pH and lipase-responsive nanocarrier-mediated dual drug delivery system to treat periodontitis in diabetic rats 治疗糖尿病大鼠牙周炎的 pH 和脂肪酶响应型纳米载体介导的双重给药系统
Lu Wang ^(a,b){ }^{\mathrm{a}, \mathrm{b}}, Yuzhou Li ^(a,c){ }^{\mathrm{a}, \mathrm{c}}, Mingxing Ren ^(b,c),Xu{ }^{\mathrm{b}, \mathrm{c}}, \mathbf{X u} Wang ^(a,c)^{\mathrm{a}, \mathrm{c}}, Lingjie Li ^(c){ }^{\mathrm{c}}, Fengyi Liu ^(a){ }^{\mathrm{a}}, Yiqing Lan ^(b,c){ }^{\mathrm{b}, \mathrm{c}}, Sheng Yang ^(a,b,c,**k^(****)){ }^{\mathrm{a}, \mathrm{b}, \mathrm{c}, \mathrm{*k}^{* *}}, Jinlin Song ^(a,b,c,^(**)){ }^{\mathrm{a}, \mathrm{b}, \mathrm{c},{ }^{*}}^(a){ }^{a} College of Stomatology, Chongqing Medical University, Chongqing, China ^(a){ }^{a} 重庆医科大学口腔医学院，重庆，中国^(b){ }^{\mathrm{b}} Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing, China ^(b){ }^{\mathrm{b}} 重庆市口腔疾病与生物医学重点实验室，重庆，中国^("c "){ }^{\text {c }} Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China ^("c "){ }^{\text {c }} 重庆市高等学校口腔生物医学工程市级重点实验室，重庆，中国
A R TICLE INFO A R TICLE 信息
Keywords: 关键词：
Diabetes mellitus 糖尿病
Periodontitis 牙周炎
Bone loss 骨质流失
PAMAM
Drug delivery 药物输送
Abstract 摘要
Precise and controlled drug delivery to treat periodontitis in patients with diabetes remains a significant clinical challenge. Nanoparticle-based drug delivery systems offer a potential therapeutic strategy; however, the low loading efficiency, non-responsiveness, and single effect of conventional nanoparticles hinder their clinical application. In this study, we designed a novel self-assembled, dual responsive, and dual drug-loading nanocarrier system, which comprised two parts: the hydrophobic lipid core formed by 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG) loaded with alpha-lipoic acid (ALA); and a hydrophilic shell comprising a poly (amidoamine) dendrimer (PAMAM) that electrostatically adsorbed minocycline hydrochloride (Mino). This unique design allows the controlled release of antioxidant/ALA under lipase stimulation from periodontal pathogens and antimicrobial/Mino under the low pH of the inflammatory microenvironment. In vivo and in vitro studies confirmed that this dual nanocarrier could inhibit the formation of subgingival microbial colonies while promoting osteogenic differentiation of cells under diabetic pathological conditions, and ameliorated periodontal bone resorption. This effective and versatile drug-delivery strategy has good potential applications to inhibit diabetes-associated periodontal bone loss. 精确、可控地给药治疗糖尿病患者的牙周炎仍然是一项重大的临床挑战。基于纳米颗粒的给药系统提供了一种潜在的治疗策略；然而，传统纳米颗粒的低负载效率、非响应性和单一效应阻碍了其临床应用。在这项研究中，我们设计了一种新型自组装、双响应、双载药纳米载体系统，它由两部分组成：疏水性脂质核心由 1，2-二硬脂酰-sn-甘油-3-磷脂乙醇胺-聚（乙二醇）（DSPE-PEG）构成，内含α-硫辛酸（ALA）；亲水性外壳由聚（氨基胺）树枝状聚合物（PAMAM）构成，静电吸附盐酸米诺环素（Mino）。这种独特的设计可以在牙周病原体的脂肪酶刺激下控制抗氧化剂/ALA 的释放，并在炎症微环境的低 pH 值下控制抗菌剂/米诺的释放。体内和体外研究证实，这种双重纳米载体可以抑制龈下微生物菌落的形成，同时促进糖尿病病理条件下细胞的成骨分化，并改善牙周骨吸收。这种高效、多用途的给药策略在抑制糖尿病相关牙周骨质流失方面具有良好的应用前景。
1. Introduction 1.导言
Diabetes mellitus (DM) and its related complications are a serious threat to global health [1,2]. Periodontitis, considered as the sixth complication of diabetes, is negatively affected by metabolic disorders and related pathological status in DM [3,4]. It has been reported that DM potentiates the severity of the local inflammation induced by dental plaque [5-7], which is the initiating factor for periodontitis and permanently exists in the periodontal environment (mainly the gingiva, periodontal ligament, and alveolar bone). Moreover, hyperglycemia status and resultant advanced glycation end product (AGE) formation could induce reactive oxygen species (ROS) accumulation, exacerbate inflammation, and impair the differentiation of osteoblasts, resulting in more rapid and severe destruction of periodontal bone [8,9]. However, the conventional clinical therapies for periodontitis (mainly mechanical debridement and surgical periodontal treatment [10]) do not consider 糖尿病（DM）及其相关并发症严重威胁着全球健康[1,2]。牙周炎被认为是糖尿病的第六大并发症，受到糖尿病代谢紊乱和相关病理状态的负面影响[3,4]。据报道，糖尿病会加剧牙菌斑诱发的局部炎症的严重程度[5-7]，而牙菌斑是牙周炎的始发因素，长期存在于牙周环境（主要是牙龈、牙周韧带和牙槽骨）中。此外，高血糖状态及由此导致的高级糖化终产物（AGE）的形成可诱导活性氧（ROS）的积累，加剧炎症反应，损害成骨细胞的分化，从而导致牙周骨质更快、更严重的破坏[8,9]。然而，传统的牙周炎临床疗法（主要是机械性清创和外科牙周治疗[10]）并没有考虑到以下因素
the specific microenvironment of diabetes, and are limited in achieving satisfactory therapeutic effects. Therefore, there is an urgent need to develop a novel strategy to implement personalized measures to treat periodontitis under diabetic conditions. 糖尿病的特殊微环境，在取得令人满意的治疗效果方面受到限制。因此，亟需开发一种新策略，实施个性化措施来治疗糖尿病条件下的牙周炎。
Nanoparticle (NP)-based drug delivery systems, mainly including liposomes [11-13], polymeric NPs [14,15], metallic NPs [16,17], and inorganic NPs [18,19], are considered as a promising and effective treatment strategy for bone regeneration [20,21]. It is well known that with the advantages of improving drug solubility, decreasing toxicity, increasing drug stability, and reducing drug decomposition, nano-drug delivery systems can utilize special microenvironmental changes to achieve controlled and programmed drug release, and play an important role in immune regulation, antibacterial and anti-inflammatory activities [22-25]. For example, Zhao et al. designed a novel nano neuro-immune blocker capsule that could exploit the photothermal effect under near infrared (NIR) irradiation to enhance the innate immune 基于纳米粒子（NP）的给药系统，主要包括脂质体[11-13]、聚合物 NPs [14,15]、金属 NPs [16,17]和无机 NPs [18,19]，被认为是一种前景广阔的有效骨再生治疗策略[20,21]。众所周知，纳米给药系统具有提高药物溶解度、降低毒性、增加药物稳定性、减少药物分解等优点，可利用特殊的微环境变化实现药物的可控和程序化释放，在免疫调节、抗菌消炎等方面发挥重要作用[22-25]。例如，Zhao 等人设计了一种新型纳米神经免疫阻断剂胶囊，该胶囊可利用近红外（NIR）照射下的光热效应来增强先天性免疫功能。
Under diabetic conditions, dental plaque and the inflammatory microenvironment are both acidic ( pH4.5-6.5\mathrm{pH} 4.5-6.5 ) [28-31], providing a potential stimulus and an ideal target for pH -responsive carriers. Many studies reported that Poly (amidoamine) (PAMAM) is pH-sensitive because of its loose structure under weakly acidic conditions [32,33], which could be exploited to trigger drug release, and thus could be widely used for responsive release in acidic environments. In addition, PAMAM is an excellent nanosized platform for drug delivery in numerous applications [34-36] because of its specific structure, such as the interior hydrophobic region, a large number of end amino groups, and narrow size distribution [37-41]. Moreover, many bacterial enzymes (e.g., esterase, lipase, and gelatinase) exist in dental plaque [29, 42,43 ] and can be exploited to achieve enzyme-mediated drug release. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG), a PEGylated lipid polymer, is sensitive to bacterial enzymes (e.g., lipase). It can also increase the solubility of a poorly soluble drug and improve the drug loading capacity [44,45]. 在糖尿病条件下，牙菌斑和炎症微环境均呈酸性（ pH4.5-6.5\mathrm{pH} 4.5-6.5 ）[28-31]，这为 pH 响应载体提供了潜在的刺激和理想的靶点。许多研究报告指出，聚（氨基胺）（PAMAM）在弱酸性条件下结构疏松，对 pH 值敏感[32,33]，可利用其触发药物释放，因此可广泛用于酸性环境中的响应性释放。此外，PAMAM 具有内部疏水区、大量末端氨基和窄尺寸分布等特殊结构[34-36]，因此在许多应用中都是极佳的纳米级药物递送平台[37-41]。此外，牙菌斑中还存在许多细菌酶（如酯酶、脂肪酶和明胶酶）[29, 42,43 ]，可以利用这些酶来实现酶介导的药物释放。1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE-PEG) 是一种 PEG 化的脂质聚合物，对细菌酶（如脂肪酶）敏感。它还能增加溶解性差的药物的溶解度，提高药物负载能力 [44,45]。
Inspired by the features of diabetic periodontal microenvironment (pathogens, low pH , high level of ROS, and bone loss)and the superior features of DSPE-PEG and PAMAM, we have developed a novel pH and lipase dual response nanocarrier system constructed from two components: a hydrophobic lipid core (DSPE-PEG) loaded with alpha-lipoic acid (ALA), which is an anti-oxidative, anti-inflammatory, and osteoinductive agent [46], and a hydrophilic dendrimer shell (PAMAM) encapsulating minocycline hydrochloride (Mino), a broad-spectrum antibiotic commonly used in clinical periodontal treatment. These two-part block copolymers were named as DPPLM NPs. Upon exposure to acidic conditions at the presence of lipase in vitro, a pH -responsive 受糖尿病牙周微环境特征（病原体、低 pH 值、高水平 ROS 和骨质流失）以及 DSPE-PEG 和 PAMAM 优越特性的启发，我们开发了一种新型 pH 值和脂肪酶双响应纳米载体系统，该系统由两部分组成：疏水性脂质内核（DSPE-PEG）装载有α-硫辛酸（ALA），这是一种抗氧化、抗炎和诱导骨生成的物质[46]；亲水性树枝状聚合物外壳（PAMAM）封装有盐酸米诺环素（Mino），这是一种临床牙周治疗中常用的广谱抗生素。这些由两部分组成的嵌段共聚物被命名为 DPPLM NPs。在体外有脂肪酶存在的酸性条件下，当暴露在酸性条件下时，会产生 pH 响应。
release of Mino and a lipase-responsive release of ALA were triggered. Moreover, the functions of enhanced osteogenic, antioxidant, anti-inflammatory, and antibacterial activities of the DPPLM NPs were determined in vitro. Furthermore, these NPs exhibited outstanding performance in destroying dental plaque and further preventing periodontal bone loss in an experimental periodontitis model in DM rats (Scheme 1). Overall, this study provided an effective therapeutic strategy to inhibit periodontal bone loss in DM. 此外，还在体外测定了 DPPLM NPs 增强的成骨、抗氧化、抗炎和抗菌活性。此外，还在体外测定了 DPPLM NPs 增强的成骨、抗氧化、抗炎和抗菌活性。此外，在 DM 大鼠牙周炎实验模型中，这些 NPs 在消灭牙菌斑和进一步防止牙周骨质流失方面表现突出（方案 1）。总之，这项研究为抑制 DM 大鼠牙周骨质流失提供了一种有效的治疗策略。
2. Materials and methods 2.材料和方法
2.1. Reagents and materials 2.1.试剂和材料
A fourth-generation poly (amidoamine) dendrimer (G4.0 PAMAM)) was obtained from Chenyuan Molecular (Weihai, Shandong, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N -[succinimidyl (polyethylene glycol)-2000] (DSPE-PEG 2000 -NHS) was obtained from Ponsure Biotechnology (Shanghai, China). The Cell Counting Kit 8 (CCK8), and Lyso-Tracker Green were supplied by Beyotime Biotechnology (Shanghai, China). The Alizarin Red Staining Kit was bought from Solarbio Science & Technology Co. (Beijing, China). Fluorescein isothiocyanate (FITC), Nile Red, Rhodamine B (Rho B), ALA, dimethyl sulfoxide (DMSO), and Streptozotocin (STZ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The collagen membrane was obtained from Geistlich Pharma AG (Wolhusen, Switzerland). AGE glycated bovine serum albumin (AGE-BSA) was bought from Abcam (Cambridge, MA, USA), Mino was purchased from AbMole Bioscience (Houston, TX, USA). Primary antibody for inducible nitrous oxide synthase (iNOS) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The TRIzol reagent and reverse transcription kit were bought from Invitrogen (Carlsbad, CA, USA) and Takara Shuzo (Kyoto, Japan). All other reagents and products for cell culture were bought from GIBCO (Gaithersburg, MD, USA) unless otherwise stated. 第四代聚（氨基胺）树枝状聚合物（G4.0 PAMAM）来自 Chenyuan Molecular 公司（中国山东威海）。1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N - [succinimidyl (polyethylene glycol)-2000] (DSPE-PEG 2000 -NHS)由 Ponsure Biotechnology（中国上海）提供。细胞计数试剂盒 8（CCK8）和溶菌酶追踪绿（Lyso-Tracker Green）由贝奥天美生物科技（上海）有限公司提供。茜素红染色试剂盒购自索拉生物科技有限公司（北京）。异硫氰酸荧光素（FITC）、尼罗红、罗丹明 B（Rho B）、ALA、二甲基亚砜（DMSO）和链脲佐菌素（STZ）购自 Sigma-Aldrich (St. Louis, MO, USA)。胶原蛋白膜购自 Geistlich Pharma AG（瑞士 Wolhusen）。AGE 糖化牛血清白蛋白（AGE-BSA）购自 Abcam 公司（美国马萨诸塞州剑桥），Mino 购自 AbMole Bioscience 公司（美国德克萨斯州休斯顿）。诱导型一氧化氮合酶（iNOS）的一抗购自 Santa Cruz Biotechnology (Santa Cruz, CA, USA)。TRIzol 试剂和反转录试剂盒分别购自 Invitrogen 公司（Carlsbad, CA, USA）和 Takara Shuzo 公司（Kyoto, Japan）。除非另有说明，用于细胞培养的所有其他试剂和产品均购自 GIBCO（Gaithersburg, MD, USA）。
2.2. Synthesis and characterization of DPP and DPPLM NPs 2.2.DPP 和 DPPLM NPs 的合成和表征
The blank polymers were synthesized using the diafiltration method. Briefly, DSPE-PEG _(2000){ }_{2000}-NHS was dissolved in DMSO ( 2 mL ) and then added dropwise into 8 mL of deionized water under magnetic stirring at 25^(@)C25{ }^{\circ} \mathrm{C} for 4 h . PAMAM was dissolved in 2 mL of deionized water. Then, the DSPE-PEG and PAMAM solutions were mixed and stirred gently at 25^(@)C25{ }^{\circ} \mathrm{C} overnight. The primary amino groups on the surface of PAMAM reacted specifically with the terminal NHS group of the PEG derivative (groups were covalently conjugated to the DSPE-PEG _(2000){ }_{2000}-NHS through 空白聚合物采用重滤法合成。简而言之，将 DSPE-PEG _(2000){ }_{2000} -NHS 溶于二甲基亚砜（2 mL）中，然后在 25^(@)C25{ }^{\circ} \mathrm{C} 的磁力搅拌下滴加到 8 mL 去离子水中 4 小时。将 PAMAM 溶于 2 mL 去离子水中。然后，将 DSPE-PEG 和 PAMAM 溶液混合，并在 25^(@)C25{ }^{\circ} \mathrm{C} 下轻轻搅拌过夜。PAMAM 表面的伯氨基与 PEG 衍生物的末端 NHS 基团发生特异性反应（基团通过 _(2000){ }_{2000} 共价键合到 DSPE-PEG _(2000){ }_{2000} -NHS 上）。
Scheme 1. Schematic illustration of engineering for DPPLM NPs. The nanocarrier system could simultaneously and efficiently carry and intelligently release two specific active drugs at periodontal bone defect sites of diabetic rats, and subsequently destroy dental plaque and ameliorate periodontal bone resorption. ALA, alphalipoic acid; Mino, minocycline hydrochloride; DPPLM NPs, DSPE-PEG-PAMAM/ALA/Mino nanoparticles. 方案 1.DPPLM NPs 工程示意图。该纳米载体系统可在糖尿病大鼠牙周骨缺损部位同时高效地携带并智能释放两种特异性活性药物，从而破坏牙菌斑并改善牙周骨吸收。ALA，硫辛酸；Mino，盐酸米诺环素；DPPLM NPs，DSPE-PEG-PAMAM/ALA/Mino 纳米颗粒。
an effective Michael addition reaction to obtain the final modified dendrimers). After the reaction was completed, the reaction solution was dialyzed in a dialysis bag (molecular weight cut off (( MWCO) =50=50 KDa ) against deionized water for 72 h at 25^(@)C25^{\circ} \mathrm{C}. The water was changed every 3 h . Finally, the DSPE-PEG-PAMAM nanoparticles, named DPP NPs for short, were obtained after freeze-drying, and stored at -20^(@)C-20^{\circ} \mathrm{C}. The drug-loaded, Nile red-loaded, and Rho B-loaded nanoparticles were prepared using the same procedure described above. ALA was added at weight ratios of ALA:PAMAM = 4:24.26, 8:24.26, and 16:24.26. Mino was added at a weight ratio of PAMAM:Mino =24.26:16=24.26: 16. The grafting ratio of DSPE-PEG on the exterior of the PAMAM was measured using the following formula based on ^(1)H{ }^{1} \mathrm{H} nuclear magnetic resonance (NMR) spectra in deuterium oxide ( D_(2)O\mathrm{D}_{2} \mathrm{O} ): axxn//b=c,p=n//64 xx100%\mathrm{a} \times \mathrm{n} / \mathrm{b}=\mathrm{c}, \mathrm{p}=\mathrm{n} / 64 \times 100 \%, where a is the average number of selected characteristic-H on PEG, b is the number of selected characteristic-H on PAMAM, c is the characteristic peak integral ratio of DPP, n is the average number of DSPE-PEG moieties successfully conjugated to PAMAM, and p is the PEGylation proportion of PAMAM. The size and morphology of empty and drug-loaded nanoparticles were monitored using dynamic light scattering (DLS, Brookhaven NanoBrook Omni, Brookhaven Instruments Corporation, Holtsville, NY, USA) and transmission electron microscopy (TEM, Hitachi-7500, Tokyo, Japan). The successful synthesis of nanoparticles was further confirmed by Fourier Transform Infrared spectroscopy (FTIR, Thermo Scientific Nicolet iS50, Thermo Scientific, Waltham, MA, USA). 在此基础上进行有效的迈克尔加成反应，得到最终的改性树枝状聚合物）。反应完成后，将反应溶液装入透析袋（截留分子量 (( MWCO） =50=50 KDa），在 25^(@)C25^{\circ} \mathrm{C} 条件下用去离子水透析 72 小时。每 3 小时换一次水。最后，经冷冻干燥得到 DSPE-PEG-PAMAM 纳米颗粒，简称 DPP NPs，储存于 -20^(@)C-20^{\circ} \mathrm{C} 。药物负载、尼罗河红负载和 Rho B 负载纳米颗粒的制备过程与上述步骤相同。以 ALA:PAMAM = 4:24.26、8:24.26 和 16:24.26 的重量比加入 ALA。米诺以 PAMAM:Mino =24.26:16=24.26: 16 的重量比加入。根据 ^(1)H{ }^{1} \mathrm{H} 在氧化氘（ D_(2)O\mathrm{D}_{2} \mathrm{O} ）中的核磁共振（NMR）光谱，使用下式测量 PAMAM 外部的 DSPE-PEG 接枝率： axxn//b=c,p=n//64 xx100%\mathrm{a} \times \mathrm{n} / \mathrm{b}=\mathrm{c}, \mathrm{p}=\mathrm{n} / 64 \times 100 \% 其中，a 是 PEG 上所选特征-H 的平均数量，b 是 PAMAM 上所选特征-H 的数量，c 是 DPP 的特征峰积分比，n 是成功共轭到 PAMAM 的 DSPE-PEG 分子的平均数量，p 是 PAMAM 的 PEG 化比例。使用动态光散射（DLS，Brookhaven NanoBrook Omni，Brookhaven Instruments Corporation，Holtsville，NY，USA）和透射电子显微镜（TEM，Hitachi-75，Tokyo，Japan）监测了空纳米颗粒和药物负载纳米颗粒的尺寸和形态。傅立叶变换红外光谱（FTIR，Thermo Scientific Nicolet iS50，Thermo Scientific，Waltham，MA，USA）进一步证实了纳米粒子的成功合成。
2.3. In vitro drug loading 2.3.体外药物负载
The drug loading content = (DLC, weight of loaded drug/weight of the drug loaded nanoparticles) xx100%\times 100 \%, and drug loading efficiency == (DLE, weight of drug incorporated in assembled nanoparticles/weight of drug used in the fabrication) xx100%\times 100 \% were determined using a UV-Vis scanning spectrophotometer. The calibration curves were acquired with different Mino concentrations at 280 nm in water and different ALA concentrations at 330 nm in ethanol. 使用紫外-可见扫描分光光度计测定了药物负载含量=（DLC，负载药物的重量/负载药物的纳米颗粒的重量） xx100%\times 100 \% 和药物负载效率 == （DLE，组装纳米颗粒中药物的重量/制造过程中使用的药物的重量） xx100%\times 100 \% 。不同浓度的 Mino 在 280 纳米波长的水中和不同浓度的 ALA 在 330 纳米波长的乙醇中分别获得校准曲线。
2.4. In vitro drug release study 2.4.体外药物释放研究
The drug responsive release characteristics were determined. Briefly, DPPLM ( 2mg,1mg//mL2 \mathrm{mg}, 1 \mathrm{mg} / \mathrm{mL} ) in a dialysis bag ( MWCO=50KDa\mathrm{MWCO}=50 \mathrm{KDa} ) was immersed in phosphate buffered saline (PBS; 20 mL ) at pH7.4,pH5.5\mathrm{pH} 7.4, \mathrm{pH} 5.5, pH 7.4 with 1mg//mL1 \mathrm{mg} / \mathrm{mL} lipase, and pH 5.5 with 1mg//mL1 \mathrm{mg} / \mathrm{mL} lipase, respectively. The release study was conducted in a thermotank under gentle shaking at 37^(@)C37^{\circ} \mathrm{C}. Next, at certain times ( 0,1,2,4,6,8,12,240,1,2,4,6,8,12,24, and 48 h ), 2.0 mL of dialysis fluid outside the bag was sampled and an equal amount of fresh release medium was supplied immediately to keep the same volume. The release amount was determined using a UV-Vis scanning spectrophotometer. 测定了药物响应释放特性。简言之，将装在透析袋（ MWCO=50KDa\mathrm{MWCO}=50 \mathrm{KDa} ）中的 DPPLM（ 2mg,1mg//mL2 \mathrm{mg}, 1 \mathrm{mg} / \mathrm{mL} ）浸入磷酸盐缓冲盐水（PBS；20 mL）中，分别在 pH7.4,pH5.5\mathrm{pH} 7.4, \mathrm{pH} 5.5 、pH 7.4（含 1mg//mL1 \mathrm{mg} / \mathrm{mL} 脂肪酶）和 pH 5.5（含 1mg//mL1 \mathrm{mg} / \mathrm{mL} 脂肪酶）条件下进行。释放研究是在 37^(@)C37^{\circ} \mathrm{C} 温和振荡的恒温槽中进行的。然后，在特定时间（ 0,1,2,4,6,8,12,240,1,2,4,6,8,12,24 和48小时），从透析袋外取样2.0 mL透析液，并立即加入等量的新鲜释放介质，以保持相同的体积。使用紫外可见扫描分光光度计测定释放量。
2.5. Cell culture 2.5.细胞培养
Bone marrow mesenchymal stem cells (BMSCs) were isolated from the long bones of 3 to 4 -week-old female Sprague-Dawley (SD) rats. Following euthanasia using carbon dioxide inhalation suffocation, the femurs and tibiae were aseptically dissected and washed with PBS. The ends of the bones were cut off below the end of the bone marrow cavity and the marrow was flushed out from the bones using a 5 mL syringe with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%10 \% fetal bovine serum (FBS) at 37^(@)C37{ }^{\circ} \mathrm{C} in a 5%CO_(2)5 \% \mathrm{CO}_{2} atmosphere. Cells were centrifuged at 200 xx g200 \times g for 5 min and resuspended in fresh DMEM. Cells were cultured in a humidified atmosphere with 5%CO_(2)5 \% \mathrm{CO}_{2} at 37^(@)C37{ }^{\circ} \mathrm{C}. Non-adherent cells were discarded and the medium was exchanged for the first time after 48 h . The adherent cells were detached using 0.25%0.25 \% trypsin/EDTA at day 7. At approximately 80%80 \% confluence, the cells were trypsinized and subcultured. The cells at passages 3-5 were used in the following experiments. 骨髓间充质干细胞（BMSCs）是从3至4周大的雌性Sprague-Dawley（SD）大鼠的长骨中分离出来的。用二氧化碳吸入窒息法安乐死大鼠后，无菌解剖大鼠的股骨和胫骨，并用PBS洗净。在骨髓腔末端以下切断骨骼末端，用 5 mL 注射器在 5%CO_(2)5 \% \mathrm{CO}_{2} 的气氛中以 37^(@)C37{ }^{\circ} \mathrm{C} 补充 10%10 \% 胎牛血清 (FBS) 的 Dulbecco 改良老鹰培养基 (DMEM) 冲洗骨骼中的骨髓。细胞在 200 xx g200 \times g 条件下离心 5 分钟，然后重悬在新鲜的 DMEM 中。细胞在 5%CO_(2)5 \% \mathrm{CO}_{2} 和 37^(@)C37{ }^{\circ} \mathrm{C} 的湿润环境中培养。48 小时后，丢弃不粘附的细胞，第一次交换培养基。第 7 天，使用 0.25%0.25 \% 胰蛋白酶/EDTA 使粘附的细胞脱离。在大约 80%80 \% 汇合时，对细胞进行胰蛋白酶处理和再培养。第 3-5 层的细胞用于以下实验。
2.6. In vitro cellular uptake 2.6.体外细胞摄取
Before the experiment, round slides were disinfected and placed in a 24 -well plate. BMSCs were seeded into the 24 -well plate at a density of 5000 cells per well and cultured for 24 h . Then, the medium was removed, and the cells were cultured with starvation medium ( 99%99 \% basal medium and 1%1 \% penicillin-streptomycin) for 12 h . Thereafter, the cells were incubated with 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP-FITC/Nile red and DPP-FITC/ Rho B for another 2 h and 24 h , respectively. Thereafter, the cells were washed with PBS three times and fixed with 4%4 \% (v/v) paraformaldehyde. The nuclei were stained with 4^('),64^{\prime}, 6-diamidino-2-phenylindole (DAPI). Then, all cells were observed using confocal laser scanning microscopy (CLSM, Leica TCS SP8, Wetzlar, Germany). 实验前，将圆形载玻片消毒并放入 24 孔板中。将 BMSCs 以每孔 5000 个细胞的密度播种到 24 孔板中，培养 24 小时。然后，去除培养基，用饥饿培养基（ 99%99 \% 基础培养基和 1%1 \% 青霉素-链霉素）培养细胞 12 小时。然后，分别用 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP-FITC/Nile red 和 DPP-FITC/ Rho B 培养细胞 2 小时和 24 小时。然后，用 PBS 冲洗细胞三次，并用 4%4 \% （v/v）多聚甲醛固定。细胞核用 4^('),64^{\prime}, 6 -二脒基-2-苯基吲哚（DAPI）染色。然后用激光共聚焦扫描显微镜（CLSM，Leica TCS SP8，Wetzlar，Germany）观察所有细胞。
2.7. In vitro cytotoxicity study 2.7.体外细胞毒性研究
The in vitro cytotoxicity of DPPLM NPs was investigated using the CCK8 kit. Briefly, BMSCs were seeded in 96 -well plates at 5000 cells per well with 100 muL100 \mu \mathrm{~L} of DMEM medium and cultured at 37^(@)C37^{\circ} \mathrm{C} for 24 h . Then, the culture medium was replaced with 100 muL100 \mu \mathrm{~L} DMEM containing DPPLM NPs at different concentrations. After 24h,10 muL24 \mathrm{~h}, 10 \mu \mathrm{~L} of CCK8 solution were added into each well. The absorbency of the solution was measured on a TECAN microplate reader at 450 nm (Tecan Group Ltd., Männedorf, Switzerland). The relative cytotoxicity rate was evaluated using the following formula: 使用 CCK8 试剂盒研究了 DPPLM NPs 的体外细胞毒性。简言之，将 BMSCs 按每孔 5000 个细胞的数量接种到 96 孔板中，加入 100 muL100 \mu \mathrm{~L} DMEM 培养基，在 37^(@)C37^{\circ} \mathrm{C} 条件下培养 24 小时。然后，用含有不同浓度 DPPLM NPs 的 100 muL100 \mu \mathrm{~L} DMEM 更换培养基。每孔加入 24h,10 muL24 \mathrm{~h}, 10 \mu \mathrm{~L} CCK8 溶液。用 TECAN 微孔板阅读器（Tecan Group Ltd., Männedorf, Switzerland）在 450 纳米波长下测量溶液的吸光度。相对细胞毒性率用以下公式进行评估：
Cell viability (%)=(A-B)//(C-B)xx100%(\%)=(\mathrm{A}-\mathrm{B}) /(\mathrm{C}-\mathrm{B}) \times 100 \% 细胞活力 (%)=(A-B)//(C-B)xx100%(\%)=(\mathrm{A}-\mathrm{B}) /(\mathrm{C}-\mathrm{B}) \times 100 \%
where A is the absorbance of the experimental group (containing cells, culture medium, CCK-8 solution, and nanoparticles solution), B is the absorbance of the blank group (with medium, CCK-8 solution, without cells, nanoparticles), and C is the absorbance of the control group (containing cells, culture medium, CCK-8 solution, without nanoparticles). 其中，A 是实验组（含细胞、培养基、CCK-8 溶液和纳米颗粒溶液）的吸光度，B 是空白组（含培养基、CCK-8 溶液，不含细胞和纳米颗粒）的吸光度，C 是对照组（含细胞、培养基、CCK-8 溶液，不含纳米颗粒）的吸光度。
2.8. Subcellular localization 2.8.亚细胞定位
BMSCs were seeded into confocal dishes and incubated at 37^(@)C37^{\circ} \mathrm{C} for 24 h , and then incubated with 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP/Nile red NPs for 20min,220 \mathrm{~min}, 2 h , and 24 h at 37^(@)C37^{\circ} \mathrm{C}. Next, the medium was removed and cells were washed with cold PBS three times, followed by staining with LysoTracker Green for another 15 min . Hoechst 33342 was used to stain the nuclei. Then, all cells were observed using CLSM. 将 BMSCs 接种到共聚焦培养皿中，在 37^(@)C37^{\circ} \mathrm{C} 条件下培养 24 小时，然后与 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP/Nile red NPs 培养 20min,220 \mathrm{~min}, 2 小时，在 37^(@)C37^{\circ} \mathrm{C} 条件下培养 24 小时。然后，除去培养基，用冷 PBS 冲洗细胞三次，再用溶菌酶追踪绿染色 15 分钟。细胞核用 Hoechst 33342 染色。然后用 CLSM 观察所有细胞。
2.9. In vitro flow cytometry 2.9.体外流式细胞仪
BMSCs were seeded in 6 -well dishes at 2xx10^(5)2 \times 10^{5} cells per well and incubated at 37^(@)C37{ }^{\circ} \mathrm{C} for 24 h . The medium was discarded and replaced with new DMEM containing 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP-FITC NPs and incubated for 24 h . Then, the medium was discarded and the cells were rinsed with PBS three times. The cells were trypsinized, collected by centrifugation, and then analyzed on a flow cytometer (BD FACSAria III, BD Biosciences, San Jose, CA, USA). Cells without any treatment were used as the blank control and named as the PBS group. 将 BMSCs 以每孔 2xx10^(5)2 \times 10^{5} 个的数量接种到 6 孔培养皿中，并在 37^(@)C37{ }^{\circ} \mathrm{C} 条件下培养 24 小时。弃去培养基，换上含有 10 mug//mL10 \mu \mathrm{~g} / \mathrm{mL} DPP-FITC NPs 的新 DMEM，培养 24 小时。然后，弃去培养基，用 PBS 冲洗细胞三次。将细胞胰蛋白酶化，离心收集，然后在流式细胞仪（BD FACSAria III，BD Biosciences，San Jose，CA，USA）上进行分析。未作任何处理的细胞作为空白对照，命名为 PBS 组。
2.10. Hard tissue penetration of PAMAM 2.10.PAMAM 的硬组织渗透
Fluorescently labeled PAMAM (PAMAM-FITC) was injected under the subcutaneous fascia of the calvarial bone of rats. The rats were sacrificed 5 days later and fluorescence scanning images of the bone were observed under a confocal microscope. To further determine that whether PAMAM could penetrate into the tooth root, the intact root was immersed in PAMAM-FITC solution for 5 min . After washing three times, the surface and cross section of the root were observed under a confocal microscope and a fluorescence microscope, respectively. 在大鼠腓骨皮下筋膜下注射荧光标记的 PAMAM（PAMAM-FITC）。5 天后将大鼠处死，在共聚焦显微镜下观察骨的荧光扫描图像。为了进一步确定 PAMAM 是否能渗入牙根，将完整的牙根浸泡在 PAMAM-FITC 溶液中 5 分钟。清洗三次后，分别在共聚焦显微镜和荧光显微镜下观察牙根的表面和横截面。
2.11. Osteogenic differentiation and detection 2.11.成骨细胞的分化和检测
To investigate the effects of DPPLM NPs on the osteogenic differentiation of AGE-induced BMSCs, mineral deposition was detected. BMSCs were seeded in 24 -well plates at a density of 5xx10^(4)5 \times 10^{4} cells per well. At approximately 80%80 \% confluence, the medium was discarded and replaced with osteogenic medium (OM; 10mMbeta10 \mathrm{mM} \beta-glycerophosphate, 1 mM dexamethasone, and 50 mug//mL50 \mu \mathrm{~g} / \mathrm{mL} l-ascorbic acid-2-phosphate in complete medium). After 14 days of incubation in OM, Alizarin Red staining was performed to assess mineral deposits. For the quantitative analysis of mineralization, deposited calcium was eluted using 10%10 \% (w/v) cetylpyridinium chloride (Sigma-Aldrich), and the OD value was measured at 562 nm . 为了研究 DPPLM NPs 对 AGE 诱导的 BMSCs 成骨分化的影响，我们检测了矿物质沉积。将 BMSCs 以每孔 5xx10^(4)5 \times 10^{4} 个的密度播种在 24 孔板中。在大约 80%80 \% 汇合时，弃去培养基，换上成骨培养基（OM；完全培养基中的 10mMbeta10 \mathrm{mM} \beta -磷酸甘油、1 mM地塞米松和 50 mug//mL50 \mu \mathrm{~g} / \mathrm{mL} l-抗坏血酸-2-磷酸）。在 OM 中培养 14 天后，进行茜素红染色以评估矿化沉积。为了对矿化进行定量分析，使用 10%10 \% （w/v）氯化十六烷基吡啶（Sigma-Aldrich）洗脱沉积的钙，并在 562 纳米波长下测量 OD 值。
Total RNA was extracted using the Trizol reagent (Takara) and converted to cDNA using a PrimeScriptRT reagent kit (Takara). Then, the cDNA was used as the template for quantitative real-time PCR (qPCR) using SYBR Premix ExTaq II (Takara) on the CFX96PCR system (Bio-Rad, Hercules, CA, USA) to quantify gene expression. After incubation for 7 days, the osteogenic related genes including Alp (alkaline phosphatase), Runx2 (runt-related transcription factor 2), Ocn (osteocalcin), Opn (osteopontin), and Osx (osterix) in BMSCs were measured using qRT-PCR. Sequences of the primers are presented in Table 2 (Supporting Information). 使用 Trizol 试剂（Takara）提取总 RNA，并使用 PrimeScriptRT 试剂盒（Takara）将其转化为 cDNA。然后，以 cDNA 为模板，在 CFX96PCR 系统（Bio-Rad, Hercules, CA, USA）上使用 SYBR Premix ExTaq II（Takara）进行实时定量 PCR（qPCR），以量化基因表达。培养 7 天后，使用 qRT-PCR 检测 BMSCs 中的成骨相关基因，包括 Alp（碱性磷酸酶）、Runx2（runt 相关转录因子 2）、Ocn（骨钙素）、Opn（骨通素）和 Osx（osterix）。引物序列见表 2（佐证信息）。
For ROS staining, the fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) (Beyotime Institute of Biotechnology, Jiangsu, China) was used to test the level of ROS. Briefly, the cells were incubated with DCFH-DA (diluted to a final concentration of 10 muM10 \mu \mathrm{M} ) in PBS for 30 min in the dark. After rinsing with PBS three times, the cells were observed under a fluorescence microscope. 在ROS染色中，使用荧光探针2,7-二氯荧光素二乙酸酯（DCFH-DA）（中国江苏贝因美生物技术研究所）来检测ROS水平。简言之，将细胞与 DCFH-DA（在 PBS 中稀释至 10 muM10 \mu \mathrm{M} 的最终浓度）在黑暗中孵育 30 分钟。用 PBS 冲洗三次后，在荧光显微镜下观察细胞。
2.14. Immunofluorescence staining 2.14.免疫荧光染色
After starvation for 12 h , BMSCs were incubated with serum-free medium containing 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} AGE in the presence or absence of 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} DPPLM NPs for 24 h . Then, the medium was discarded and cells were washed with PBS three times. Cells were fixed with 4%4 \% paraformaldehyde solution for 10 min , permeabilized with 0.2%0.2 \% TritonX-100 for 10 min , and blocked with 5%5 \% donkey serum at 37^(@)C37^{\circ} \mathrm{C} for 1 h . After each step, the cells were rinsed with PBS three times. Then, mouse monoclonal anti-iNOS (1:300; sc-7271; Santa Cruz Biotechnology) antibodies were incubated with the cells at 4^(@)C4^{\circ} \mathrm{C} for 12 h , and a fluorescence-conjugated anti-mouse secondary antibody (Alexa Fluor ^(®){ }^{\circledR} 647; 1:500; Beyotime, Shanghai, China) was subsequently added to bind with the primary antibody. The nuclei were stained with DAPI. Finally, the cells were observed under a confocal laser scanning microscope. 饥饿 12 小时后，用含有 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} AGE 的无血清培养基在 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} DPPLM NPs 存在或不存在的情况下培养 BMSCs 24 小时。然后，弃去培养基，用 PBS 冲洗细胞三次。细胞用 4%4 \% 多聚甲醛溶液固定 10 分钟，用 0.2%0.2 \% TritonX-100 透化 10 分钟，用 5%5 \% 驴血清在 37^(@)C37^{\circ} \mathrm{C} 下阻滞 1 小时。每个步骤后，用 PBS 冲洗细胞三次。然后，小鼠单克隆抗 iNOS（1:300；sc-7271；Santa Cruz Biotechnology）抗体与细胞在 4^(@)C4^{\circ} \mathrm{C} 下孵育 12 小时，随后加入荧光结合的抗小鼠二抗（Alexa Fluor ^(®){ }^{\circledR} 647；1:500；贝奥天美，中国上海）与一抗结合。细胞核用 DAPI 染色。最后，在共聚焦激光扫描显微镜下观察细胞。
2.15. In vitro antibacterial testing of DPPLM NPs 2.15.DPPLM NPs 的体外抗菌测试
Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were chosen to evaluate the antibacterial ability using the broth microdilution method as described previously [42]. 100 muL100 \mu \mathrm{~L} of bacterial suspension was seeded in a 96 -well plate at a concentration of 10^(5)10^{5} colony forming units (CFU)/mL and incubated with 100 muL100 \mu \mathrm{~L} of DPPLM NPs at serial concentrations from 0 to 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} at 37^(@)C37^{\circ} \mathrm{C} for 24 h . The OD value of the suspensions in each well was measured using a TECAN microplate reader at 600 nm at defined time points. The minimum inhibitory concentration (MIC) was taken as the lowest concentration at which the well was clear. After spreading these test dilutions for MIC determination on agar, the minimum bactericidal concentration (MBC) was 选择金黄色葡萄球菌（S. aureus）和大肠杆菌（E. coli）来评估肉汤微稀释法的抗菌能力，具体方法如前所述[42]。将 100 muL100 \mu \mathrm{~L} 浓度为 10^(5)10^{5} 菌落形成单位（CFU）/毫升的细菌悬浮液接种到 96 孔板中，然后与 100 muL100 \mu \mathrm{~L} 浓度为 0 至 100 mug//mL100 \mu \mathrm{~g} / \mathrm{mL} 的 DPPLM NPs 在 37^(@)C37^{\circ} \mathrm{C} 下培养 24 小时。在规定的时间点，使用 TECAN 微孔板阅读器在 600 纳米波长下测量每个孔中悬浮液的 OD 值。最低抑菌浓度 (MIC) 取为孔内清澈的最低浓度。将这些试验稀释液涂抹在琼脂上进行 MIC 测定后，得出最低杀菌浓度 (MBC)
determined as the lowest concentration that yielded no bacterial colonies on the plate. 以平板上无细菌菌落的最低浓度为准。
A bacterial suspension at a concentration of 10^(5)CFU//mL10^{5} \mathrm{CFU} / \mathrm{mL} was added to a 6-well plate covered with sterile square slides and incubated at 37^(@)C37^{\circ} \mathrm{C} for 24 h under stationary conditions. Thereafter, the medium was removed and the bacterial pellets were rinsed with PBS three times. DPPLM NPs ( 2 mL ) at a concentration of 200 mug//mL200 \mu \mathrm{~g} / \mathrm{mL} were added carefully to the wells. The plate was cultured for another 24 h . Thereafter, the bacterial pellets were rinsed with PBS three times and fixed with 4%4 \% (v/v) paraformaldehyde sputter-coated with gold. Finally, the morphological changes to the bacteria were observed using scanning electron microscopy (SEM). 将浓度为 10^(5)CFU//mL10^{5} \mathrm{CFU} / \mathrm{mL} 的细菌悬浮液加入铺有无菌方形载玻片的 6 孔板中，在 37^(@)C37^{\circ} \mathrm{C} 静止条件下培养 24 小时。之后，除去培养基，用 PBS 冲洗细菌颗粒三次。将浓度为 200 mug//mL200 \mu \mathrm{~g} / \mathrm{mL} 的 DPPLM NPs（2 mL）小心地加入孔中。平板再培养 24 小时。之后，用 PBS 冲洗细菌颗粒三次，并用 4%4 \% （v/v）多聚甲醛溅射镀金固定。最后，使用扫描电子显微镜（SEM）观察细菌的形态变化。
2.16. Live/dead biofilm staining 2.16.活/死生物膜染色
The bacterial suspension was added to a 24 -well plate at the concentration of 10^(5)CFU//mL10^{5} \mathrm{CFU} / \mathrm{mL} and incubated for 24 h . Thereafter, the bacterial suspension was discarded, and the wells were washed with PBS three times to remove planktonic bacteria. DPPLM NPs were added and incubated for 12 h . Bacteria were stained with a L7007 LIVE/DEAD BacLight Staining Kit (Thermo Fisher Scientific) for 15 min in the dark at room temperature. All bacteria were stained with SYTO9 to produce green fluorescence, and dead bacteria with damaged membranes were stained with propidium iodide to produce red fluorescence. A fluorescence microscope was used to capture the images of each sample. 将浓度为 10^(5)CFU//mL10^{5} \mathrm{CFU} / \mathrm{mL} 的细菌悬浮液加入 24 孔板中，培养 24 小时。然后，弃去细菌悬浮液，用 PBS 洗孔三次以去除浮游细菌。加入 DPPLM NPs 并培养 12 小时。用 L7007 LIVE/DEAD BacLight 染色试剂盒（赛默飞世尔科技公司）在室温暗处对细菌染色 15 分钟。所有细菌都用 SYTO9 染色以产生绿色荧光，而膜受损的死亡细菌则用碘化丙啶染色以产生红色荧光。使用荧光显微镜拍摄每个样本的图像。
2.17. Establishment of the DM rat model 2.17.建立 DM 大鼠模型
Preparation of the High-Fat Diet (HFD)/STZ-Induced Diabetic Rats: Male Sprague Dawley (SD) rats (200-250 g) were purchased from Experimental Animal Center of Chongqing Medical University. To establish a type 2 diabetic (T2DM) rat model, rats were fed with HFD (60% fat; Research Diets, D12492) for 2 weeks. After consumption of HFD for 2 weeks, rats were injected intraperitoneally with STZ dissolved in sodium citrate buffer ( pH 4.5 , prepared immediately before use) at a dose of 35mg//kg35 \mathrm{mg} / \mathrm{kg}. The rats’ blood glucose levels were measured every week using a Roche glucose meter, and the rats with >= 16.7mmol//L\geq 16.7 \mathrm{mmol} / \mathrm{L} random blood glucose levels were considered diabetic and offered HFD till the end of the study. An oral glucose tolerance test (OGTT) was performed to confirm the successful establishment of the diabetic rat model. Briefly, rats were fasted for 12 h and then received an intragastric administration of glucose ( 2g//kg2 \mathrm{~g} / \mathrm{kg} ). Their blood glucose was tested at defined time points. The animal handling and surgical procedures were conducted in accordance with protocols approved by the Research Ethics Committee of the Stomatological Hospital of Chongqing Medical University (CQHS-REC-2021 (LSNo. 28)). 制备高脂饮食（HFD）/STZ诱导的糖尿病大鼠：雄性Sprague Dawley（SD）大鼠（200-250克）购自重庆医科大学实验动物中心。为了建立 2 型糖尿病（T2DM）大鼠模型，大鼠被喂食 HFD（60% 脂肪；Research Diets，D12492）2 周。喂食 HFD 2 周后，给大鼠腹腔注射溶解在柠檬酸钠缓冲液（pH 4.5，使用前立即配制）中的 STZ，剂量为 35mg//kg35 \mathrm{mg} / \mathrm{kg} 。每周使用罗氏血糖仪测量大鼠的血糖水平，随机血糖水平 >= 16.7mmol//L\geq 16.7 \mathrm{mmol} / \mathrm{L} 的大鼠被视为糖尿病大鼠，并提供高纤维食物直至研究结束。进行口服葡萄糖耐量试验（OGTT）以确认糖尿病大鼠模型的成功建立。简而言之，大鼠禁食 12 小时，然后胃内注射葡萄糖（ 2g//kg2 \mathrm{~g} / \mathrm{kg} ）。在规定的时间点检测它们的血糖。动物处理和手术过程均按照重庆医科大学附属口腔医院研究伦理委员会批准的方案进行（CQHS-REC-2021 (LSNo. 28)）。
2.18. Establishment of experimental periodontitis model in diabetic rats (DMEP) and animal treatments 2.18.糖尿病大鼠牙周炎实验模型（DMEP）的建立和动物治疗方法
DM rats were used to establish an experimental periodontitis (EP) model. Briefly, DM rats were anesthetized with intraperitoneal injections of 10%10 \% chloral hydrate ( 20mg//kg20 \mathrm{mg} / \mathrm{kg} of body weight), and then a ligature wire with a diameter of 0.2 mm was placed around the maxillary first molars for 2 weeks. Subsequently, before surgery, the collagen membrane (CM) was sterilized in UV for 20 min and immersed in 1000 mug//mL\mu \mathrm{g} / \mathrm{mL} DPPLM solution or a mixture of 136 mug//mL136 \mu \mathrm{~g} / \mathrm{mL} ALA and 172 mug//mL172 \mu \mathrm{~g} / \mathrm{mL} Mino (free drug) for 30 min . Twenty-four diabetic rats with periodontitis (DMEP rats) were randomly divided into four groups ( n=6\mathrm{n}=6 per group), and the CMs were placed into the interproximate space between the maxillary first molars and the maxillary secondary molars: (1) Untreated group (diabetic periodontitis rats without treatment), (2) CM group, (3) CM+\mathrm{CM}+ free drug group, (4) CM + DPPLM group. Animals were sacrificed after 2 weeks and their maxillaries were collected, fixed in 4%4 \% paraformaldehyde for 2 days, and then rinsed with PBS for further analyses. Microcomputed tomography (micro-CT) analysis was used to examine 利用DM大鼠建立实验性牙周炎（EP）模型。简单地说，DM大鼠腹腔注射 10%10 \% 水合氯醛（ 20mg//kg20 \mathrm{mg} / \mathrm{kg} 体重）麻醉，然后在上颌第一磨牙周围放置直径为0.2毫米的结扎线2周。随后，在手术前，将胶原膜（CM）在紫外线下消毒 20 分钟，并在 1000 mug//mL\mu \mathrm{g} / \mathrm{mL} DPPLM 溶液或 136 mug//mL136 \mu \mathrm{~g} / \mathrm{mL} ALA 和 172 mug//mL172 \mu \mathrm{~g} / \mathrm{mL} Mino（游离药物）的混合物中浸泡 30 分钟。将 24 只患有牙周炎的糖尿病大鼠（DMEP 大鼠）随机分为四组（每组 n=6\mathrm{n}=6 ），并将 CM 置入上颌第一磨牙和上颌第二磨牙之间的近端间隙：(1) 未治疗组（未治疗的糖尿病牙周炎大鼠），(2) CM 组，(3) CM+\mathrm{CM}+ 游离药物组，(4) CM + DPPLM 组。动物2周后处死，收集上颌骨，在 4%4 \% 多聚甲醛中固定2天，然后用PBS冲洗，进行进一步分析。显微计算机断层扫描（micro-CT）分析用于检查
the amounts of bone loss. The vertical bone loss was determined by measuring the distance between cemento-enamel junction (CEJ) of the maxillary molars and the alveolar bone crest (ABC). After micro-CT, the samples were decalcified in 10% EDTA for 4 weeks, dehydrated in gradient alcohol, embedded in paraffin, and sectioned at 5mum5 \mu \mathrm{~m} thickness for hematoxylin and eosin (H&E) staining and Masson’s staining. The morphological characteristics of bone healing were observed under a light microscope (IX71-400X, OLYMPUS, Tokyo, Japan). 骨质流失量。通过测量上颌磨牙骨水泥-釉质交界处（CEJ）与牙槽骨嵴（ABC）之间的距离来确定垂直骨质流失量。显微计算机断层扫描后，样本在10% EDTA中脱钙4周，在梯度酒精中脱水，包埋在石蜡中，并以 5mum5 \mu \mathrm{~m} 厚度切片，进行苏木精和伊红（H&E）染色和马森氏染色。在光学显微镜（IX71-400X，OLYMPUS，日本东京）下观察骨愈合的形态特征。
2.19. In vivo antibacterial testing of DPPLM NPs 2.19.DPPLM NPs 的体内抗菌测试
The tooth was carefully extracted from the alveolar bone and the plaque biofilms covering the tooth surface were observed using SEM. 从牙槽骨中小心翼翼地拔出牙齿，使用扫描电子显微镜观察覆盖在牙齿表面的菌斑生物膜。
Standard plate counting: The dental plaque was collected from the tooth surface using a cotton swab, which was soaked in sterile deionized water, and then ultrasonically shaken to obtain a bacterial suspension. The bacterial suspension was then diluted by 10 -fold and 100 muL100 \mu \mathrm{~L} of the dilution was plated on LB agar and cultured at 37^(@)C37^{\circ} \mathrm{C} for 24 h . 标准平板计数：用棉签从牙齿表面收集牙菌斑，将其浸泡在无菌去离子水中，然后用超声波振荡，获得细菌悬浮液。然后将细菌悬浮液稀释 10 倍，将稀释液中的 100 muL100 \mu \mathrm{~L} 液滴在 LB 琼脂上，在 37^(@)C37^{\circ} \mathrm{C} 条件下培养 24 小时。
2.20. Statistical analysis 2.20.统计分析
All experimental data are presented as the mean +-\pm standard deviation (SD), and the results were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA analysis among three or more groups followed by Tukey’s post hoc test. A value of P < 0.05P<0.05 was considered statistically significant ( **P < 0.05,^(****)P < 0.01,^(******)P < 0.001* P<0.05,{ }^{* *} P<0.01,{ }^{* * *} P<0.001, ****P < 0.0001<0.0001 ). At least three independent experiments were performed in this study. 所有实验数据均以平均值 +-\pm 标准差（SD）表示，结果采用单因素方差分析（ANOVA）或三组或更多组间的双因素方差分析，然后进行Tukey事后检验。 P < 0.05P<0.05 值被认为具有统计学意义（ **P < 0.05,^(****)P < 0.01,^(******)P < 0.001* P<0.05,{ }^{* *} P<0.01,{ }^{* * *} P<0.001 , ****P < 0.0001<0.0001 ）。本研究至少进行了三次独立实验。
3. Results 3.成果
3.1. Design, preparation, and characterization of DPPLM NPs 3.1.DPPLM NPs 的设计、制备和表征
DSPE-PEG-NHS was self-assembled in water, and then covalently linked to the amino group on the surface of PAMAM by an amidation reaction between succinic anhydride and the amino-ended PAMAM at different molar ratios ( 76.8 : 1,32:1,6.4:11,32: 1,6.4: 1 ) to obtain the amphiphilic copolymer, named DSPE-PEG-PAMAM (DPP) (Fig. 1A, Figs. S2 and S3). DSPE-PEG-NHS在水中自组装，然后通过琥珀酸酐与氨基端PAMAM以不同的摩尔比（76.8 : 1,32:1,6.4:11,32: 1,6.4: 1 ）发生酰胺化反应，与PAMAM表面的氨基共价连接，得到两亲共聚物，命名为DSPE-PEG-PAMAM（DPP）（图1A，图S2和图S3）。
When the molar ratio of DSPE-PEG and PAMAM was 6.4:1, DPP NPs had a relatively high drug loading content, because the drug loading content increased as the content of PAMAM increased, while there were no significant differences in the size and surface charge of the three kinds of DPP NPs. Therefore, the composition of DSPE-PEG and PAMAM in DPP NPs was fixed at 6.4:1 for subsequent experiments. 当 DSPE-PEG 和 PAMAM 的摩尔比为 6.4:1 时，DPP NPs 的载药量相对较高，因为载药量随着 PAMAM 含量的增加而增加，而三种 DPP NPs 的尺寸和表面电荷没有明显差异。因此，在随后的实验中，DPP NPs 中 DSPE-PEG 和 PAMAM 的比例固定为 6.4:1。
As shown in the ^(1)H{ }^{1} \mathrm{H} NMR spectrum (Fig. S1), the characteristic peaks of PAMAM appeared in the spectra at 2.42, 2.62, 2.72, 2.81, 3.23, and 3.28 ppm , corresponding to H6-H1\mathrm{H} 6-\mathrm{H} 1. The characteristic peaks of PEG (-CH_(2)O-)\left(-\mathrm{CH}_{2} \mathrm{O}-\right) and DSPE (-CH_(2)-)\left(-\mathrm{CH}_{2}-\right) were detected at 3.69 ppm and 1.29 ppm , respectively. After conjugation, the spectrum of DPP exhibited characteristic chemical shifts of hydrogens in DSPE-PEG and PAMAM, which confirmed that PAMAM was successfully introduced into the DSPE-PEGNHS molecules. According to the peak area of DSPE-PEG and PAMAM, the molar ratio of DSPE-PEG and PAMAM in the product was approximately 4. The FTIR spectra of the raw materials are presented in Fig. 1C. PAMAM presented characteristic peaks of amide I and amide II at 1632 cm^(-1)\mathrm{cm}^{-1} and 1543cm^(-1)1543 \mathrm{~cm}^{-1}, respectively. The characteristic peaks of PAMAM at 3074cm^(-1)3074 \mathrm{~cm}^{-1} and 3276cm^(-1)3276 \mathrm{~cm}^{-1} corresponded to the strong vibration of N-HN-\mathrm{H} in the peripheral amino group. The peaks at 2830cm^(-1)2830 \mathrm{~cm}^{-1} and 2934 cm^(-1)\mathrm{cm}^{-1} were attributed to C-H stretching. For DSPE-PEG-NHS, the characteristic peaks located at 1097cm^(-1)1097 \mathrm{~cm}^{-1} were attributed to C-O-C\mathrm{C}-\mathrm{O}-\mathrm{C} stretching vibrations, the absorption peaks corresponding to 1342cm^(-1)1342 \mathrm{~cm}^{-1} belonged to C-H\mathrm{C}-\mathrm{H} wagging vibration. After the conjugation reaction between DSPE-PEG-NHS and PAMAM, the characteristic peaks for nuC-O-C\nu \mathrm{C}-\mathrm{O}-\mathrm{C}(1097cm^(-1)),omegaC-H(1342cm^(-1))\left(1097 \mathrm{~cm}^{-1}\right), \omega \mathrm{C}-\mathrm{H}\left(1342 \mathrm{~cm}^{-1}\right) of DSPE-PEG, and nuC-O(1632cm^(-1))\nu \mathrm{C}-\mathrm{O}\left(1632 \mathrm{~cm}^{-1}\right) of PAMAM were clearly shown in the FTIR spectrum of DSPE-PEGPAMAM. These results suggested that PAMAM was successfully conjugated to DSPE-PEG. 如 ^(1)H{ }^{1} \mathrm{H} NMR 光谱（图 S1）所示，PAMAM 的特征峰出现在 2.42、2.62、2.72、2.81、3.23 和 3.28 ppm 处，对应于 H6-H1\mathrm{H} 6-\mathrm{H} 1 。PEG (-CH_(2)O-)\left(-\mathrm{CH}_{2} \mathrm{O}-\right) 和 DSPE (-CH_(2)-)\left(-\mathrm{CH}_{2}-\right) 的特征峰分别出现在 3.69 ppm 和 1.29 ppm 处。共轭后，DPP 的光谱显示出 DSPE-PEG 和 PAMAM 中氢原子的特征化学位移，这证实 PAMAM 已成功引入 DSPE-PEGNHS 分子中。根据 DSPE-PEG 和 PAMAM 的峰面积，产品中 DSPE-PEG 和 PAMAM 的摩尔比约为 4。 原料的傅立叶变换红外光谱见图 1C。PAMAM 在 1632 cm^(-1)\mathrm{cm}^{-1} 和 1543cm^(-1)1543 \mathrm{~cm}^{-1} 处分别出现了酰胺 I 和酰胺 II 的特征峰。PAMAM 在 3074cm^(-1)3074 \mathrm{~cm}^{-1} 和 3276cm^(-1)3276 \mathrm{~cm}^{-1} 处的特征峰与外围氨基中 N-HN-\mathrm{H} 的强振动相对应。 2830cm^(-1)2830 \mathrm{~cm}^{-1} 和 2934 cm^(-1)\mathrm{cm}^{-1} 处的峰归因于 C-H 伸展。对于 DSPE-PEG-NHS， 1097cm^(-1)1097 \mathrm{~cm}^{-1} 处的特征峰属于 C-O-C\mathrm{C}-\mathrm{O}-\mathrm{C} 伸缩振动， 1342cm^(-1)1342 \mathrm{~cm}^{-1} 处的吸收峰属于 C-H\mathrm{C}-\mathrm{H} 摆动振动。DSPE-PEG-NHS与PAMAM发生共轭反应后，DSPE-PEG的 nuC-O-C\nu \mathrm{C}-\mathrm{O}-\mathrm{C}(1097cm^(-1)),omegaC-H(1342cm^(-1))\left(1097 \mathrm{~cm}^{-1}\right), \omega \mathrm{C}-\mathrm{H}\left(1342 \mathrm{~cm}^{-1}\right) 和PAMAM的 nuC-O(1632cm^(-1))\nu \mathrm{C}-\mathrm{O}\left(1632 \mathrm{~cm}^{-1}\right) 特征峰清晰地显示在DSPE-PEGPAMAM的傅立叶红外光谱中。这些结果表明，PAMAM 与 DSPE-PEG 成功共轭。
Then, ALA-loaded DSPE-PEG NPs were prepared using the nanoprecipitation method, and Mino-loaded PAMAM was synthesized via electrostatic interaction. To optimize the drug loading content (DLC) and drug loading efficiency (DLE), we further investigated the drugloaded NPs by varying the weight ratios of polymeric DPP/drug. The DLC of ALA increased from 7% to 16.83%16.83 \% and the DLE declined from 53.72%53.72 \% to 34.71%34.71 \% when the drug to DSPE-PEG (DP) ratio increased from 2:152: 15 to 8:158: 15. The DLC and DLE of ALA used in this study was 13.61%13.61 \% and 51.88%, respectively. Furthermore, ALA and Mino were coencapsulated into micelles, and the maximal DLC of Mino that could be achieved was 17.17%17.17 \%, and the DLE of Mino was 33.37%33.37 \% in this study (Table 1). 然后，我们采用纳米沉淀法制备了 ALA 负载的 DSPE-PEG NPs，并通过静电作用合成了 Mino-loaded PAMAM。为了优化载药量（DLC）和载药效率（DLE），我们通过改变聚合物 DPP/药物的重量比进一步研究了载药 NPs。当药物与DSPE-PEG（DP）的重量比从 2:152: 15 增加到 8:158: 15 时，ALA的DLC从7%增加到 16.83%16.83 \% ，DLE从 53.72%53.72 \% 下降到 34.71%34.71 \% 。本研究中使用的 ALA 的 DLC 和 DLE 分别为 13.61%13.61 \% 和 51.88%。此外，本研究还将 ALA 和 Mino 共包囊到胶束中，Mino 的最大 DLC 为 17.17%17.17 \% ，Mino 的 DLE 为 33.37%33.37 \% （表 1）。
We also measured the zeta potential of DPP and DPPLM NPs. DSPEPEG NPs presented negatively charged zeta potentials (-2.02+-0.61(-2.02 \pm 0.61 mV ) (Fig. S3). However, the zeta potential was neutral or positive after the conjugation with PAMAM, and the zeta potential of DPPLM NPs was 1.02+-0.35mV1.02 \pm 0.35 \mathrm{mV} (Fig. 1E), making NPs more likely to adhere to negatively charged bacteria via electrostatic interactions. TEM imaging (Fig. 1B) indicated that the morphology of both blank DPP and DPPLM NPs was irregular spheres. DLS analysis (Fig. 1D) revealed that the size of DPPLM NPs was approximately 12.78 nm , which was slightly larger than the blank DPP NPs (approximately 11.58 nm ). 我们还测量了 DPP 和 DPPLM NPs 的 zeta 电位。DSPEPEG NPs呈现出带负电荷的zeta电位 (-2.02+-0.61(-2.02 \pm 0.61 mV ）（图 S3）。然而，与 PAMAM 共轭后，zeta 电位呈中性或正值，而 DPPLM NPs 的 zeta 电位为 1.02+-0.35mV1.02 \pm 0.35 \mathrm{mV} （图 1E），这使得 NPs 更有可能通过静电作用附着在带负电荷的细菌上。TEM 成像（图 1B）表明，空白 DPP 和 DPPLM NPs 的形态均为不规则球形。DLS 分析（图 1D）显示，DPPLM NPs 的尺寸约为 12.78 nm，比空白 DPP NPs（约 11.58 nm）略大。
3.2. Drug release from DPPLM NPs was enhanced in an acidic and lipasecontaining environment 3.2.DPPLM NPs 的药物释放在酸性和含脂环境中得到增强
Meanwhile, we addressed the NPs’ response to pH and bacterial enzymes using TEM and DLS in vitro (Fig. 1B, D). The DLS results 同时，我们在体外使用 TEM 和 DLS 研究了 NPs 对 pH 值和细菌酶的反应（图 1B 和 D）。DLS 结果
Table 1 表 1
Drug loading content (DLC) and drug loading efficiency (DLE). 载药量（DLC）和载药效率（DLE）。
DLC of Mino 米诺的 DLC
DLE of Mino 米诺的 DLE
DLC of ALA ALA 的 DLC
DLE of ALA ALA 的 DLE
DPPL _(4)_{4}
-
-
7%\mathbf{7 \%}
53.72%\mathbf{5 3 . 7 2 \%}
DPPL _(8)_{8}
-
-
13.61%\mathbf{1 3 . 6 1 \%}
51.88%\mathbf{5 1 . 8 8 \%}
DPPL _(16)_{16}
-
-
16.83%\mathbf{1 6 . 8 3 \%}
34.71%\mathbf{3 4 . 7 1 \%}
DPPL _(8)M_(16)_{8} \mathbf{M}_{16}
17.17%\mathbf{1 7 . 1 7 \%}
33.37%\mathbf{3 3 . 3 7 \%}
-
-
DLC of Mino DLE of Mino DLC of ALA DLE of ALA
DPPL _(4) - - 7% 53.72%
DPPL _(8) - - 13.61% 51.88%
DPPL _(16) - - 16.83% 34.71%
DPPL _(8)M_(16) 17.17% 33.37% - -| | DLC of Mino | DLE of Mino | DLC of ALA | DLE of ALA |
| :--- | :--- | :--- | :--- | :--- |
| DPPL $_{4}$ | - | - | $\mathbf{7 \%}$ | $\mathbf{5 3 . 7 2 \%}$ |
| DPPL $_{8}$ | - | - | $\mathbf{1 3 . 6 1 \%}$ | $\mathbf{5 1 . 8 8 \%}$ |
| DPPL $_{16}$ | - | - | $\mathbf{1 6 . 8 3 \%}$ | $\mathbf{3 4 . 7 1 \%}$ |
| DPPL $_{8} \mathbf{M}_{16}$ | $\mathbf{1 7 . 1 7 \%}$ | $\mathbf{3 3 . 3 7 \%}$ | - | - |
Table 2 表 2
The primer sequences of all genes used in qPCR. qPCR 中使用的所有基因的引物序列。
Gene 基因
Forward primer (5^(')∼3^('))\left(5^{\prime} \sim 3^{\prime}\right) 前向引物 (5^(')∼3^('))\left(5^{\prime} \sim 3^{\prime}\right)
Reverse primer (5^(')∼3^('))\left(5^{\prime} \sim 3^{\prime}\right) 反向引物 (5^(')∼3^('))\left(5^{\prime} \sim 3^{\prime}\right)
indicated that the DPPLM NPs swelled slightly (approximately 15.54 nm ) at pH 5.5 . When the NPs were incubated at pH 5.5 with lipase, the DLS results showed a significant increase to 180 nm in diameter, which might be ascribed to the disassembly and re-agglomeration of the NPs. The results were consistent with the morphological changes of NPs observed by TEM. We also measured the zeta potential (Fig. 1E) of DPPLM NPs at pH 5.5 , with or without the lipase, which showed that when DPPLM NPs were incubated with the lipase, their zeta potential was reduced to -1.95+-0.10mV-1.95 \pm 0.10 \mathrm{mV}, which was similar to that of DSPE-PEG (Fig. S3). The size changes revealed that the DPPLM NPs facilitated the on-demand release of drug in the acidic microenvironment and in the presence of lipase. 结果表明，DPPLM NPs 在 pH 值为 5.5 时轻微膨胀（约 15.54 nm）。当 NPs 在 pH 5.5 条件下与脂肪酶一起孵育时，DLS 结果显示其直径显著增加到 180 nm，这可能是由于 NPs 的分解和重新聚集。这一结果与 TEM 观察到的 NPs 形态变化一致。我们还测量了 DPPLM NPs 在 pH 值为 5.5 时的 zeta 电位（图 1E），结果表明当 DPPLM NPs 与脂肪酶共培养时，其 zeta 电位降低到 -1.95+-0.10mV-1.95 \pm 0.10 \mathrm{mV} ，与 DSPE-PEG 的 zeta 电位相似（图 S3）。尺寸变化表明，DPPLM NPs 在酸性微环境和脂肪酶存在的情况下促进了药物的按需释放。
Next, we investigated the accumulated release of ALA and Mino from DPPLM NPs in response to pH or in the presence of lipase, respectively. Fig. 2A shows that in the presence of lipase, DPPLM NPs exhibited an enhanced release behavior of ALA compared to that without enzymes, which was ascribed to the catalyzed degradation of the DSPE-PEG cores and the resultant disintegration of the DPPLM NPs. As shown in Fig. 2B, the release of Mino at pH 5.5 was a little faster than that at pH 7.4 , which could be attributed to the weakened electrostatic interactions between Mino and PAMAM under acidic conditions. In summary, these results indicated that the acid and lipase promoted drug release feature will endow the system with a preferential drug release profile. 接下来，我们研究了 DPPLM NPs 在 pH 值或脂肪酶存在下分别累积释放 ALA 和 Mino 的情况。图 2A 显示，在有脂肪酶存在的情况下，DPPLM NPs 的 ALA 释放行为比没有酶的情况下更强，这归因于 DSPE-PEG 核心的催化降解和 DPPLM NPs 的解体。如图 2B 所示，在 pH 值为 5.5 时，Mino 的释放速度略快于 pH 值为 7.4 时，这可能是由于在酸性条件下 Mino 与 PAMAM 之间的静电相互作用减弱所致。总之，这些结果表明，酸和脂肪酶促进药物释放的特性将使该系统具有优先药物释放特性。
We also determined the penetration and retention of PAMAM into hard tissue. As shown in Fig. S5A, the fluorescent signal was visible at the cortical layer of the bone along the direction of diffusion at 5 days after administration, indicating that PAMAM-FITC was able to penetrate into the superficial layer of bone. Furthermore, the penetration of PAMAM into the root was determined using confocal microscopy and fluorescence microscopy. Figs. S5B and C shows fluorescence signals in the cementum layer of the root, which demonstrated that PAMAM could penetrate slightly into the cementum. Collectively, these results indicated that the small-sized PAMAM NPs were possibly able to penetrate into the hard tissue to deliver the drugs to a certain extent. 我们还测定了 PAMAM 在硬组织中的渗透和保留情况。如图 S5A 所示，给药 5 天后，荧光信号在骨皮质层沿扩散方向可见，表明 PAMAM-FITC 能够渗透到骨表层。此外，还使用共聚焦显微镜和荧光显微镜测定了 PAMAM 对牙根的渗透情况。图 S5B 和 C 显示了牙根骨水泥层的荧光信号，这表明 PAMAM 可以轻微渗入骨水泥层。总之，这些结果表明，小尺寸的 PAMAM NPs 有可能在一定程度上渗透到硬组织中输送药物。
3.3. DPPLM NPs had good biocompatibility and were effectively endocytosed by BMSCs 3.3.DPPLM NPs 具有良好的生物相容性，可被 BMSCs 有效内吞
The cytotoxicity of drug-loaded nanoparticles against BMSCs were measured using a CCK8 assay. The results indicated that DPPLM NPs showed low cytotoxicity after incubation for 24 h , even at relatively high doses (Fig. 2C). 使用 CCK8 检测法测量了载药纳米粒子对 BMSCs 的细胞毒性。结果表明，即使在相对较高的剂量下，DPPLM NPs 在孵育 24 小时后也表现出较低的细胞毒性（图 2C）。
To investigate cellular uptake behavior of NPs, we labeled DPP NPs with FITC. Meanwhile, Nile red, a hydrophobic fluorescent dye, and Rhodamine B, a hydrophilic fluorescent dye, were encapsulated in FITClabeled NPs as model drug substitutes for ALA and Mino, respectively. The cellular uptake of DPP-FITC NPs in BMSCs was confirmed using flow cytometry (Fig. 2D). Furthermore, CLSM images (Fig. 2E and F) showed that the intensity of red and green fluorescence in the BMSCs increased gradually from 2 to 24 h after incubation with fluorescently-labeled NPs, which indicated the successfully internalization of NPs by cells. 为了研究 NPs 的细胞摄取行为，我们用 FITC 标记了 DPP NPs。同时，我们在 FIT 标记的 NPs 中封装了疏水性荧光染料尼罗红和亲水性荧光染料罗丹明 B，分别作为 ALA 和 Mino 的模型药物替代物。流式细胞术证实了 BMSC 细胞对 DPP-FITC NPs 的吸收（图 2D）。此外，CLSM 图像（图 2E 和 F）显示，荧光标记 NPs 培养 2 至 24 h 后，BMSCs 中的红色和绿色荧光强度逐渐增加，这表明细胞成功内化了 NPs。
Furthermore, we investigated the subcellular localization of NPs in BMSCs using CLSM (Fig. 2G). The lysosomes were labeled by LysoTracker Green. The red fluorescence was very weak at an incubation time of 20 min , indicating that few NPs were absorbed into cells at this 此外，我们还使用 CLSM 研究了 NPs 在 BMSCs 中的亚细胞定位（图 2G）。溶酶体用 LysoTracker Green 标记。在孵育 20 分钟时，红色荧光非常微弱，表明此时细胞中吸收的 NPs 很少。
A large amount of evidence suggests that increased production of AGE, which are the products of nonenzymatic glycation of macromolecules (proteins, lipids, and nucleic acids) accumulates in bone tissue during DM [9]. Thus, we further established a model to mimic the diabetic environment using AGE-BSA in vitro, which is modified by a variety of AGE structures and resembles in vivo generated forms. 大量证据表明，AGE 是大分子（蛋白质、脂类和核酸）非酶糖化的产物，在糖尿病期间骨组织中 AGE 的生成增加 [9]。因此，我们进一步建立了一个模拟糖尿病环境的体外 AGE-BSA 模型，该模型由多种 AGE 结构修饰而成，与体内生成的形式相似。
First, we verified that ALA had a good ability to promote osteogenic 首先，我们验证了 ALA 具有良好的促进成骨的能力。
differentiation of BMSCs using Alizarin Red staining and quantitative analysis. The results shown in Fig. 3A and B indicated that ALA-treated BMSCs had over two-fold more calcium deposition and mineralization nodules than did the control group. Then, we investigated the osteoinductive properties of DPPLM NPs. As shown in Fig. 3C and D, the DPPLM group promoted the formation of mineralized nodules significantly, while the negative effect of AGE-BSA on the osteogenic differentiation of BMSCs was reversed in the AGE + DPPLM group. Additionally, qRT-PCR (Fig. 3E-I) revealed that the mRNA expression of osteogenic genes [Alp (alkaline phosphatase), Opn (osteopontin), Osx (osterix), Ocn (osteocalcin), and Runx2 (runt-related transcription factor 2)] were downregulated in the AGE-BSA group, but recovered in the AGE + DPPLM 利用茜素红染色和定量分析对 BMSCs 进行分化。图 3A 和 B 中的结果表明，ALA 处理的 BMSCs 的钙沉积和矿化结节是对照组的两倍多。然后，我们研究了 DPPLM NPs 的骨诱导特性。如图 3C 和 D 所示，DPPLM 组显著促进了矿化结节的形成，而 AGE-BSA 对 BMSCs 成骨分化的负面影响在 AGE + DPPLM 组得到了逆转。此外，qRT-PCR（图 3E-I）显示，成骨基因[Alp（碱性磷酸酶）、Opn（骨通蛋白）、Osx（osterix）、Ocn（骨钙蛋白）和 Runx2（runt 相关转录因子 2）]的 mRNA 表达在 AGE-BSA 组中下调，但在 AGE + DPPLM 组中恢复。
group after incubation for 7 days, indicating that treatment with DPPLM NPs enhanced the osteogenic differentiation and mineralization of BMSCs in a diabetic pathological environment. 这表明，在糖尿病病理环境中，DPPLM NPs 可促进 BMSCs 的成骨分化和矿化。
To determine the underlying mechanisms of DPPLM NP-mediated osteogenesis, we subsequently examined the intracellular ROS and iNOS levels. As shown in Fig. 3J, the AGE group presented increased numbers of positive cells compared with those in the other groups. However, marked suppression of ROS generation was detected in the DPPLM + AGE and DPPLM groups. The immunofluorescence staining data showed that the expression of iNOS in AGE-induced BMSCs was markedly enhanced, while the expression of iNOS in DPPLM + AGE group was reduced (Fig. 3K), which showed a similar trend to the intracellular ROS 为了确定 DPPLM NP 介导成骨的潜在机制，我们随后检测了细胞内 ROS 和 iNOS 的水平。如图 3J 所示，与其他组相比，AGE 组的阳性细胞数量有所增加。然而，在 DPPLM + AGE 组和 DPPLM 组检测到 ROS 生成明显受到抑制。免疫荧光染色数据显示，AGE 诱导的 BMSCs 中 iNOS 的表达明显增强，而 DPPLM + AGE 组 iNOS 的表达减少（图 3K），这与细胞内 ROS 的变化趋势相似。
We selected E. coli (a typical gram-negative bacteria) and S. aureus (a typical gram-positive bacteria) to evaluate the antibacterial activity of the DPPLM NPs. The broth microdilution method was used to evaluate the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the DPPLM NPs. The MIC value of DPPLM was 我们选择大肠杆菌（典型的革兰氏阴性菌）和金黄色葡萄球菌（典型的革兰氏阳性菌）来评估 DPPLM NPs 的抗菌活性。肉汤微稀释法用于评估 DPPLM NPs 的最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。DPPLM 的 MIC 值为
A rat experimental periodontitis model of type 2 diabetes mellitus (T2DM) was established to evaluate the antibacterial ability of DPPLM NPs and their periodontal regeneration ability in vivo (Fig. 5A). An oral glucose tolerance test (OGTT) was performed to confirm that the T2DM rat model was successfully established [47]. Fig. S6 shows that the blood glucose levels in both DM and control groups increased during the initial 30 min and gradually decreased over the following 120 min , whereas the sustained high glucose level in the DM group suggested the successful establishment of hyperglycemia. When the blood glucose of the rats was over 16.7mmol//L16.7 \mathrm{mmol} / \mathrm{L}, we created the experimental periodontitis model in these rats and then carried out the experiments. 为了评估 DPPLM NPs 的抗菌能力及其体内牙周再生能力，我们建立了 2 型糖尿病（T2DM）大鼠实验性牙周炎模型（图 5A）。进行口服葡萄糖耐量试验（OGTT）以确认 T2DM 大鼠模型的成功建立 [47]。图 S6 显示，DM 组和对照组的血糖水平在最初的 30 分钟内均有所升高，并在随后的 120 分钟内逐渐降低，而 DM 组的血糖水平持续较高，表明高血糖的成功建立。当大鼠血糖超过 16.7mmol//L16.7 \mathrm{mmol} / \mathrm{L} 时，我们在这些大鼠中建立了实验性牙周炎模型，然后进行实验。
To evaluate the antibacterial effect in vivo, we performed plate counting methods and SEM. We counted the bacterial colonies originally derived from the teeth surface (Fig. 4E). Only a few bacterial colonies were seen in CM + DPPLM group, which was less than in the CM + free drug group, while there were a large amount of bacteria in the Untreated group and CM group (Fig. 4E). These results indicated that the antibacterial effect of the CM + DPPLM group was better than that of the CM+\mathrm{CM}+ free drug group, and much better than that of the Untreated group and CM group. Next, we observed the morphology of the dental plaque that was attached to the surfaces of the teeth after DPPLM treatment using SEM. Fig. 4F shows that a large amount of bacteria were closely packed and interlaced as a plaque biofilm in the Untreated group, which was similar to the images exhibited in CM group. Small amounts of bacteria were scattered in the CM + DPPLM group, indicating that the DPPLM NPs had a more effective antibacterial activity. 为了评估体内的抗菌效果，我们采用了平板计数法和扫描电镜法。我们对最初来自牙齿表面的细菌菌落进行了计数（图 4E）。在 CM + DPPLM 组中只看到少量细菌菌落，少于 CM + 游离药物组，而在未处理组和 CM 组中则有大量细菌（图 4E）。这些结果表明，CM + DPPLM 组的抗菌效果优于 CM+\mathrm{CM}+ 游离药物组，更优于未处理组和 CM 组。接着，我们用扫描电镜观察了 DPPLM 治疗后附着在牙齿表面的牙菌斑的形态。图 4F 显示，在未处理组中，大量细菌紧密排列并交错成牙菌斑生物膜，这与 CM 组的图像相似。在 CM + DPPLM 组中，少量细菌分散，这表明 DPPLM NPs 具有更有效的抗菌活性。
3.7. DPPLM NPs alleviated periodontal bone loss in DM rats 3.7.DPPLM NPs 可减轻 DM 大鼠牙周骨质流失
To investigate the osteogenesis of DPPLM NPs under DM conditions in vivo, micro-CT was conducted and the distance between the CEJ-ABC on the palatal side of the first maxillary molar was measured. To ensure the consistency of the measurement standards, we viewed the micro-CT reconstructed images from the same angle and adjusted all tooth cusps on the same plane. No significant difference in the vertical distance between the CEJ-ABC was found in the DM group and in the healthy group, while the CEJ-ABC distance in the periodontitis group increased, and the CEJ-ABC distance in the diabetic periodontitis group was significantly enhanced compared with that of the healthy group (Figs. S7 and S8). These results indicated that the diabetic periodontitis model was successfully established, and that DM led to the increased alveolar bone loss. As shown in Fig. 5B and C, the collagen membrane (CM) group showed no bone healing effects, the CEJ-ABC distance of which was equal to the Untreated group, indicating that the CM had no influence on bone regeneration. The CEJ-ABC distance of the CM + free drug group showed a slight reduction; however, the difference was not statistically significant compared with that of the Untreated group, indicating that the free drug insufficiently promoted osteogenesis. The CEJ-ABC distance in the CM + DPPLM group was significantly reduced, by an average of 0.357 mm , compared with that of the Untreated group, indicating a positive effect of DPPLM on delaying alveolar bone loss. 为了研究DPPLM NPs在DM条件下的体内成骨情况，我们进行了显微CT测量，并测量了上颌第一磨牙腭侧CEJ-ABC之间的距离。为确保测量标准的一致性，我们从同一角度观察显微 CT 重建图像，并将所有牙尖调整在同一平面上。DM组与健康组的CEJ-ABC垂直距离无明显差异，而牙周炎组的CEJ-ABC距离有所增加，糖尿病牙周炎组的CEJ-ABC距离较健康组明显增加（图S7和S8）。这些结果表明，糖尿病牙周炎模型的建立是成功的，DM 导致了牙槽骨丧失的增加。如图 5B 和 C 所示，胶原膜（CM）组没有骨愈合效果，其 CEJ-ABC 距离与未处理组相等，表明胶原膜对骨再生没有影响。CM+游离药物组的CEJ-ABC距离略有减少，但与未处理组相比差异无统计学意义，表明游离药物对骨生成的促进作用不足。与未处理组相比，CM + DPPLM 组的 CEJ-ABC 距离明显缩小，平均缩小了 0.357 毫米，这表明 DPPLM 对延缓牙槽骨流失有积极作用。
To further evaluate the inflammatory status and periodontium regeneration, we performed H&E staining and Masson’s staining of the alveolar bone. We observed unattached and more disordered periodontal ligaments between the maxillary first molars and maxillary second molars in the Untreated group, CM group, and CM + free drug group, while the ligaments in the DPPLM group were denser and well arranged (Fig. 5D and E). In addition, the DPPLM group showed a shorter CEJ-ABC distance, which indicated that DPPLM could inhibit periodontal destruction. In summary, the results indicated that the DPPLM NPs effectively inhibited periodontal bone loss. 为了进一步评估炎症状况和牙周再生情况，我们对牙槽骨进行了 H&E 染色和 Masson 染色。我们观察到，未治疗组、CM 组和 CM + 游离药物组的上颌第一磨牙和上颌第二磨牙之间的牙周韧带未附着且较为紊乱，而 DPPLM 组的韧带较为致密且排列整齐（图 5D 和 E）。此外，DPPLM 组的 CEJ-ABC 距离更短，这表明 DPPLM 可抑制牙周破坏。总之，研究结果表明，DPPLM NPs 能有效抑制牙周骨质流失。
4. Discussion 4.讨论
Advanced nanoscale systems of a distinct nature have exhibited promising prospects by taking advantage of special microenvironmental changes to achieve controlled drug release for bone regeneration. Efforts to produce superior nanoscale systems are ongoing. In particular, a great deal of research has been carried out on stimuli-responsive nanoscale systems for drug delivery to ensure the therapeutic effect of their cargoes at the site of infection. In the present study, a pH//\mathrm{pH} / lipase-responsive dualdrug delivery system was designed to exert antibacterial, antioxidant, anti-inflammatory, and osteoinductive functions in diabetic microenvironments to inhibit alveolar bone loss under DM conditions. 通过利用特殊的微环境变化来实现骨再生药物的可控释放，具有独特性质的先进纳米级系统展现出了广阔的前景。生产高级纳米级系统的努力仍在继续。特别是，人们对用于给药的刺激响应纳米级系统进行了大量研究，以确保其货物在感染部位的治疗效果。在本研究中，我们设计了一种 pH//\mathrm{pH} / 脂肪酶响应型双药物递送系统，在糖尿病微环境中发挥抗菌、抗氧化、抗炎和骨诱导功能，以抑制 DM 条件下的牙槽骨流失。
It has been reported that the biofilm microenvironment is relatively acidic ( pH4.5-6.5\mathrm{pH} 4.5-6.5 ) because of anaerobic fermentation [31]. Some molecules and groups can be protonated under acidic conditions and are deprotonated under alkaline conditions. Thus, their hydrophilicity will undergo a sharp reversal to change the structure and morphology of their assemblies [25]. Several studies have demonstrated that PAMAM-based nanocarriers can be developed to respond to pH changes, attributed to the conformational changes from a “dense core” at high pH to a “dense shell” at low pH [32]. In the present study, the PAMAM-based nanocarrier system responded to pH and accelerated the release of the transported drug (Fig. 2A and B). Moreover, at the cellular level, because of the proton sponge effect of PAMAM, the acidic microenvironment could promote the escape of the nanocarriers from the lysosomes into the cytoplasm, which was confirmed by subcellular localization experiments (Fig. 2G). In addition, the PAMAM nanoarchitectures designed in the present study could improve drug solubility and drug permeation. It is well documented that some specific enzymes (e.g., esterase, lipase, and gelatinase) are overexpressed in biofilm-associated infections [29,42], and can be exploited to achieve enzyme-mediated drug release. In this work, the incorporation of DSPE-PEG not only introduced the capability to respond to lipase, but also improved the pharmacokinetics of PAMAMs, reduced their toxicity, and increased the solubility and enhanced the bioavailability of hydrophobic drugs. Overall, the DPPLM NPs incorporated the positive attributes of PAMAM and DSPE-PEG to overcome the disadvantages of current drug delivery systems and simultaneously incorporated the benefits of both carrier systems. The hydrophilic blocks of PAMAM formed the outer shell, which protected Mino from direct damage by the outside environment, such as hydrolysis and enzymatic degradation, to enhance its stability and prolong its circulation time in the body. The hydrophobic blocks of DSPE-PEG constituted the inner core and encapsulated the poorly soluble drug, ALA, which improved the drug’s solubility and stability. 据报道，由于厌氧发酵，生物膜微环境相对呈酸性（ pH4.5-6.5\mathrm{pH} 4.5-6.5 ）[31]。一些分子和基团在酸性条件下会质子化，在碱性条件下会去质子化。因此，它们的亲水性会发生急剧逆转，从而改变其组合物的结构和形态 [25]。一些研究表明，基于 PAMAM 的纳米载体可以对 pH 值的变化做出反应，这归因于其构象从高 pH 值时的 "致密核心 "变为低 pH 值时的 "致密外壳"[32]。在本研究中，基于 PAMAM 的纳米载体系统对 pH 值做出了反应，并加速了转运药物的释放（图 2A 和 B）。此外，在细胞水平上，由于 PAMAM 的质子海绵效应，酸性微环境可促进纳米载体从溶酶体逸出进入细胞质，这一点在亚细胞定位实验中得到了证实（图 2G）。此外，本研究中设计的 PAMAM 纳米结构还能提高药物的溶解度和渗透性。有资料表明，一些特定的酶（如酯酶、脂肪酶和明胶酶）在生物膜相关感染中过度表达[29,42]，可以利用这些酶来实现酶介导的药物释放。在这项研究中，DSPE-PEG 的加入不仅引入了对脂肪酶的反应能力，还改善了 PAMAMs 的药代动力学，降低了其毒性，增加了疏水性药物的溶解度并提高了其生物利用度。总之，DPPLM NPs 融合了 PAMAM 和 DSPE-PEG 的积极特性，克服了现有药物递送系统的缺点，同时兼具两种载体系统的优点。 PAMAM 的亲水块构成外壳，保护 Mino 免受水解和酶降解等外界环境的直接破坏，从而提高其稳定性并延长其在体内的流通时间。DSPE-PEG 的疏水性嵌段构成内核，将溶解性较差的药物 ALA 包裹起来，提高了药物的溶解性和稳定性。
According to the results of TEM, DLS, and zeta potential analysis, the DPPLM NPs were small in size and positively charged. Size and charge can influence the penetration behavior of nanoparticles in biofilms. Gao and co-workers reported that azithromycin (AZM)-conjugated clustered nanoparticles (denoted as AZM-DA NPs) with a small size and positive surface charge exhibited excellent penetration and retention capability inside biofilms, both in vitro and in vivo [31]. Notably, small-sized NPs are particularly beneficial for drug delivery because they can reduce diffusional hindrance, accumulate and penetrate deeper into the inner regions [48]. Similarly, in our study, the small size and positive surface charge endowed the DPPLM NPs with the ability of penetration and retention in local infected tissue. In addition, the bacterial membrane is generally negatively charged [31]. Thus, the positive surface charge of DPPLM promoted interactions with the negatively charged bacterial wall, and led to increased nanoparticle uptake in both Gram-positive and Gram-negative bacteria. 根据 TEM、DLS 和 zeta 电位分析结果，DPPLM NPs 的尺寸较小，且带正电荷。尺寸和电荷会影响纳米粒子在生物膜中的渗透行为。Gao 及其合作者报告说，阿奇霉素（AZM）共轭簇状纳米粒子（称为 AZM-DA NPs）尺寸小且表面带正电荷，在体外和体内生物膜中都表现出极佳的穿透和滞留能力[31]。值得注意的是，小尺寸的 NPs 特别有利于给药，因为它们可以减少扩散阻碍，积聚并深入内部区域 [48]。同样，在我们的研究中，小尺寸和正表面电荷赋予了 DPPLM NPs 在局部感染组织中的渗透和保留能力。此外，细菌膜一般带负电荷 [31]。因此，DPPLM 的正表面电荷促进了与带负电的细菌壁的相互作用，从而增加了革兰氏阳性菌和革兰氏阴性菌对纳米粒子的吸收。
Previous studies have demonstrated that DM increases the risk of periodontitis for the reason of increased AGE deposition, increased ROS production, and subsequently exaggerated inflammation in the periodontal tissues. It is widely known that ROS produced by immune cells have antibacterial effects [49,50[49,50 ] by reacting with their essential 以往的研究表明，糖尿病会增加牙周炎的风险，原因是 AGE 沉积增加、ROS 生成增加，进而导致牙周组织炎症加剧。众所周知，免疫细胞产生的 ROS 具有抗菌作用 [49,50[49,50 ]。
macromolecules such as DNA, RNA, and pathogenic proteins, damaging the cell membrane and cell wall and causing the pathogen’s death [51]. However, the relatively sudden release of ROS produced by immune cells has become known as the “oxidative burst” [52,53] or “respiratory burst” [54]. While a transient burst of ROS is important for the elimination of pathogens, overproduction of ROS that overwhelms the antioxidant defense system will lead to oxidative stress, interfere with cell cycle progression, and impair osteogenesis [55], which might be more pernicious for periodontal bone regeneration. Therefore, the reduction of ROS overproduction and inflammation, along with the promotion of osteogenic differentiation, are beneficial for periodontal bone regeneration under DM conditions. Hence, transporting a feasible therapeutic agent to periodontal tissues is of great significance to achieve this ambitious goal. ALA, a natural ingredient of the human body, not only acts as a powerful antioxidant, but also displays anti-inflammatory, immunomodulatory, and osteoinductive effects [46,56,57]. We confirmed that ALA promotes osteogenic differentiation of BMSCs using Alizarin Red staining (Fig. 3A). Moreover, ALA has emerging roles in the prevention of diabetes complications and bone loss, as well as in the inhibition of periapical bone loss [46,56,58]. Therefore, we choose ALA as one of the model drugs for the treatment of diabetic periodontitis. Although ALA cannot be applied locally because of its poor aqueous dispersibility, difficulty in permeating into periodontal tissue, possible side effect, the drug-loaded nanoparticles effectively solved these problems. In our study, ALA was encapsulated in the inner core of the hydrophobic blocks of DSPE-PEG using the nanoprecipitation method and was released responsively from the nanosystem under lipase conditions (Fig. 2A). 免疫细胞产生的 ROS 会破坏 DNA、RNA 和病原蛋白等大分子，破坏细胞膜和细胞壁，导致病原体死亡 [51]。然而，免疫细胞产生的相对突然的 ROS 释放被称为 "氧化猝灭"[52,53] 或 "呼吸猝灭"[54]。虽然一过性的 ROS 暴发对消灭病原体很重要，但过度产生的 ROS 使抗氧化防御系统不堪重负，会导致氧化应激，干扰细胞周期的进展，损害成骨过程[55]，这对牙周骨再生可能更为有害。因此，在 DM 条件下，减少 ROS 过度产生和炎症，同时促进成骨分化，有利于牙周骨再生。因此，向牙周组织输送可行的治疗剂对于实现这一宏伟目标具有重要意义。ALA是人体的一种天然成分，不仅是一种强大的抗氧化剂，还具有抗炎、免疫调节和诱导骨生成的作用[46,56,57]。我们利用茜素红染色法证实了 ALA 可促进 BMSCs 的成骨分化（图 3A）。此外，ALA 在预防糖尿病并发症和骨质流失以及抑制根尖周骨质流失方面也有新的作用 [46,56,58]。因此，我们选择 ALA 作为治疗糖尿病牙周炎的模型药物之一。虽然 ALA 因其水溶性分散性差、难以渗透至牙周组织、可能产生副作用而无法局部应用，但药物载体纳米粒子有效地解决了这些问题。 在我们的研究中，使用纳米沉淀法将 ALA 包封在 DSPE-PEG 疏水块的内核中，并在脂肪酶条件下从纳米系统中释放出来（图 2A）。
Moreover, prolonged exposure to ROS in DM will be beneficial for bacteria and can induce protective functions [49], resulting in opportunistic pathogens being retained and accumulating over time. Periodontitis treatment under a DM environment also requires antibiotics to control bacterial infections. In this work, DPP NPs showed no antibacterial activity; however, after loading with antibacterial/Mino, they exhibited strong antibacterial activity. 此外，在 DM 环境中长期暴露于 ROS 会对细菌有利，并能诱发保护功能[49]，从而导致机会性病原体被长期保留和积累。DM 环境下的牙周炎治疗也需要抗生素来控制细菌感染。在这项研究中，DPP NPs 没有显示出抗菌活性；但在负载抗菌/Mino 后，它们显示出了很强的抗菌活性。
5. Conclusion 5.结论
The present study has some limitations. Firstly, we have not fully explored the underlying therapeutic mechanism by which the drugloaded nanoparticles regulate inflammation metabolism and oxidative stress. In addition, we did not consider the general condition of the whole body in DM. In the future, we will find solutions to the above problems and further focus on making the most of the diabetic microenvironment (e.g., high glucose and high level of ROS) to develop redoxresponsive or glucose-responsive nanomaterials for the controlled release of drugs at the indicated points. 本研究存在一些局限性。首先，我们没有充分探讨载药纳米颗粒调节炎症代谢和氧化应激的潜在治疗机制。此外，我们也没有考虑到 DM 患者全身的一般状况。今后，我们将针对上述问题寻找解决方案，并进一步关注如何充分利用糖尿病微环境（如高血糖和高水平的 ROS），开发出氧化还原响应型或葡萄糖响应型纳米材料，以实现药物在指定点的控制释放。
In conclusion, we constructed a novel drug delivery system based on DSPE-PEG-PAMAM, which was able to effectively co-deliver the antimicrobial/Mino and the antioxidant/ALA, disrupt dental-plaque biofilms, and suppress periodontal bone loss. This dual-drug delivery system has several advantages: (1) The ability to penetrate and be retained in local infected tissue, which is strongly dependent on their small size and surface charge. (2) High biocompatibility because DSPEPEG has a similar structure to the cytomembrane. (3) High loading efficiency resulting from the unique core-shell and radiate dendritic structure harboring a large void space for the drug. (4) Multiple functions, mainly via loading with two kinds of specific and efficient drugs simultaneously. (5) pH and lipase-responsive abilities obtained by exploiting the specificity of the microenvironment. Considering these advantages, this drug delivery system implements precise measures and offers a promising strategy to treat periodontitis under DM conditions. 总之，我们构建了一种基于 DSPE-PEG-PAMAM 的新型给药系统，该系统能够有效地联合给药抗菌剂/Mino 和抗氧化剂/ALA，破坏牙菌斑生物膜并抑制牙周骨质流失。这种双重给药系统有以下几个优点：（1）能够穿透并保留在局部感染组织中，这与它们的小尺寸和表面电荷有很大关系。(2）生物相容性高，因为 DSPEPEG 具有与细胞膜相似的结构。(3) 独特的核壳和辐射树枝状结构为药物提供了较大的空隙，因而具有较高的装载效率。(4) 多种功能，主要是同时装载两种特异性高效药物。(5) 利用微环境的特异性，获得 pH 和脂肪酶响应能力。考虑到这些优点，该给药系统实施了精确的措施，为在 DM 条件下治疗牙周炎提供了一种前景广阔的策略。
Data availability 数据可用性
The authors declare that all data supporting the findings of this study 作者声明，支持本研究结果的所有数据
are available within the paper and Supplementary Information. 可在论文和补充资料中查阅。
Lu Wang: Methodology, Formal analysis, Software, Analysis using software, Investigation, Writing - original draft. Yuzhou Li: Writing review & editing, Resources. Mingxing Ren: Formal analysis, Visualization. Xu Wang: Formal analysis, Visualization. Linjie Li: Resources, Data curation. Fengyi Liu: Validation, Data curation. Yiqing Lan: Validation, Data curation. Sheng Yang: Supervision, Project administration, Conceptualization, Funding acquisition. Jinlin Song: Supervision, Project administration, Conceptualization, Funding acquisition. Lu Wang：方法论、形式分析、软件、利用软件进行分析、调查、写作 - 原稿。李玉洲：写作审阅和编辑、资源。Mingxing Ren：形式分析、可视化。王旭形式分析、可视化李林杰：资源、数据整理。刘凤仪：验证、数据整理。Yiqing Lan：验证、数据整理：验证、数据整理。杨胜监督、项目管理、构思、获取资金。宋金林监督、项目管理、构思、资金获取。
Declaration of competing interest 利益冲突声明
The authors have no conflicts of interest to declare. 作者无利益冲突需要声明。
Acknowledgements 致谢
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 82171010, 81771082, 31971282, 82001103); the Basic Research and Frontier Exploration Grant of Chongqing Science and Technology Commission (Grant Nos. cstc2021jcyj-jqX0028, cstc2019jcyj-msxmX0366, cstc2019jcyjbshX0005); the “Associate Doctoral Supervisor” Cultivation Fund of the Stomatological Hospital of Chongqing Medical University (Grant No. KQFBD002); the Project of the Scientific and Technological Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201900441); and the Chongqing Graduate Tutor Team (Grant No. dstd201903). 本研究得到国家自然科学基金资助（批准号：82171010、81771082、31971282、82001103）；重庆市科学技术委员会基础研究与前沿探索资助（批准号：cstc2021jcyj-jqX0028、cstc2019jcyj-msxmX0366、cstc2019jcyjbshX0005cstc2021jcyj-jqX0028、cstc2019jcyj-msxmX0366、cstc2019jcyjbshX0005）；重庆医科大学附属口腔医院 "副博士生导师 "培养基金（批准号：KQFBD002）。KQFBD002）；重庆市教委科技攻关项目（批准号：KJQN201900441）；重庆市研究生导师团队（批准号：dstd201903）。
Appendix A. Supplementary data 附录 A.补充数据
Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioactmat.2022.02.008. 本文的补充数据可在线查阅：https://doi. org/10.1016/j.bioactmat.2022.02.008。
References 参考资料
[1] X. Sun, Z.J. Ma, X. Zhao, W.J. Jin, C.Y. Zhang, J. Ma, L. Qiang, W.H. Wang, Q. Deng, H. Yang, J.Z. Zhao, Q.Q. Liang, X.J. Zhou, L. Li, J. Wang, Threedimensional bioprinting of multicell-laden scaffolds containing bone morphogenic protein-4 for promoting M2 macrophage polarization and accelerating bone defect repair in diabetes mellitus, Bioact Mater (3) (2020 Sep 25 6) 757-769. [1] X. Sun，Z.J. Ma，X. Zhao，W.J. Jin，C.Y. Zhang，J. Ma，L. Qiang，W.H. Wang，Q. Deng，H. Yang，J.Z. Zhao，Q.Q. Liang，X.J. Zhou，L. Li，J. Wang，Threedimensional bioprinting of multicell-laden scaffolds containing bone morphogenic protein-4，for promoting M2 macrophage polarization and accelating bone defect repair in diabetesellitus，J.含骨形态发生蛋白-4的多细胞支架的三维生物打印促进 M2 巨噬细胞极化并加速糖尿病骨缺损修复，Bioact Mater (3) (2020 Sep 25 6) 757-769.
[2] Y. Zheng, S.H. Ley, F.B. Hu, Global aetiology and epidemiology of type 2 diabetes mellitus and its complications, Nat. Rev. Endocrinol. 14 (2) (2018) 88-98. [2] Y. Zheng、S.H. Ley、F.B. Hu，《2 型糖尿病及其并发症的全球病因学和流行病学》，Nat.Rev. Endocrinol.14 (2) (2018) 88-98.
[3] T. Kocher, J. Konig, W.S. Borgnakke, C. Pink, P. Meisel, Periodontal complications of hyperglycemia/diabetes mellitus: epidemiologic complexity and clinical challenge, Periodontol 78 (1) (2000) 59-97, 2018. [3] T. Kocher、J. Konig、W.S. Borgnakke、C. Pink、P. Meisel，《高血糖/糖尿病的牙周并发症：流行病学的复杂性和临床挑战》，Periodontol 78 (1) (2000) 59-97，2018.
[4] M. Zheng, C. Wang, A. Ali, Y.A. Shih, Q. Xie, C. Guo, Prevalence of periodontitis in people clinically diagnosed with diabetes mellitus: a meta-analysis of epidemiologic studies, Acta Diabetol. (2021). [4] M. Zheng, C. Wang, A. Ali, Y.A. Shih, Q. Xie, C. Guo, Prevalence of periodontitis in people clinically diagnosed with diabetes mellitus: a meta-analysis of epidemiologic studies, Acta Diabetol.(2021).
[5] D.T. Graves, Z. Ding, Y. Yang, The impact of diabetes on periodontal diseases, Periodontol 82 (1) (2000) 214-224, 2020. [5] D.T. Graves、Z. Ding、Y. Yang，《糖尿病对牙周病的影响》，Periodontol 82 (1) (2000) 214-224, 2020。
[6] D. Polak, L. Shapira, An update on the evidence for pathogenic mechanisms that may link periodontitis and diabetes, J. Clin. Periodontol. 45 (2) (2018) 150-166. [6] D. Polak, L. Shapira, An update on the evidence for pathogenic mechanisms that may link periodontitis and diabetes, J. Clin.Periodontol.45 (2) (2018) 150-166.
[7] A.I. Khumaedi, D. Purnamasari, I.P. Wijaya, Y. Soeroso, The relationship of diabetes, periodontitis and cardiovascular disease, Diabetes Metab Syndr 13 (2) (2019) 1675-1678. [7] A.I. Khumaedi，D. Purnamasari，I.P. Wijaya，Y. Soeroso，糖尿病、牙周炎与心血管疾病的关系，Diabetes Metab Syndr 13 (2) (2019) 1675-1678。
[8] M. Yamamoto, T. Sugimoto, Advanced glycation end products, diabetes, and bone strength, Curr. Osteoporos. Rep. 14 (6) (2016) 320-326. [8] M. Yamamoto, T. Sugimoto, Advanced glycation end products, diabetes, and bone strength, Curr.Osteoporos.14 (6) (2016) 320-326。
[9] K. Asadipooya, E.M. Uy, Advanced glycation end products (AGEs), receptor for AGEs, diabetes, and bone: review of the literature, J Endocr Soc 3 (10) (2019) 1799-1818. [9] K. Asadipooya, E.M. Uy, 高级糖化终产物（AGEs）、AGEs受体、糖尿病和骨骼：文献综述，J Endocr Soc 3 (10) (2019) 1799-1818。
[10] T.C. Simpson, J.C. Weldon, H.V. Worthington, I. Needleman, S.H. Wild, D.R. Moles, B. Stevenson, S. Furness, Z. Iheozor-Ejiofor, Treatment of periodontal disease for glycaemic control in people with diabetes mellitus, Cochrane Database Syst. Rev. 11 (2015) CD004714. [10] T.C. Simpson, J.C. Weldon, H.V. Worthington, I. Needleman, S.H. Wild, D.R. Moles, B. Stevenson, S. Furness, Z. Iheozor-Ejiofor, Treatment of periodontal disease for glycaemic control in people with diabetes mellitus, Cochrane Database Syst.Rev. 11 (2015) CD004714。
[11] Y. Fan, M. Marioli, K. Zhang, Analytical characterization of liposomes and other lipid nanoparticles for drug delivery, J. Pharm. Biomed. Anal. 192 (2021) 113642. [11] Y. Fan, M. Marioli, K. Zhang, Analytical characterization of liposomes and other lipid nanoparticles for drug delivery, J. Pharm. Biomeded.Biomed.Anal.192 (2021) 113642.
[12] L.M. Ickenstein, P. Garidel, Lipid-based nanoparticle formulations for small molecules and RNA drugs, Expet Opin. Drug Deliv. 16 (11) (2019) 1205-1226. [12] L.M. Ickenstein, P. Garidel, Lipid-based nanoparticle formulations for small molecules and RNA drugs, Expet Opin.Drug Deliv.16 (11) (2019) 1205-1226.
[13] Y. Liu, K.M. Castro Bravo, J. Liu, Targeted liposomal drug delivery: a nanoscience and biophysical perspective, Nanoscale Horiz 6 (2) (2021) 78-94.
[14] Z. Wan, R. Zheng, P. Moharil, Y. Liu, J. Chen, R. Sun, X. Song, Q. Ao, Polymeric micelles in cancer immunotherapy, Molecules 26 (5) (2021).
[15] W. Zhang, A. Mehta, Z. Tong, L. Esser, N.H. Voelcker, Development of polymeric nanoparticles for blood-brain barrier transfer-strategies and challenges, Adv. Sci. 8 (10) (2021) 2003937.
[16] I. Aksoy, H. Kucukkececi, F. Sevgi, O. Metin, I. Hatay Patir, Photothermal antibacterial and antibiofilm activity of black phosphorus/gold nanocomposites against pathogenic bacteria, ACS Appl. Mater. Interfaces 12 (24) (2020) 26822-26831. [16] I. Aksoy、H. Kucukkececi、F. Sevgi、O. Metin、I. Hatay Patir，黑磷/金纳米复合材料对病原菌的光热抗菌和抗生物膜活性，ACS Appl.Interfaces 12 (24) (2020) 26822-26831.
[17] Q. Zhao, J. Wang, C. Yin, P. Zhang, J. Zhang, M. Shi, K. Shen, Y. Xiao, Y. Zhao, X. Yang, Y. Zhang, Near-infrared light-sensitive nano neuro-immune blocker capsule relieves pain and enhances the innate immune response for necrotizing infection, Nano Lett. 19 (9) (2019) 5904-5914. [17] Q. Zhao, J. Wang, C. Yin, P. Zhang, J. Zhang, M. Shi, K. Shen, Y. Xiao, Y. Zhao, X. Yang, Y. Zhang, Near-infrared light-sensitive nano neuro-immune blocker capsule relieves pain and enhances the innate immune response for necrotizing infection, Nano Lett.19 (9) (2019) 5904-5914.
[18] S. Gomez-Grana, J. Perez-Juste, P. Herves, Cyclodextrins and inorganic nanoparticles: another tale of synergy, Adv. Colloid Interface Sci. 288 (2021) 102338. [18] S. Gomez-Grana、J. Perez-Juste、P. Herves，环糊精和无机纳米粒子：另一个协同作用的故事，Adv.288 (2021) 102338.
[19] X. Li, X. Liu, X. Liu, Self-assembly of colloidal inorganic nanocrystals: nanoscale forces, emergent properties and applications, Chem. Soc. Rev. 50 (3) (2021) 2074-2101. [19] X. Li, X. Liu, X. Liu, Self-assembly of colloidal inorganic nanocrystals: nanoscale forces, emergent properties and applications, Chem. Soc. Rev. 50 (3) (2021) 2074-2101.50 (3) (2021) 2074-2101.
[20] S. Vieira, S. Vial, R.L. Reis, J.M. Oliveira, Nanoparticles for bone tissue engineering, Biotechnol. Prog. 33 (3) (2017) 590-611. [20] S. Vieira、S. Vial、R.L. Reis、J.M. Oliveira，用于骨组织工程的纳米颗粒，Biotechnol.Prog.33 (3) (2017) 590-611.
[21] A. Bozorgi, M. Khazaei, M. Soleimani, Z. Jamalpoor, Application of nanoparticles in bone tissue engineering; a review on the molecular mechanisms driving osteogenesis, Biomater Sci 9 (13) (2021) 4541-4567. [21] A. Bozorgi、M. Khazaei、M. Soleimani、Z. Jamalpoor，《纳米颗粒在骨组织工程中的应用；骨生成的分子机制综述》，Biomater Sci 9 (13) (2021) 4541-4567。
[22] Y. Wang, Q. Yuan, W. Feng, W. Pu, J. Ding, H. Zhang, X. Li, B. Yang, Q. Dai, L. Cheng, J. Wang, F. Sun, D. Zhang, Targeted delivery of antibiotics to the infected pulmonary tissues using ROS-responsive nanoparticles, J. Nanobiotechnol. 17 (1) (2019) 103. [22] Y. Wang, Q. Yuan, W. Feng, W. Pu, J. Ding, H. Zhang, X. Li, B. Yang, Q. Dai, L. Cheng, J. Wang, F. Sun, D. Zhang, Targeted delivery of antibiotics to the infected pulmonary tissues using ROS-responsive nanoparticles, J. Nanobiotechnol.17 (1) (2019) 103.
[23] N. Jia, W. Li, D. Liu, S. Wu, B. Song, J. Ma, D. Chen, H. Hu, Tumor microenvironment stimuli-responsive nanoparticles for programmed anticancer drug delivery, Mol. Pharm. 17 (5) (2020) 1516-1526. [23] N. Jia，W. Li，D. Liu，S. Wu，B. Song，J. Ma，D. Chen，H. Hu，Tumor microenvironment stimuli-responsive nanoparticles for programmed anticancer drug delivery，Mol.Pharm.17 (5) (2020) 1516-1526.
[24] U. Kauscher, M.N. Holme, M. Bjornmalm, M.M. Stevens, Physical stimuliresponsive vesicles in drug delivery: beyond liposomes and polymersomes, Adv. Drug Deliv. Rev. 138 (2019) 259-275. [24] U. Kauscher, M.N. Holme, M. Bjornmalm, M.M. Stevens, Physical stimuliresponsive vesicles in drug delivery: beyond liposomes and polymersomes, Adv.Drug Deliv.Rev. 138 (2019) 259-275.
[25] X. Mu, S. Gan, Y. Wang, H. Li, G. Zhou, Stimulus-responsive vesicular polymer nano-integrators for drug and gene delivery, Int. J. Nanomed. 14 (2019) 5415-5434. [25] X. Mu, S. Gan, Y. Wang, H. Li, G. Zhou, Stimulus-responsive vesicular polymer nano-integrators for drug and gene delivery, Int. J. Nanomed.J. Nanomed.14 (2019) 5415-5434.
[26] P.M. Preshaw, S.M. Bissett, Periodontitis and diabetes, Br. Dent. J. 227 (7) (2019) 577-584. [26] P.M. Preshaw, S.M. Bissett, Periodontitis and diabetes, Br. Dent.J. 227 (7) (2019) 577-584.
[27] A. Bascones-Martínez, M. Muñoz-Corcuera, J. Bascones-Ilundain, Diabetes and periodontitis: a bidirectional relationship, Med. Clínica 145 (1) (2015) 31-35. [27] A. Bascones-Martínez，M. Muñoz-Corcuera，J. Bascones-Ilundain，《糖尿病与牙周炎：双向关系》，Med.Clínica 145 (1) (2015) 31-35。
[28] F. Hu, Z. Zhou, Q. Xu, C. Fan, L. Wang, H. Ren, S. Xu, Q. Ji, X. Chen, A novel pHresponsive quaternary ammonium chitosan-liposome nanoparticles for periodontal treatment, Int. J. Biol. Macromol. 129 (2019) 1113-1119. [28] F. Hu, Z. Zhou, Q. Xu, C. Fan, L. Wang, H. Ren, S. Xu, Q. Ji, X. Chen, A novel pHresponsive quaternary ammonium chitosan-liposome nanoparticles for periodontal treatment, Int. J. Biol.J. Biol.Macromol.129 (2019) 1113-1119.
[29] C.Y. Zhang, J. Gao, Z. Wang, Bioresponsive nanoparticles targeted to infectious microenvironments for sepsis management, Adv. Mater. 30 (43) (2018), e1803618. [29] C.Y. Zhang, J. Gao, Z. Wang, Bioresponsive nanoparticles targeted to infectious microenenvironments for sepsis management, Adv. Mater.30 (43) (2018), e1803618.
[30] B. Horev, M.I. Klein, G. Hwang, Y. Li, D. Kim, H. Koo, D.S. Benoit, pH-activated nanoparticles for controlled topical delivery of farnesol to disrupt oral biofilm virulence, ACS Nano 9 (3) (2015) 2390-2404. [30] B. Horev、M.I. Klein、G. Hwang、Y. Li、D. Kim、H. Koo、D.S. Benoit，pH-活化纳米颗粒用于法尼醇的受控局部递送以破坏口腔生物膜毒力，ACS Nano 9 (3) (2015) 2390-2404。
[31] Y. Gao, J. Wang, M. Chai, X. Li, Y. Deng, Q. Jin, J. Ji, Size and charge adaptive clustered nanoparticles targeting the biofilm microenvironment for chronic lung infection management, ACS Nano 14 (5) (2020) 5686-5699.
[32] Y. Liu, V.S. Bryantsev, M.S. Diallo, W.A. Goddard, PAMAM dendrimers undergo pH responsive conformational changes without swelling, J. Am. Chem. Soc. 131 (8) (2009) 2798-2799. [32] Y. Liu、V.S. Bryantsev、M.S. Diallo、W.A. Goddard，PAMAM 树枝形分子发生 pH 值响应的构象变化而不膨胀，J. Am.Chem.131 (8) (2009) 2798-2799.
[33] L. Han, R.Q. Huang, S.H. Liu, S.X. Huang, C. Jiang, Peptide-conjugated PAMAM for targeted doxorubicin delivery to transferrin receptor overexpressed tumors, Mol. Pharm. 7 (2010) 2156-2165. [33] L. Han, R.Q. Huang, S.H. Liu, S.X. Huang, C. Jiang, Peptide-conjugated PAMAM for targeted doxorubicin delivery to transferrin receptor overexpressed tumors, Mol.Pharm.7 (2010) 2156-2165.
[34] G. Jiang, S. Liu, T. Yu, R. Wu, Y. Ren, H.C. van der Mei, J. Liu, H.J. Busscher, PAMAM dendrimers with dual-conjugated vancomycin and Ag-nanoparticles do not induce bacterial resistance and kill vancomycin-resistant Staphylococci, Acta Biomater. 123 (2021) 230-243. [34] G. Jiang、S. Liu、T. Yu、R. Wu、Y. Ren、H.C. van der Mei、J. Liu、H.J. Busscher，PAMAM 树枝形分子与万古霉素和 Ag-nanoparticles 双共轭不会诱导细菌耐药性，并能杀死耐万古霉素的葡萄球菌，Acta Biomater.123 (2021) 230-243.
[35] Y. Wang, Y. Luo, Q. Zhao, Z. Wang, Z. Xu, X. Jia, An enzyme-responsive nanogel carrier based on PAMAM dendrimers for drug delivery, ACS Appl. Mater. Interfaces 8 (31) (2016) 19899-19906. [35] Y. Wang, Y. Luo, Q. Zhao, Z. Wang, Z. Xu, X. Jia, An enzyme-responsive nanogel carrier based on PAMAM dendrimers for drug delivery, ACS Appl.Interfaces 8 (31) (2016) 19899-19906.
[36] Y. Zhang, S. Ma, X. Liu, Y. Xu, J. Zhao, X. Si, H. Li, Z. Huang, Z. Wang, Z. Tang, W. Song, X. Chen, Supramolecular assembled programmable nanomedicine as in [36] Y. Zhang，S. Ma，X. Liu，Y. Xu，J. Zhao，X. Si，H. Li，Z. Huang，Z. Wang，Z. Tang，W. Song，X. Chen，Supramolecular assembled programmable nanomedicine as in
situ cancer vaccine for cancer immunotherapy, Adv. Mater. 33 (7) (2021), e2007293. 用于癌症免疫疗法的原位癌疫苗，Adv. Mater.33 (7) (2021), e2007293.
[37] A.S. Chauhan, Dendrimers for drug delivery, Molecules 23 (4) (2018). [37] A.S. Chauhan，Dendrimers for drug delivery，Molecules 23 (4) (2018)。
[38] H. Kheraldine, O. Rachid, A.M. Habib, A.E. Al Moustafa, I.F. Benter, S. Akhtar, Emerging innate biological properties of nano-drug delivery systems: a focus on PAMAM dendrimers and their clinical potential, Adv. Drug Deliv. Rev. 178 (2021) 113908. [38] H. Kheraldine, O. Rachid, A.M. Habib, A.E. Al Moustafa, I.F. Benter, S. Akhtar, Emerging innate biological properties of nano-drug delivery systems: a focus on PAMAM dendrimers and their clinical potential, Adv.Drug Deliv.Rev. 178 (2021) 113908.
[39] L. Aranda-Lara, B.E.O. Garcia, K. Isaac-Olive, G. Ferro-Flores, L. Melendez-Alafort, E. Morales-Avila, Drug delivery systems-based dendrimers and polymer micelles for nuclear diagnosis and therapy, Macromol. Biosci. 21 (3) (2021), e2000362. [39] L. Aranda-Lara、B.E.O. Garcia、K. Isaac-Olive、G. Ferro-Flores、L. Melendez-Alafort、E. Morales-Avila，基于树枝状分子和聚合物胶束的核诊断和治疗药物输送系统，Macromol.Biosci.21 (3) (2021), e2000362.
[40] Z. Zeng, D. Qi, L. Yang, J. Liu, Y. Tang, H. Chen, X. Feng, Stimuli-responsive selfassembled dendrimers for oral protein delivery, J. Contr. Release 315 (2019) 206-213. [40] Z. Zeng, D. Qi, L. Yang, J. Liu, Y. Tang, H. Chen, X. Feng, Stimuli-responsive selfassembled dendrimers for oral protein delivery, J. Contr.Release 315 (2019) 206-213.
[41] D. Huang, D. Wu, Biodegradable dendrimers for drug delivery, Mater Sci Eng C Mater Biol Appl 90 (2018) 713-727.
[42] M. Chen, S. Xie, J. Wei, X. Song, Z. Ding, X. Li, Antibacterial micelles with vancomycin-mediated targeting and pH//\mathrm{pH} / lipase-triggered release of antibiotics, ACS Appl. Mater. Interfaces 10 (43) (2018) 36814-36823. [42] M. Chen, S. Xie, J. Wei, X. Song, Z. Ding, X. Li, Antibacterial micelles with Vancomycin-mediated targeting and pH//\mathrm{pH} / lipase-triggered release of antibiotics, ACS Appl.Interfaces 10 (43) (2018) 36814-36823.
[43] V.M. Barnes, S.G. Ciancio, O. Shibly, T. Xu, W. Devizio, H.M. Trivedi, L. Guo, T. J. Jonsson, Metabolomics reveals elevated macromolecular degradation in periodontal disease, J. Dent. Res. 90 (11) (2011) 1293-1297. [43] V.M. Barnes, S.G. Ciancio, O. Shibly, T. Xu, W. Devizio, H.M. Trivedi, L. Guo, T. J. Jonsson, Metabolomics reveals elevated macromolecular degradation in periodontal disease, J. Dent. Res.90 (11) (2011) 1293-1297。
[44] J. Che, C.I. Okeke, Z.B. Hu, J. Xu, DSPE-PEG: a distinctive component in drug delivery system, Curr. Pharmaceut. Des. 21 (12) (2015) 1598-1605. [44] J. Che、C.I. Okeke、Z.B. Hu、J. Xu，DSPE-PEG：给药系统中的一种独特成分，Curr.Pharmaceut.Des.21 (12) (2015) 1598-1605.
[45] A.T. Wang, D.S. Liang, Y.J. Liu, X.R. Qi, Roles of ligand and TPGS of micelles in regulating internalization, penetration and accumulation against sensitive or resistant tumor and therapy for multidrug resistant tumors, Biomaterials 53 (2015) 160-172.
[46] B. Salehi, Y. Berkay Yilmaz, G. Antika, T. Boyunegmez Tumer, M. Fawzi Mahomoodally, D. Lobine, M. Akram, M. Riaz, E. Capanoglu, F. Sharopov, N. Martins, W.C. Cho, J. Sharifi-Rad, Insights on the use of alpha-lipoic acid for therapeutic purposes, Biomolecules 9 (8) (2019).
[47] R. Jagannathan, J.S. Neves, B. Dorcely, S.T. Chung, K. Tamura, M. Rhee, M. Bergman, The oral glucose tolerance test: 100 Years later, Diabetes Metab Syndr Obes 13 (2020) 3787-3805. [47] R. Jagannathan、J.S. Neves、B. Dorcely、S.T. Chung、K. Tamura、M. Rhee、M. Bergman，口服葡萄糖耐量试验：100 年后，糖尿病代谢综合征肥胖症 13 (2020) 3787-3805。
[48] T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, Q. Cheng, S. Huo, Z. Liang, M. Fermeglia, S. Pricl, X.J. Liang, P. Rocchi, L. Peng, Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance, Proc. Natl. Acad. Sci. U. S. A. 112 (10) (2015) 2978-2983. [48] T. Wei, C. Chen, J. Liu, C. Liu, P. Posocco, X. Liu, Q. Cheng, S. Huo, Z. Liang, M. Fermeglia, S. Pricl, X.J. Liang, P. Rocchi, L. Peng, Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance, Proc.Natl.Sci. U. S. A. 112 (10) (2015) 2978-2983.
[49] H. Van Acker, T. Coenye, The role of reactive oxygen species in antibiotic-mediated killing of bacteria, Trends Microbiol. 25 (6) (2017) 456-466. [49] H. Van Acker, T. Coenye, The role of reactive oxygen species in antibiotic-mediated killing of bacteria, Trends Microbiol.25 (6) (2017) 456-466.
[50] F. Vatansever, W.C. de Melo, P. Avci, D. Vecchio, M. Sadasivam, A. Gupta, R. Chandran, M. Karimi, N.A. Parizotto, R. Yin, G.P. Tegos, M.R. Hamblin, Antimicrobial strategies centered around reactive oxygen species-bactericidal antibiotics, photodynamic therapy, and beyond, FEMS Microbiol. Rev. 37 (6) (2013) 955-989. [50] F. Vatansever, W.C. de Melo, P. Avci, D. Vecchio, M. Sadasivam, A. Gupta, R. Chandran, M. Karimi, N.A. Parizotto, R. Yin, G.P. Tegos, M.R. Hamblin, Antimicrobial strategies centered around reactive oxygen species-bactericidal antibiotics, photodynamic therapy, and beyond, FEMS Microbiol.Rev. 37 (6) (2013) 955-989。
[51] P. Ma, H. Xiao, C. Yu, J. Liu, Z. Cheng, H. Song, X. Zhang, C. Li, J. Wang, Z. Gu, J. Lin, Enhanced cisplatin chemotherapy by iron oxide nanocarrier-mediated generation of highly toxic reactive oxygen species, Nano Lett. 17 (2) (2017) 928-937. [51] P. Ma, H. Xiao, C. Yu, J. Liu, Z. Cheng, H. Song, X. Zhang, C. Li, J. Wang, Z. Gu, J. Lin, Enhanced cisplatin chemotherapy by iron oxide nanocarrier-mediated generation of highly toxic reactive oxygen species, Nano Lett.17 (2) (2017) 928-937.
[52] M. Freitas, J.L. Lima, E. Fernandes, Optical probes for detection and quantification of neutrophils’ oxidative burst, A review, Anal Chim Acta 649 (1) (2009) 8-23. [52] M. Freitas, J.L. Lima, E. Fernandes, Optical probes for detection and quantification of neutrophils' oxidative burst, A review, Anal Chim Acta 649 (1) (2009) 8-23.
[53] Y. Chen, W.G. Junger, Measurement of oxidative burst in neutrophils, Methods Mol. Biol. 844 (2012) 115-124. [53] Y. Chen, W.G. Junger, Measurement of oxidative burst in neutrophils, Methods Mol.844 (2012) 115-124.
[54] D.C. Thomas, The phagocyte respiratory burst: historical perspectives and recent advances, Immunol. Lett. 192 (2017) 88-96. [54] D.C. Thomas, The phagocyte respiratory burst: historical perspectives and recent advances, Immunol.Lett.192 (2017) 88-96.
[55] L. Sui, J. Wang, Z. Xiao, Y. Yang, Z. Yang, K. Ai, ROS-scavenging nanomaterials to treat periodontitis, Front. Chem. 8 (2020) 595530. [55] L. Sui，J. Wang，Z. Xiao，Y. Yang，Z. Yang，K. Ai，ROS 清除纳米材料治疗牙周炎，Front.Chem.8 (2020) 595530.
[56] W. Liu, L.J. Shi, S.G. Li, The immunomodulatory effect of alpha-lipoic acid in autoimmune diseases, BioMed Res. Int. 2019 (2019) 8086257. [56] W. Liu, L.J. Shi, S.G. Li, α-硫辛酸在自身免疫性疾病中的免疫调节作用，BioMed Res. Int. 2019 (2019) 8086257.
[57] V.M. Mendoza-Nunez, B.I. Garcia-Martinez, J. Rosado-Perez, E. Santiago-Osorio, J. Pedraza-Chaverri, V.J. Hernandez-Abad, The effect of 600 mg alpha-lipoic acid supplementation on oxidative stress, inflammation, and RAGE in older adults with type 2 diabetes mellitus, Oxid. Med. Cell. Longev. (2019) 3276958, 2019. [57] V.M. Mendoza-Nunez、B.I. Garcia-Martinez、J. Rosado-Perez、E. Santiago-Osorio、J. Pedraza-Chaverri、V.J. Hernandez-Abad，《补充 600 毫克α-硫辛酸对 2 型糖尿病老年人氧化应激、炎症和 RAGE 的影响》，Oxid.Med.Cell.Longev.(2019) 3276958, 2019.
[58] U. Aksoy, G. Savtekin, A.O. Sehirli, F. Kermeoglu, A. Kalender, H. Ozkayalar, S. Sayiner, K. Orhan, Effects of alpha-lipoic acid therapy on experimentally induced apical periodontitis: a biochemical, histopathological and micro-CT analysis, Int. Endod. J. 52 (9) (2019) 1317-1326. [58] U. Aksoy, G. Savtekin, A.O. Sehirli, F. Kermeoglu, A. Kalender, H. Ozkayalar, S. Sayiner, K. Orhan, Effects of alpha-lipoic acid therapy on experimentally induced apical periodontitis: a biochemical, histopathological and micro-CT analysis, Int. Endod.J. 52 (9) (2019)J. 52 (9) (2019) 1317-1326.
Peer review under responsibility of KeAi Communications Co., Ltd. 同行评审由 KeAi Communications Co., Ltd. 负责。
Corresponding author. College of Stomatology, Chongqing Medical University, Chongqing, China. 通讯作者：重庆医科大学口腔医学院重庆医科大学口腔医学院
** Corresponding author. College of Stomatology, Chongqing Medical University, Chongqing, China. ** 通讯作者。重庆医科大学口腔医学院。
E-mail addresses: ysdentist@hospital.cqmu.edu.cn (S. Yang), songjinlin@hospital.cqmu.edu.cn (J. Song). 电子邮件地址：ysdentist@hospital.cqmu.edu.cn (S. Yang), songjinlin@hospital.cqmu.edu.cn (J. Song)。
