibit the osteogenic differentiationpotential of adipose-derived stem cells by modulating Wnt/β-cateninsignalling pathway via DNA methylation.Cell Prolif 2020,53(6),e12834.7. Li, Y.; Wang, L.; Zhang, M.; Huang, K.; Yao, Z.; Rao, P.; Cai, X.; Xiao, J., Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/β-catenin signaling pathway via DNA methylation. Cell Prolif 2020,53(6),e12834.
8.Chang,P.C.；Tsai,S.C.；Jheng,Y.H.；Lin,Y.F.；Chen,C.C.,Soft-tissuewound healing by anti-advanced glycation end-products agents.JDentRes 2014,93(4),388-93.8. Chang, P.C.; Tsai, S.C.; Jheng, Y.H.; Lin, Y.F.; Chen, C.C., Soft-tissue wound healing by anti-advanced glycation end-products agents. JDentRes 2014, 93(4), 388-93.
9.Thornalley,P.J.,Use of aminoguanidine(Pimagedine)to prevent theformation of advanced glycation endproducts.Arch Biochem Biophys 2003,419(1),31-40.9. Thornalley, P.J., Use of aminoguanidine (Pimagedine) to prevent the formation of advanced glycation endproducts. Arch Biochem Biophys 2003, 419(1), 31-40.
10.Benencia,F.；Courrèges,M.C.；Gamba,G.；Cavalieri,H.；Massouh,E.J.,Effect of aminoguanidine,a nitric oxide synthase inhibitor,on ocularinfection with herpes simplex virus in Balb/c mice.Invest Ophthalmol Vis Sci2001,42(6),1277-84.10. Benencia, F.; Courrèges, M.C.; Gamba, G.; Cavalieri, H.; Massouh, E.J., Effect of aminoguanidine, a nitric oxide synthase inhibitor, on ocular infection with herpes simplex virus in Balb/c mice. Invest Ophthalmol Vis Sci2001, 42(6), 1277-84.
11.Nilsson,B.O.,Biological effects of aminoguanidine:anupdate.Inflamm Res 1999,48(10),509-15.11. Nilsson, B.O., Biological effects of aminoguanidine: an update. Inflamm Res 1999, 48(10), 509-15.
12.Sezgin,Y.；Bilgin Cetin,M.；Bulut,S.；Alptekin,N.；
13.Wu,T.；Zhang,D.；Qiao,Q.；Qin,X.；Yang,C.；Kong,M.；Deng,H.；Zhang,Z.,Biomimetic Nanovesicles for Enhanced Antitumor Activity of CombinationalPhotothermal and Chemotherapy.MolPharm 2018,15(3),1341-1352.13. Wu, T.; Zhang, D.; Qiao, Q.; Qin, X.; Yang, C.; Kong, M.; and Chemotherapy. MolPharm 2018, 15(3), 1341-1352.
14.Li,J.；Xin,M.；Huo,Y.；Cai,A.；Yan,M.；Wang,C.；Wei,G.,Synthesis of β-cyclodextrin-PEG-G molecules to delay tumor growth and application ofβ-cyclodextrin-PEG-G aggregates as drug carrier.CarbohydrPolym 2020,229,115478.14. Li, J.; Xin, M.; Huo, Y.; Cai, A.; Yan, M.; Wang, C.; Wei, G., Synthesis of β-cyclodextrin-PEG-G molecules to delay tumor growth and application ofβ-cyclodextrin-PEG-G aggregates as drug carrier.CarbohydrPolym 2020,229,115478.
15.Chen,J.；Ding,J.；Xu,W.；Sun,T.；Xiao,H.；Zhuang,X.；Chen,X.,Receptorand Microenvironment Dual-Recognizable Nanogel for Targeted Chemotherapy ofHighly Metastatic Malignancy.Nano Lett 2017,17(7),4526-4533.15. Chen, J.; Ding, J.; Xu, W.; Sun, T.; Xiao, H.; Zhuang, X.; Chen, X., Receptor and Microenvironment Dual-Recognizable Nanogel for Targeted Chemotherapy of Highly Metastatic Malignancy. Nano Lett 2017, 17(7), 4526-4533.
16.Feng,S.-S.；Chien,S.,Chemotherapeutic engineering:Application andfurther development of chemical engineering principles for chemotherapy ofcancer and other diseases.Chemical Engineering Science 2003,58(18),4087-4114.16. Feng, S.-S.; Chien, S., Chemotherapeutic engineering: Application and further development of chemical engineering principles for chemotherapy of cancer and other diseases. Chemical Engineering Science 2003, 58(18), 4087-4114.
17.Ran,J.D.Y.T.Q.Z.C.W.M.L.P.J.J.S.G.L.L.C.X.,A Bioinspired MedicalAdhesive Derived from Skin Secretion of Andrias davidianus for Wound HealingAdvanced Functional Materials 2019,1809110.17. Ran, J.D.Y.T.Q.Z.C.W.M.L.P.J.J.S.G.L.L.C.X., A Bioinspired Medical Adhesive Derived from Skin Secretion of Andrias davidianus for Wound Healing Advanced Functional Materials 2019, 1809110.
18.Lynam,D.；Peterson,C.；Maloney,R.；Shahriari,D.；Garrison,A.；Saleh,S.；Mehrotra,S.；Chan,C.；Sakamoto,J.,Augmenting protein release from layer-by-layer functionalized agarose hydrogels.CarbohydrPolym 2014,103,377-84.18. Lynam, D.; Peterson, C.; Maloney, R.; Shahriari, D.; Garrison, A.; Saleh, S.; Mehrotra, S.; Chan, C.; Sakamoto, J., Augmenting protein release from layer-by-layer functionalized agarose hydrogels. Carbohydr Polym 2014, 103, 377-84.
19.Graves,D.T.；Nooh,N.；Gillen,T.；Davey,M.；Patel,S.；Cottrell,D.；Amar,S.,IL-1 plays a critical role in oral,but not dermal,wound healing.JImmunol2001,167(9),5316-20.19. Graves, D.T.; Nooh, N.; Gillen, T.; Davey, M.; Patel, S.; Cottrell, D.; Amar, S., IL-1 plays a critical role in oral, but not dermal, wound healing. J Immunol 2001, 167(9), 5316-20.
20.Zhang,Z.；Dai,Q.；Zhang,Y.；Zhuang,H.；Wang,E.；Xu,Q.；Ma,L.；Wu,C.；Huan,Z.；Guo,F.；Chang,J.,Design of a Multifunctional Biomaterial Inspired byAncient Chinese Medicine for Hair Regeneration in BurnedSkin.ACSApplMaterInterfaces 2020,12(11),12489-12499.20. Zhang, Z.; Dai, Q.; Zhang, Y.; Zhuang, H.; Wang, E.; .; Chang, J., Design of a Multifunctional Biomaterial Inspired by Ancient Chinese Medicine for Hair Regeneration in Burned Skin. ACS Appl Mater Interfaces 2020, 12(11), 12489-12499.
21.Shabbir,A.；Cox,A.；Rodriguez-Menocal,L.；Salgado,M.；Van Badiavas,E.,Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normaland Chronic Wound Fibroblasts,and Enhance Angiogenesis In Vitro.Stem CellsDev 2015,24(14),1635-47.21. Shabbir, A.; Cox, A.; Rodriguez-Menocal, L.; Salgado, M.; Van Badiavas, E., Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normaland Chronic Wound Fibroblasts, and Enhance Angiogenesis In Vitro. Stem Cells Dev 2015, 24(14), 1635-47.
22.Fesseha,B.K.；Abularrage,C.J.；Hines,K.F.；Sherman,R.；Frost,P.；Langan,S.；Canner,J.；Likes,K.C.；Hosseini,S.M.；Jack,G.；Hicks,C.W.；Yalamanchi,S.；Mathioudakis,N.,Association of Hemoglobin A(1c)and Wound Healing inDiabetic Foot Ulcers.Diabetes Care2018,41(7),1478-1485.22. Fesseha, B.K.; Abularrage, C.J.; Hines, K.F.; Sherman, R.; Frost, P.; Langan, S.; Canner, J.; Likes, K.C.; Hosseini, S.M.; Jack, G.; ; Yalamanchi, S.; Mathioudakis, N., Association of Hemoglobin A(1c) and Wound Healing in Diabetic Foot Ulcers. Diabetes Care 2018, 41(7), 1478-1485.
23.Long,M.；Rojo de la Vega,M.；Wen,Q.；Bharara,M.；Jiang,T.；Zhang,R.；Zhou,S.；Wong,P.K.；Wondrak,G.T.；Zheng,H.；Zhang,D.D.,An Essential Role ofNRF2in Diabetic Wound Healing.Diabetes 2016,65(3),780-93.23. Long, M.; Rojo de la Vega, M.; Wen, Q.; Bharara, M.; Jiang, T.; H.; Zhang, D.D., An Essential Role of NRF2 in Diabetic Wound Healing. Diabetes 2016, 65(3), 780-93.
24.Werner,S.；Grose,R.,Regulation ofwound healing by growth factorsand cytokines.Physiol Rev 2003,83(3),835-70.24. Werner, S.; Grose, R., Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003, 83(3), 835-70.
25.Ballestas,S.A.；Turner,T.C.；Kamalakar,A.；Stephenson,Y.C.；Willett,N.J.；Goudy,S.L.；Botchwey,E.A.,Improving hard palate wound healing usingimmune modulatory autotherapies.Acta Biomater 2019,91,209-219.25. Ballestas, S.A.; Turner, T.C.; Kamalakar, A.; Stephenson, Y.C.; Willett, N.J.; 09-219.
26.Takeo,M.；Lee,W.；Ito,M.,Wound healing and skin regeneration.ColdSpring Harb PerspectMed 2015,5(1),a023267.26. Takeo, M.; Lee, W.; Ito, M., Wound healing and skin regeneration. Cold Spring Harb PerspectMed 2015, 5(1), a023267.
27.Zhao,X.；Wu,H.；Guo,B.；Dong,R.；Qiu,Y.；Ma,P.X.,Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing withhemostasis and adhesiveness for cutaneous wound healing.Biomaterials 2017,122,34-47.27. Zhao, X.; Wu, H.; Guo, B.; Dong, R.; Qiu, Y.; Ma, P.X., Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials 2017, 122, 34-47.
28.Lucas,T.；Schafer,F.；Muller,P.；Eming,S.A.；Heckel,A.；Dimmeler,S.,Light-inducible antimiR-92a as a therapeutic strategy to promote skin repairin healing-impaired diabetic mice.Nat Commun 2017,8,15162.28. Lucas, T.; Schafer, F.; Muller, P.; Eming, S.A.; Heckel, A.; Dimmeler, S., Light-inducible antimiR-92a as a therapeutic strategy to promote skin repair in healing-impaired diabetic mice .
29.Shin,J.W.；Kwon,S.H.；Choi,J.Y.；Na,J.I.；Huh,C.H.；Choi,H.R.；Park,K.C.,Molecular Mechanisms ofDermal Aging and Antiaging Approaches.IntJMol Sci2019,20(9).29. Shin, J.W.; Kwon, S.H.; Choi, J.Y.; Na, J.I.; Huh, C.H.; Choi, H.R.; Park, K.C., Molecular Mechanisms of Dermal Aging and Antiaging Approaches.
30.Desmoulière,A.；Geinoz,A.；Gabbiani,F.；Gabbiani,G.,Transforminggrowth factor-beta 1induces alpha-smooth muscle actin expression ingranulation tissue myofibroblasts and in quiescent and growing culturedfibroblasts.JCellBiol 1993,122(1),103-11.30. Desmoulière, A.; Geinoz, A.; Gabbiani, F.; Gabbiani, G., Transforming growth factor-
31.Wang,Y.Y.；Jiang,H.；Pan,J.；Huang,X.R.；Wang,Y.C.；Huang,H.F.；To,K.F.；Nikolic-Paterson,D.J.；Lan,H.Y.；Chen,J.H.,Macrophage-to-MyofibroblastTransition Contributes to Interstitial Fibrosis in Chronic Renal AllograftInjury.JAm Soc Nephrol 2017,28(7),2053-2067.31. Wang, Y.Y.; Jiang, H.; Pan, J.; Huang, X.R.; Wang, Y.C.; Huang, H.F.; Myofibroblast Transition Contributes to Interstitial Fibrosis in Chronic Renal Allograft Injury. JAm Soc Nephrol 2017, 28(7), 2053-2067.
32.Jinno,K.；Takahashi,T.；Tsuchida,K.；Tanaka,E.；Moriyama,K.,Acceleration of palatal wound healing in Smad3-deficient mice.JDentRes 2009,88(8),757-61.32. Jinno, K.; Takahashi, T.; Tsuchida, K.; Tanaka, E.; Moriyama, K., Acceleration of palatal wound healing in Smad3-deficient mice. JDentRes 2009, 88(8), 757-61.
33.Yang,R.；Liu,F.；Wang,J.；Chen,X.；Xie,J.；Xiong,K.,Epidermal stemcells in wound healing and their clinical applications.Stem CellRes Ther2019,10(1),229.33. Yang, R.; Liu, F.; Wang, J.; Chen, X.; Xie, J.; Xiong, K., Epidermal stem cells in wound healing and their clinical applications. Stem Cell Res Ther2019, 10(1) ,229.
34.Garcin,C.L.；Ansell,D.M.,The battle of the bulge:re-evaluating hairfollicle stem cells in wound repair.Exp Dermatol 2017,26(2),101-104.34. Garcin, C.L.; Ansell, D.M., The battle of the bulge: re-evaluating hairfollicle stem cells in wound repair. Exp Dermatol 2017, 26(2), 101-104.
35.Ojeh,N.；Pastar,I.；Tomic-Canic,M.；Stojadinovic,O.,Stem Cells inSkin Regeneration,Wound Healing,and Their Clinical Applications.IntJMol Sci2015,16(10),25476-501.35. Ojeh, N.; Pastar, I.; Tomic-Canic, M.; Stojadinovic, O., Stem Cells in Skin Regeneration, Wound Healing, and Their Clinical Applications. IntJMol Sci2015, 16(10), 25476-501.
36.Spees,J.L.；Lee,R.H.；Gregory,C.A.,Mechanisms of mesenchymal stem/stromal cell function.Stem CellRes Ther 2016,7(1),125.36.Spees, J.L.; Lee, R.H.; Gregory, C.A., Mechanisms of mesenchymal stem/stromal cell function. Stem Cell Res Ther 2016, 7(1), 125.
37.Calenic,B.；Greabu,M.；Caruntu,C.；Tanase,C.；Battino,M.,Oralkeratinocyte stem/progenitor cells:specific markers,molecular signalingpathways and potential uses.Periodontol 20002015,69(1),68-82.37. Calenic, B.; Greabu, M.; Caruntu, C.; Tanase, C.; Battino, M., Oralkeratinocyte stem/progenitor cells: specific markers, molecular signaling pathways and potential uses. Periodontol 20002015, 69(1), 68-82.
38.Liesveld,J.L.；Sharma,N.；Aljitawi,O.S.,Stem cell homing:Fromphysiology to therapeutics.Stem Cells 2020,38(10),1241-1253.38. Liesveld, J.L.; Sharma, N.; Aljitawi, O.S., Stem cell homing: Fromphysiology to therapeutics. Stem Cells 2020, 38(10), 1241-1253.
39.He,X.T.；Li,X.；Xia,Y.；Yin,Y.；Wu,R.X.；Sun,H.H.；Chen,F.M.,Buildingcapacity for macrophage modulation and stem cell recruitment in high-stiffness hydrogels for complex periodontal regeneration:Experimental studiesin vitro and in rats.Acta Biomater 2019,88,162-180.39. He, X.T.; Li, X.; Xia, Y.; Yin, Y.; Wu, R.X.; Sun, H.H.; Chen, F.M., Building capacity for macrophage modulation and stem cell recruitment in high-stiffness hydrogels for complex periodontal regeneration : Experimental studies in vitro and in rats. Acta Biomater 2019,88,162-180.
40.Tomasek,J.J.；Gabbiani,G.；Hinz,B.；Chaponnier,C.；Brown,R.A.,Myofibroblasts and mechano-regulation ofconnective tissueremodelling.NatRevMol CellBiol 2002,3(5),349-63.40. Tomasek, J.J.; Gabbiani, G.; Hinz, B.; Chaponnier, C.; Brown, R.A., Myofibroblasts and mechano-regulation of connective tissue remodeling. Nat Rev Mol Cell Biol 2002, 3(5), 349-63.
41.Park,J.；Yan,G.；Kwon,K.C.；Liu,M.；Gonnella,P.A.；Yang,S.；Daniell,H.,Oral delivery ofnovel human IGF-1 bioencapsulated in lettuce cells promotesmusculoskeletal cell proliferation,differentiation and diabetic fracturehealing.Biomaterials 2020,233,119591.41. Park, J.; Yan, G.; Kwon, K.C.; Liu, M.; Gonnella, P.A.; Yang, S.; Daniell, H., Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracturehealing. Biomaterials 2020, 233, 119591.
42.Murugan Girija,D.；Kalachaveedu,M.；Ranga Rao,S.；Subbarayan,R.,Transdifferentiation of human gingival mesenchymal stem cells into functionalkeratinocytes by Acalypha indica in three-dimensionalmicroenvironment.JCellPhysiol 2018,233(11),8450-8457.42. Murugan Girija, D.; Kalachaveedu, M.; Ranga Rao, S.; Subbarayan, R., Transdifferentiation of human gingival mesenchymal stem cells into functional keratinocytes by Acalypha indica in three-dimensional microenvironment. JCellPhysiol 2018, 233(11),8450 -8457.
43.Bergoglio,V.；Larcher,F.；Chevallier-Lagente,O.；Bernheim,A.；Danos,O.；Sarasin,A.；Rio,M.D.；Magnaldo,T.,Safe selection ofgenetically manipulatedhuman primary keratinocytes with very high growth potential using CD24.MolTher 2007,15(12),2186-93.43. Bergoglio, V.; Larcher, F.; Chevallier-Lagente, O.; Bernheim, A.; Danos, O.; Sarasin, A.; very high growth potential using CD24. MolTher 2007, 15(12), 2186-93.
44.Tancharoen,S.；Gando,S.；Binita,S.；Nagasato,T.；Kikuchi,K.；Nawa,Y.；Dararat,P.；Yamamoto,M.；Narkpinit,S.；Maruyama,I.,HMGB1 Promotes IntraoralPalatal Wound Healing through RAGE-Dependent Mechanisms.IntJMol Sci 2016,17(11).44. Tancharoen, S.; Gando, S.; Binita, S.; Nagasato, T.; Kikuchi, K.; .,HMGB1 Promotes Intraoral Palatal Wound Healing through RAGE-Dependent Mechanisms.IntJMol Sci 2016,17(11).
45.Ghazawi,F.M.；Cyr,J.；Darwich,R.；Le,M.；Rahme,E.；Moreau,L.；Netchiporouk,E.；Zubarev,A.；Roshdy,O.；Glassman,S.J.；Sasseville,D.；Litvinov,I.V.,Cutaneous malignant melanoma incidence and mortality trends in Canada:Acomprehensive population-based study.J Am AcadDermatol 2019,80(2),448-459.45. Ghazawi, F.M.; Cyr, J.; Darwich, R.; Le, M.; Rahme, E.; Moreau, L.; Sasseville, D.; Litvinov, I.V., Cutaneous malignant melanoma incidence and mortality trends in Canada: A comprehensive population-based study. J Am AcadDermatol 2019, 80(2), 448-459.
46.Ascierto,P.A.；Accorona,R.；Botti,G.；Farina,D.；Fossati,P.；Gatta,G.；Gogas,H.；Lombardi,D.；Maroldi,R.；Nicolai,P.；Ravanelli,M.；Vanella,V.,Mucosalmelanoma ofthe head and neck.CritRev OncolHematol 2017,112,136-152.46. Ascierto, P.A.; Accorona, R.; Botti, G.; Farina, D.; Fossati, P.; Gatta, G.; Gogas, H.; Lombardi, D.; Maroldi, R.; Nicolai, P. ; Ravanelli, M.; Vanella, V., Mucosalmelanoma of the head and neck. Crit Rev Oncol Hematol 2017, 112, 136-152.
47.Lopez,F.；Rodrigo,J.P.；Cardesa,A.；Triantafyllou,A.；Devaney,K.O.；Mendenhall,W.M.；Haigentz,M.,Jr.；Strojan,P.；Pellitteri,P.K.；Bradford,C.R.；Shaha,A.R.；Hunt,J.L.；de Bree,R.；Takes,R.P.；Rinaldo,A.；Ferlito,A.,Update onprimary head and neck mucosal melanoma.HeadNeck 2016,38(1),147-55.47. Lopez, F.; Rodrigo, J.P.; Cardesa, A.; Triantafyllou, A.; Devaney, K.O.; Shaha, A.R.; Hunt, J.L.; de Bree, R.; Takes, R.P.; Rinaldo, A.; Ferlito, A., Update on primary head and neck mucosal melanoma.
48.Mohammadi,M.；Jaafari,M.R.；Mirzaei,H.R.；Mirzaei,H.,Mesenchymal stemcell:a new horizon in cancer gene therapy.Cancer Gene Ther 2016,23(9),285-6.48. Mohammadi, M.; Jaafari, M.R.; Mirzaei, H.R.; Mirzaei, H., Mesenchymal stemcell: a new horizon in cancer gene therapy. Cancer Gene Ther 2016, 23(9), 285-6.
49.Francois,S.；Usunier,B.；Forgue-Lafitte,M.E.；L'Homme,B.；Benderitter,M.；Douay,L.；Gorin,N.C.；Larsen,A.K.；Chapel,A.,Mesenchymal Stem CellAdministration Attenuates Colon Cancer Progression by Modulating the ImmuneComponent within the Colorectal Tumor Microenvironment.Stem Cells TranslMed2019,8(3),285-300.49. Francois, S.; Usunier, B.; Forgue-Lafitte, M.E.; L'Homme, B.; Benderitter, M.; Douay, L.; Gorin, N.C.; Larsen, A.K.; Cell Administration Attenuates Colon Cancer Progression by Modulating the Immune Component within the Colorectal Tumor Microenvironment. Stem Cells TranslMed2019, 8(3), 285-300.
50.Guerrero,S.；Inostroza-Riquelme,M.；Contreras-Orellana,P.；Diaz-Garcia,V.；Lara,P.；Vivanco-Palma,A.；Cardenas,A.；Miranda,V.；Robert,P.；Leyton,L.；Kogan,M.J.；Quest,A.F.G.；Oyarzun-Ampuero,F.,Curcumin-loaded nanoemulsion:anew safe and effective formulation to prevent tumor reincidence andmetastasis.Nanoscale 2018,10(47),22612-22622.50. Guerrero, S.; Inostroza-Riquelme, M.; Contreras-Orellana, P.; Diaz-Garcia, V.; Lara, P.; Robert, P.; Leyton, L.; Kogan, M.J.; Quest, A.F.G.; Oyarzun-Ampuero, F., Curcumin-loaded nanoemulsion: a new safe and effective formulation to prevent tumor recurrence and metastasis. Nanoscale 2018, 10(47), 22612 -22622.
51.Firat,E.T.；
52.Aiala,G.F.；Oliveira,A.M.；Costa,F.O.；Fialho,D.L.；Cunha,A.S.,Jr.；Oliveira,P.A.,Effect of local application of aminoguanidine on thebiomechanical retention of implants in rats with induceddiabetes.IntJOralMaxillofac Implants 2013,28(5),1272-7.52. Aiala, G.F.; Oliveira, A.M.; Costa, F.O.; Fialho, D.L.; Cunha, A.S., Jr.; Oliveira, P.A., Effect of local application of aminoguanidine on the biomechanical retention of implants in rats with induceddiabetes. 2013,28 (5), 1272-7.
53.Farhad,A.R.；Razavi,S.M.；Rozati,A.R.；Shekarchizade,N.；Manshaei,M.,Selective nitric oxide synthase inhibitor promotes bone healing.DentRes J(Isfahan)2017,14(5),306-313.53. Farhad, A.R.; Razavi, S.M.; Rozati, A.R.; Shekarchizade, N.; Manshaei, M., Selective nitric oxide synthase inhibitor promotes bone healing. DentRes J (Isfahan) 2017, 14(5), 306-313.
54.Wang,X.；Lv,F.；Li,T.；Han,Y.；Yi,Z.；Liu,M.；Chang,J.；Wu,C.,ElectrospunMicropatterned Nanocomposites Incorporated with Cu(2)S Nanoflowers for SkinTumor Therapy and Wound Healing.ACSNano 2017,11(11),11337-11349.54. Wang, X.; Lv, F.; Li, T.; Han, Y.; Yi, Z.; Liu, M.; Chang, J.; Wu, C., Electrospun Micropatterned Nanocomposites Incorporated with Cu(2) S Nanoflowers for SkinTumor Therapy and Wound Healing. ACSNano 2017, 11(11), 11337-11349.
Claims (10)
Translated fromChineseApplications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2021113071734 | 2021-11-05 | ||
| CN202111307173 | 2021-11-05 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114984233A CN114984233A (en) | 2022-09-02 |
| CN114984233Btrue CN114984233B (en) | 2023-07-14 |
Family
ID=83032431
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210643002.7AActiveCN114984233B (en) | 2021-11-05 | 2022-06-08 | A local drug delivery system based on salamander skin secretion and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114984233B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119868628A (en)* | 2023-05-10 | 2025-04-25 | 重庆医科大学附属口腔医院 | Sponge material based on giant salamander skin secretion and used for stopping bleeding and promoting bones |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335288A (en)* | 2010-07-20 | 2012-02-01 | 陕西理工学院 | Giant salamander skin mucus scalding ointment |
| CN112107723A (en)* | 2019-06-20 | 2020-12-22 | 重庆医科大学附属口腔医院 | Medical water-based adhesive and using method thereof |
| CN112121065A (en)* | 2020-10-09 | 2020-12-25 | 重庆医科大学附属口腔医院 | Medical styptic powder and application thereof |
- 2022
- 2022-06-08CNCN202210643002.7Apatent/CN114984233B/enactiveActive
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335288A (en)* | 2010-07-20 | 2012-02-01 | 陕西理工学院 | Giant salamander skin mucus scalding ointment |
| CN112107723A (en)* | 2019-06-20 | 2020-12-22 | 重庆医科大学附属口腔医院 | Medical water-based adhesive and using method thereof |
| CN112121065A (en)* | 2020-10-09 | 2020-12-25 | 重庆医科大学附属口腔医院 | Medical styptic powder and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| A Bioinspired Medical Adhesive Derived from skin secretion of Andrias davidianus for Wound Healing;Jun Deng et al.;《Advanced Functional Materials》;第29卷(第31期);1809110* |
| A Natural Hydrogel with Prohealing Properties Enhances Tendon Regeneration;Ruyi Dang et al.;《Small》;第18卷(第36期);e2105255* |
| Bioinspired Andrias davidianus-Derived wound dressings for localized drug-elution;Xiang Liu et al.;《Bioactive Materials》;482-494* |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114984233A (en) | 2022-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Summa et al. | A biocompatible sodium alginate/povidone iodine film enhances wound healing | |
| Borbolla-Jiménez et al. | Films for wound healing fabricated using a solvent casting technique | |
| Dam et al. | Wound healing strategies based on nanoparticles incorporated in hydrogel wound patches | |
| Namazi et al. | Strategies for prevention of scars: what can we learn from fetal skin? | |
| Li et al. | Porous chitosan bilayer membrane containing TGF-β1 loaded microspheres for pulp capping and reparative dentin formation in a dog model | |
| Chang et al. | Investigation of the skin repair and healing mechanism of N‐carboxymethyl chitosan in second‐degree burn wounds | |
| Bhatnagar et al. | Delivery systems for platelet derived growth factors in wound healing: A review of recent developments and global patent landscape | |
| Watanabe et al. | Oral mucosal adhesive films containing royal jelly accelerate recovery from 5-fluorouracil–induced oral mucositis | |
| Pan et al. | Revisited and innovative perspectives of oral ulcer: from biological specificity to local treatment | |
| Momeni et al. | In vitro and in vivo investigation of a novel amniotic‐based chitosan dressing for wound healing | |
| Zhao et al. | An injectable hydrogel scaffold with IL-1β-activated MSC-derived exosomes for the treatment of endometritis | |
| Wang et al. | Micro-environment triple-responsive hyaluronic acid hydrogel dressings to promote antibacterial activity, collagen deposition, and angiogenesis for diabetic wound healing | |
| Yu et al. | Multifunctional hyaluronic acid/gelatin methacryloyl core-shell microneedle for comprehensively treating oral mucosal ulcers | |
| Xing et al. | A Multifunctional Nanocomposite Hydrogel Delivery System Based on Dual‐Loaded Liposomes for Scarless Wound Healing | |
| Wang et al. | Substance P&dimethyloxallyl glycine‐loaded carboxymethyl chitosan/gelatin hydrogel for wound healing | |
| CN114984233B (en) | A local drug delivery system based on salamander skin secretion and its application | |
| Li et al. | Zinc-induced photocrosslinked konjac glucomannan/glycyrrhizic acid hydrogel promotes skin wound healing in diabetic mice through immune regulation | |
| Chummun et al. | Assessing the mechanisms of action of natural molecules/extracts for phase-directed wound healing in hydrogel scaffolds | |
| Rathna et al. | Advancements in wound healing: integrating biomolecules, drug delivery carriers, and targeted therapeutics for enhanced tissue repair | |
| CN118126401A (en) | Preparation method of natural glycopeptide hydrogel | |
| You et al. | Nanosilver alleviates foreign body reaction and facilitates wound repair by regulating macrophage polarization | |
| Yang et al. | Antioxidant-enriched autologous biogel promoted diabetic wound healing by remodeling inherent posttraumatic inflammatory patterning and restoring compromised microenvironment homeostasis | |
| Zhou et al. | Promoting the healing of diabetic wounds with an antimicrobial gel containing AgNPs with anti-infective and anti-inflammatory properties | |
| Aldaghi et al. | Doxycycline‐loaded carboxymethyl cellulose/sodium alginate/gelatin hydrogel: An approach for enhancing pressure ulcer healing in a rat model | |
| CN118557509B (en) | Hydrogel carrier and its application in preparing preparation for treating diabetic wounds |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |