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Linyuan Gui,1,* Qingyuan Ye,2,* Lu Yu,3 Geng Dou,1 Yang Zhou,1 Yang Liu,4 Yanqi Zhang,5 Xiaoshan Yang,1,6 Fang Jin,5 Shiyu Liu,1 Yan Jin,1 Lili Ren1 1State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China; 2State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Key Laboratory of Stomatology, Digital Dentistry Center, School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China; 3Department of Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, Shandong, 250012, People’s Republic of China; 4Department of Anesthesiology and Perioperative Medicine, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China; 5State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Clinical Research Center for Oral Diseases, Department of Orthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China; 6Stomatological Hospital, School of Stomatology, Southern Medical University, Guangzhou, Guangdong, 510280, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lili Ren; Yan Jin, State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, The Fourth Military Medical University, Xi’an, Shaanxi, 710032, People’s Republic of China, Email lanyangddgy@qq.com; yanjin@fmmu.edu.cnBackground: Osteoporosis is a highly prevalent disease that causes fractures and loss of motor function. Current drugs targeted for osteoporosis often have inevitable side effects. Bone marrow mesenchymal stem cell (BMSCs)-derived apoptotic extracellular vesicles (ApoEVs) are nanoscale extracellular vesicles, which has been shown to promote bone regeneration with low immunogenicity and high biological compatibility. However, natural ApoEVs cannot inherently target bones, and are often eliminated by macrophages in the liver and spleen. Thus, our study aimed to reconstruct ApoEVs to enhance their bone-targeting capabilities and bone-promoting function and to provide a new method for osteoporosis treatment.Methods: We conjugated a bone-targeting peptide, (Asp-Ser-Ser)6 ((DSS)6), onto the surface of ApoEVs using standard carbodiimide chemistry with DSPE-PEG-COOH serving as the linker. The bone-targeting ability of (DSS)6-ApoEVs was determined using an in vivo imaging system and confocal laser scanning microscopy (CLSM). We then loaded ubiquitin ligase RING finger protein146 (RNF146) into BMSCs via adenovirus transduction to obtain functional ApoEVs. The bone-promoting abilities of (DSS)6-ApoEVs and (DSS)6-ApoEVsRNF146 were measured in vitro and in vivo.Results: Our study successfully synthesized bone-targeting and gained functional (DSS)6-ApoEVsRNF146 and found that engineered ApoEVs could promote osteogenesis in vitro and exert significant bone-targeting and osteogenesis-promoting effects to alleviate osteoporosis in a mouse model.Conclusion: To promote the bone-targeting ability of natural ApoEVs, we successfully synthesized engineered ApoEVs, (DSS)6-ApoEVsRNF146 and found that they could significantly promote osteogenesis and alleviate osteoporosis compared with natural ApoEVs, which holds great promise for the treatment of osteoporosis. Keywords: osteoporosis, mesenchymal stem cells, apoptotic extracellular vesicles, bone-targeting, bone regeneration

Jie Li,1 Jiangtao Long,1 Zheng Zhao,2 Qianqian Wang,1 Wang Bo,2 Liang Ren,1 Yan Fan,3 Peng Wang,1 Yi Cheng,1 Binbin Liu,1 Xinkui Cheng,1 Hongwei Xi2 1Department of Orthopedics, Children’s Hospital Affiliated to Shanxi Medical University, Taiyuan, People’s Republic of China; 2Department of General Surgery, Children’s Hospital Affiliated to Shanxi Medical University, Taiyuan, People’s Republic of China; 3Department of Burn and Plastic Surgery, Children’s Hospital affiliated to Shanxi Medical University, Taiyuan, People’s Republic of ChinaCorrespondence: Hongwei Xi, Email xihongwei148@sina.comBackground: Multiple stages including hemostasis, inflammation, proliferation, and remodeling were involved in the wound healing process. The increase in nanomaterials in recent years has extended the scope of tools for wound healing; however, it is still difficult to achieve the four multistage procedures simultaneously.Materials and Methods: In this study, graphene-spiky silica heterostructured nanoparticles (GS) were synthesized for the procedural acceleration of the multistage in wound healing process. The nanobridge effect of GS was analyzed through the adhesion of two skins, the antibacterial effect was assessed in Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria, cell proliferation and migration were investigated in mouse embryonic fibroblast (NIH-3T3) cells, and the in vivo wound healing effect was examined in female BALB/c mice with a cutting wound and E. coli or S. aureus bacteria infection on the back.Results: First, GS has a strong nanobridge effect on the rapid closure of wounds because the spiky architecture on the surface of GS facilitates the adhesion of skins, promoting the hemostasis stage. Second, graphene exhibits antimicrobial activities both in chemical and physical interactions, especially under simulated sunlight irradiation. Third, graphene plays an important role in scaffolding function, together with the spiky topographical architecture of GS, accelerating the proliferation and maturation stages.Conclusion: By periodically promoting every stage of wound healing, GS combined with simulated sunlight irradiation could significantly accelerate wound healing. With a simple composition and compact structure but multiple functions, this strategy will be the guideline for the development of ideal wound-healing nanomaterials.Keywords: graphene-spiky silica heterostructured nanoparticles, wound healing, multistage

Yaning Li,1 Junfang Nie,1 Jie Dai,1 Jun Yin,1 Binbin Huang,1 Jia Liu,1 Guoguang Chen,1 Lili Ren1,2 1School of Pharmacy, Nanjing Tech University, Nanjing, People’s Republic of China; 2Department of Microbiology and Immunology, Stanford University, Stanford, CA, USACorrespondence: Guoguang Chen; Lili Ren, School of Pharmacy, Nanjing Tech University, Nanjing, People’s Republic of China, Tel +86-2558-139-416, Email ggchen@njtech.edu.cn; renlili@njtech.edu.cnPurpose: Nanotechnology has been widely used in antitumor research. The complex physiological environment has brought significant challenges to the field of antitumor micelles. The ideal micelles must not only have an invisible surface to extend the circulation time but must also enhance the retention of drugs and cellular internalization at the tumor.Methods: A graded response micelle (RPPssD@IR780/DOC) was designed to self-assemble by cRGD-poly(β-amino esters)-polyethylene glycol-ss-distearoyl phosphatidylethanolamine (cRGD-PBAE-PEG-ss-DSPE) loaded with docetaxel (DOC) and IR-780 iodide (IR780). The micelles were designed to allow the PEG shell to prolong the blood circulation time in the body and effectively accumulate in the tumor. Subsequently, the acidic microenvironment of the tumor could transform the PBAE to hydrophilic, thereby increasing the size of micelles and exposing cyclic Arg-Gly-Asp (cRGD) peptides to increase the retention and cellular internalization of micelles in the tumor. After tumor cells had captured micelles, the high expression of glutathione in the cells prompted the release of DOC and IR780. Subsequently, the IR780 was stimulated by an 808-nm laser to generate local heat and reactive oxygen species (ROS) to synergize with DOC to treat the tumor.Results: In vitro and in vivo experimental results suggested that RPPssD@IR780/DOC was a potential photochemical effective for the treatment of tumors with non-negligible antitumor activity and good biocompatibility.Conclusion: A dual-response pH/redox delivery system with on-demand RGD exposure was designed to achieve photochemotherapy of tumors with good biosafety and antitumor effects.Graphical Abstract: Keywords: micelles, drug delivery, ligand hidden, size variable, photo-chemotherapy

Guanglin Li,1,2 Chenxu Shang,3 Qingqing Li,4 Lifang Chen,1,2 Zejun Yue,1,2 Lingxuan Ren,3 Jianjun Yang,3 Jiye Zhang,4 Weirong Wang1,2 1Department of Medical Laboratory Animal Science, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, People’s Republic of China; 2Institute of Cardiovascular Science, Translational Medicine Institute, Xi’an Jiaotong University Health Science Center, Xi’an, People’s Republic of China; 3Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, People’s Republic of China; 4School of Pharmacy, Xi’an Jiaotong University Health Science Center, Xi’an, People’s Republic of ChinaCorrespondence: Weirong Wang, Xi’an Jiaotong University Health Science Center, No. 76 Yanta West Road, Xi’an, 710061, People’s Republic of China, Tel/Fax +86 29 82655362, Email szb2013072@xjtu.edu.cnIntroduction: Shikonin is well known for its anti-inflammatory activity in cardiovascular diseases. However, the application of shikonin is limited by its low water solubility and poor bioavailability. Methoxy poly (ethylene glycol)-b-poly (ϵ-caprolactone) (MPEG-PCL) is considered a promising delivery system for hydrophobic drugs. Therefore, in this study, we prepared shikonin-loaded MPEG-PCL micelles and investigated their effect on endothelial-to-mesenchymal transition (EndMT) induced by inflammatory cytokines.Methods: Shikonin was encapsulated in MPEG-PCL micelles using an anti-solvent method and the physiochemical characteristics of the micelles (particle size, zeta potential, morphology, critical micelle concentration (CMC), drug loading and encapsulation efficiency) were investigated. Cellular uptake of micelles in human umbilical vein endothelial cells (HUVECs) was evaluated using fluorescence microscopy. In vitro EndMT inhibition was explored in HUVECs by quantitative real-time PCR analysis.Results: The average particle size of shikonin-loaded MPEG-PCL micelles was 54.57± 0.13 nm and 60 nm determined by dynamic light scattering and transmission electron microscopy, respectively. The zeta potential was − 6.23± 0.02 mV. The CMC of the micelles was 6.31× 10− 7mol/L. The drug loading and encapsulation efficiency were 0.88± 0.08% and 43.08± 3.77%, respectively. The MPEG-PCL micelles significantly improved the cellular uptake of cargo with low water solubility. Real-time PCR analysis showed that co-treatment with TNF-α and IL-1β successfully induced EndMT in HUVECs, whereas this process was significantly inhibited by shikonin and shikonin-loaded MPEG-PCL micelles, with greater inhibition mediated by the shikonin-loaded MPEG-PCL micelles.Conclusion: Shikonin-loaded MPEG-PCL micelles significantly improved the EndMT-inhibiting effect of the free shikonin. MPEG-PCL is suitable for use more generally as a lipophilic drug carrier.Keywords: shikonin, shikonin-loaded MPEG-PCL micelles, inflammation, EndMT

Rui-Qin Yang,1– 3 Min Chen,3,4 Qiang Zhang,5 Yi-Yang Gao,1– 3 Kang-Liang Lou,1– 3 Tong-Tong Lin,5 Wen-He Huang,1,2 Yun-Zhu Zeng,6 Yong-Qu Zhang,1– 3 Yong-Ying Dang,1– 3 Lei Ren,5,* Guo-Jun Zhang1,2,4,* 1Cancer Center & Department of Breast and Thyroid Surgery, Xiang’an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian, People’s Republic of China; 2Key Laboratory for Endocrine-Related Cancer Precision Medicine of Xiamen, Xiang’an Hospital of Xiamen University, Xiamen, Fujian, People’s Republic of China; 3Clinical Central Research Core, Xiang’an Hospital of Xiamen University, Xiamen, Fujian, People’s Republic of China; 4Cancer Research Center, School of Medicine, Xiamen University, Xiamen, Fujian, People’s Republic of China; 5State Key Lab of Physical Chemistry of Solid Surface, College of Materials, Xiamen University, Xiamen, Fujian, People’s Republic of China; 6The Pathology department, Cancer Hospital, Shantou University Medical College, Shantou, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Guo-Jun Zhang, Tel +86-592-2889988, Fax +86-592-2889202, Email gjzhang@xah.xmu.edu.cnPurpose: Tumor-free surgical margin is crucial but challenging in breast-conserving surgery (BCS). Fluorescence imaging is a promising strategy for surgical navigation that can reliably assist the surgeon with visualization Of the tumor in real-time. Notably, finding an optimized fluorescent probe has been a challenging research topic. Herein, we developed a novel near-infrared (NIR) fluorescent probe based on tailored Hepatitis B Core virus-like protein (HBc VLP) and presented the preclinical imaging-guided surgery.Methods: The RGD-HBc160 VLP was synthesized by genetic engineering followed encapsulation of ICG via disassembly-reassembly. The applicability of the probe was tested for cell and tissue binding capacities through cell-based plate assays, xenograft mice model, and MMTV-PyVT mammary tumor transgenic mice. Subsequently, the efficacy of RGD-HBc160/ICG-guided surgery was evaluated in an infiltrative tumor-bearing mouse model. The protein-induced body’s immune response was further assessed.Results: The prepared RGD-HBc160/ICG showed outstanding integrin αvβ3 targeting ability in vitro and in vivo. After intravenous administration of probe, the fluorescence guidance facilitated more complete tumor resection and improved overall survival Of the infiltrative tumor-bearing mice. The probe also showed the excellent capability to differentiate between benign and malignant breast tissues in the mammary tumor transgenic mice. Interestingly, the ingenious tailoring of HBc VLP could not only endow its tumor-targeting ability towards integrin αvβ3 but also significantly reduce the humoral and cellular immune response.Conclusion: The RGD-HBc160/ICG holds promise as an effective tool to delineate tumor margin. These results have translational potential to achieve margin-negative resection and improve the stratification of patients for a potentially curative.Graphical Abstract: Keywords: breast Cancer, tumor margin, NIR fluorescence imaging, imaging-guided surgery, Hepatitis B core virus-like particles

Qinghui Zhao1 12 23 3,* Hongming Tang1 12 23 3,* Lishu Ren4 4, Jie Wei4 4 1School of Life Sciences and Technology, Tongji University, Shanghai 200092, People’s Republic of China; 2Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200123, People’s Republic of China; 3Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai 200123, People’s Republic of China; 4Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qinghui Zhao Tel +86-21-38804518Email qinghui_zhao@126.comPurpose: A bioactive and degradable scaffold of ternary composite with good biocompatibility and osteogenesis was developed for bone tissue repair.Materials and Methods: Polybutylene succinate (PS:50 wt%), magnesium phosphate (MP:40 wt%) and wheat protein (WP:10 wt%) composite (PMWC) scaffold was fabricated, and the biological performances of PMWC were evaluated both in vitro and vivo in this study.Results: PMWC scaffold possessed not only interconnected macropores (400 μm to 600 μm) but also micropores (10 μm ∼ 20 μm) on the walls of macropores. Incorporation of MP into composite improved the apatite mineralization (bioactivity) of PMWC scaffold in simulated body fluid (SBF), and addition of WP into composite further enhanced the degradability of PMWC in PBS compared with the scaffold of PS (50 wt%)/MP (50 wt%) composite (PMC) and PS alone. In addition, the PMWC scaffold containing MP and WP significantly promoted the proliferation and differentiation of mouse pre-osteoblastic cell line (MC3T3-E1) cells. Moreover, the images from synchrotron radiation microcomputed tomography (SRmCT) and histological sections of the in vivo implantation suggested that the PMWC scaffold containing MP and WP prominently improved the new bone formation and ingrowth compared with PMC and PS. Furthermore, the immunohistochemical analysis further confirmed that the PMWC scaffold obviously promoted osteogenesis and vascularization in vivo compared with PMC and PS.Conclusion: This study demonstrated that the biocompatible PMWC scaffold with improved bioactivity and degradability significantly promoted the osteogenesis and vascularization in vivo, which would have a great potential to be applied for bone tissue repair.Keywords: polybutylene succinate, composite scaffold, cytocompatibility, osteogenesis, vascularization

Yunfei Niu,1 Lieping Guo,2 Fangyong Hu,3 Lishu Ren,4 Qirong Zhou,1 Jiangying Ru,3 Jie Wei4 1Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, People’s Republic of China; 2Department of Oncology, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai 200438, People’s Republic of China; 3Department of Orthopaedics, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou 225009, People’s Republic of China; 4Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of ChinaCorrespondence: Yunfei NiuDepartment of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, People’s Republic of ChinaEmail nyunfei1219@163.comJiangying RuDepartment of Orthopaedics, The Affiliated Hospital of Yangzhou University, Yangzhou University, Yangzhou 225009, People’s Republic of ChinaTel +86-21-311 61690Fax +86-21-31161016Email rujiangying@163.comPurpose: To improve the surface bio-properties of polyetheretherketone (PEEK)/nano magnesium silicate (n-MS) composite (PC).Materials and Methods: The surface of PC was firstly treated by particle impact (PCP) and subsequently modified by concentrated sulfuric acid (PCPS).Results: PCPS surface exhibited not only macropores with sizes of about 150 μm (fabricated by particle impact) but also micropores with sizes of about 2 μm (created by sulfonation of PEEK) on the macroporous walls, and sulfonic acid (-SO3H) groups were introduced on PCPS surface. In addition, many n-MS nanoparticles were exposed on the microporous walls, which formed micro-nano structures. Moreover, the surface roughness and hydrophilicity of PCPS were obviously enhanced as compared with PC and PCP. Moreover, the apatite mineralization of PCPS in simulated body fluid (SBF) was obviously improved as compared with PC. Furthermore, compared with PC and PCP, PCPS exhibited antibacterial performances due to the presence of -SO3H groups. In addition, the responses (eg, adhesion and proliferation as well as differentiation) of bone marrow mesenchymal stem cell of rat to PCPS were significantly promoted as compared with PC and PCP.Conclusion: PCPS with macro-microporous surface containing -SO3H groups and micro-nano structures exhibited antibacterial activity and induced cell responses, which might possess large potential for bone substitute and repair.Keywords: PEEK based composite, sulfonation, macro-microporous surface, antibacterial performances, cell responses

Yamin Yang,1 Yue Hu,2 Henry Du,3 Lei Ren,4 Hongjun Wang5 1Department of Biomedical Engineering, Nanjing University of Aeronautics and Astronautics, Nanjing, Jiangsu, China; 2Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA; 3Department of Chemical Engineering and Materials Sciences, Stevens Institute of Technology, Hoboken, NJ, USA; 4Department of Biomaterials, College of Materials, Xiamen University, Xiamen, Fujian, China; 5Department of Biomedical Engineering, Chemistry and Biological Sciences, Stevens Institute of Technology, Hoboken, NJ, USA Introduction: In recognition of the potentials of gold nanoparticles (Au NPs) in enhanced photodynamic therapy (PDT) for cancer, it is desirable to further understand the shape-dependent surface plasmonic resonance (SPR) properties of various gold nanostructures and evaluate their performances in PDT. Materials and methods: Monodispersed colloidal spherical solid Au NPs were synthesized by UV-assisted reduction using chloroauric acid and sodium citrate, and hollow gold nanorings (Au NRs) with similar outer diameter were synthesized based on sacrificial galvanic replacement method. The enhanced electromagnetic (EM) field distribution and their corresponding efficiency in enhancing singlet oxygen (1O2) generation of both gold nanostructures were investigated based on theoretical simulation and experimental measurements. Their shape-dependent SPR response and resulted cell destruction during cellular PDT in combination with 5-aminolevulinic acid (5-ALA) were further studied under different irradiation conditions. Results: With comparable cellular uptake, more elevated formation of 1O2 in 5-ALA-enabled PDT was detected with the presence of Au NRs than that with Au NPs under broadband light irradiation in both cell-free and intracellular conditions. As a result of the unique morphological attributes, exhibiting plasmonic effect of Au NRs was still achievable in the near infrared (NIR) region, which led to an enhanced therapeutic efficacy of PDT under NIR light irradiation. Conclusion: Shape-dependent SPR response of colloidal Au NPs and Au NRs and their respective effects in promoting PDT efficiency were demonstrated in present study. Our innovative colloidal Au NRs with interior region accessible to surrounding photosensitizers would serve as efficient enhancers of PDT potentially for deep tumor treatment. Keywords: gold nanoparticles, colloidal gold nanorings, surface plasmonic resonance, photodynamic therapy

Lei Wang,1,* Guoyuan Li,1,* Ling Ren,2,* Xiangdong Kong,1 Yugang Wang,1 Xiuguo Han,1 Wenbo Jiang,3 Kerong Dai,1 Ke Yang,2 Yongqiang Hao11Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, 2Special Materials and Device Research Department, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, 3Medical 3D Printing Innovation Research Center, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague–Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing. Keywords: nano-sized copper, lysyl oxidase, osteogenesis, fracture healing, callus evolution

Sunkui Ke,1 Tong Zhou,2 Peiyan Yang,3 Yange Wang,2 Peng Zhang,2 Keman Chen,2 Lei Ren,2 Shefang Ye2 1Department of Thoracic Surgery, Zhongshan Hospital of Xiamen University, 2Department of Biomaterials, College of Materials, Xiamen University, 3Department of Surgery, First Affiliated Hospital of Xiamen University, Xiamen University, Xiamen, People’s Republic of China Abstract: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic receptors have been identified as highly promising antitumor agents preferentially eliminating cancer cells with minimal damage, the emergence of TRAIL resistance in most cancers may contribute to therapeutic failure. Thus, there is an urgent need for new approaches to overcome TRAIL resistance. Gold nanoparticles (AuNPs) are one of the most promising nanomaterials that show immense antitumor potential via targeting various cellular and molecular processes; however, the effects of AuNPs on TRAIL sensitivity in cancer cells remain unclear. In this study, we found that AuNPs combined with TRAIL exhibited a greater potency in promoting apoptosis in non-small-cell lung cancer (NSCLC) cells compared with TRAIL alone, suggesting that AuNPs sensitize cancer cells to TRAIL. Further experiments demonstrated that the combination of TRAIL and AuNPs was more effective in causing excessive mitochondrial fragmentation in cancer cells accompanied by a dramatic increase in mitochondrial recruitment of dynamin-related protein 1 (Drp1), mitochondrial dysfunctions, and enhancement of autophagy induction. Small interfering RNA (siRNA) silencing of Drp1 or inhibition of autophagy could effectively alleviate apoptosis in cells exposed to TRAIL combined with AuNPs. In vivo studies revealed that AuNPs augmented TRAIL sensitivity in tumor-bearing mice. Our data indicated that AuNPs potentiate apoptotic response to TRAIL in NSCLC cells through Drp1-dependent mitochondrial fission, and TRAIL combined with AuNPs can be a potential chemotherapeutic strategy for the treatment of NSCLC. Keywords: AuNPs, TRAIL, mitochondrial dynamics, Drp1, autophagy/mitophagy

Lihua Ren,1,2 Jin Zhang,1,2 Yang Zou,1,2 Lianshuang Zhang,1,2 Jialiu Wei,1,2 Zhixiong Shi,1,2 Yanbo Li,1,2 Caixia Guo,2 Zhiwei Sun,1,2 Xianqing Zhou1,21Department of Toxicology and Hygienic Chemistry, School of Public Health, 2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing, People’s Republic of ChinaAbstract: The reproductive toxicity of silica nanoparticles (SiNPs) is well known, but the underlying mechanism is still not clear. To investigate the toxic mechanism of SiNPs on spermatogenic cells, 60 C57 male mice were randomly and equally divided into three groups (the control group, the saline control group, and the SiNPs group) with two observed time points (45 days and 75 days). The mice in the SiNPs group were administered with SiNPs 2 mg/kg diluted in normal saline, and the mice of the saline control group were given equivoluminal normal saline by tracheal perfusion every 3 days for 45 days (in total 15 times). The control group mice were bred without treatment. In each group, a half number of the mice were sacrificed on the 45th day after the first dose, and the remaining half were sacrificed on the 75th day. The results showed that SiNPs increased the malformation of sperms and decreased the motility and concentration of sperms in epididymis on the 45th day after the first dose. SiNPs induced oxidative stress in testis and led to apoptosis and necroptosis of the spermatogenic cells. Furthermore, SiNPs increased the expression of Fas/FasL/RIPK1/FADD/caspase-8/caspase-3 and RIPK3/MLKL on the 45th day after the first dose. However, compared with the saline control group, the index of sperms and the expression of Fas/FasL/RIPK1/FADD/caspase-8/caspase-3/RIPK3/MLKL showed no significant changes in the SiNPs group on the 75th day after the first dose. These data suggested that SiNPs could induce apoptosis and necroptosis in the spermatogenic cells by activating the RIPK1 pathway resulting from oxidative stress in male mice. SiNPs-induced damage recovered on the 75th day after the first dose, which suggested that SiNPs-induced toxicity is reversible.Keywords: silica nanoparticles, RIPK1 signal pathway, apoptosis, necroptosis, spermatogenic cells, reversible

Lei Wang,1,* Ling Ren,2,* Tingting Tang,1 Kerong Dai,1 Ke Yang,2 Yongqiang Hao1 1Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, People’s Republic of China; 2Institute of Metal Research, Chinese Academy of Sciences, Shenyang, People’s Republic of China *These authors contributed equally to this work Abstract: Foreign body reaction induced by biomaterials is a serious problem in clinical applications. Although 317L-Cu stainless steel (317L-Cu SS) is a new type of implant material with antibacterial ability and osteogenic property, the foreign body reaction level still needs to be assessed due to its Cu2+ releasing property. For this purpose, two macrophage cell lines were selected to detect cellular proliferation, apoptosis, mobility, and the secretions of inflammatory cytokines with the influence of 317L-Cu SS. Our results indicated that 317L-Cu SS had no obvious effect on the proliferation and apoptosis of macrophages; however, it significantly increased cellular migration and TNF-α secretion. Then, C57 mice were used to assess foreign body reaction induced by 317L-Cu SS. We observed significantly enhanced recruitment of inflammatory cells (primarily macrophages) with increased TNF-α secretion and apoptosis level in tissues around the materials in the early stage of implantation. With tissue healing, both inflammation and apoptosis significantly decreased. Further, we discovered that NF-κB pathway and Caspase 3 played important roles in 317L-Cu SS induced inflammation and apoptosis. We concluded that 317L-Cu SS could briefly promote the inflammation and apoptosis of surrounding tissues by regulating the activity of NF-κB pathway and Caspase 3. All these discoveries demonstrated that 317L-Cu SS has a great potential for clinical application. Keywords: nano-copper, foreign body reaction, inflammation, apoptosis, NF-κB, Caspase 3

Shefang Ye,1 Min Chen,1 Yuanqin Jiang,1,2 Mingliang Chen,3 Tong Zhou,1 Yange Wang,1 Zhenqing Hou,1 Lei Ren11Department of Biomaterials, Research Center of Biomedical Engineering, College of Materials, Xiamen University, Xiamen, People's Republic of China; 2First Affiliated Hospital of Xiamen University, Xiamen, People's Republic of China; 3Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, People's Republic of ChinaAbstract: Polyhydroxylated derivatives of fullerene C60, named fullerenols (C60[OH]n), have stimulated great interest because of their potent antioxidant properties in various chemical and biological systems, which enable them to be used as a new promising pharmaceutical for the future treatment of oxidative stress-related diseases, but the details remain unknown. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a principal transcription factor that regulates expression of several antioxidant genes via binding to the antioxidant response element and plays a crucial role in cellular defence against oxidative stress. In this study we investigated whether activation of the Nrf2/antioxidant response element pathway contributes to the cytoprotective effects of C60(OH)24. Our results showed that C60(OH)24 enhanced nuclear translocation of Nrf2 and upregulated expression of phase II antioxidant enzymes, including heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1, and γ-glutamate cysteine ligase in A549 cells. Treatment with C60(OH)24 resulted in phosphorylation of p38 mitogen-activated protein kinases (p38 MAPK), extracellular signal-regulated kinases, and c-Jun-N-terminal kinases. By using inhibitors of cellular kinases, we showed that pretreatment of A549 cells with SB203580, a specific inhibitor of p38 MAPK, abolished nuclear translocation of Nrf2 and induction of HO-1 protein induced by C60(OH)24, indicating an involvement of p38 MAPK in Nrf2/HO-1 activation by C60(OH)24. Furthermore, pretreatment with C60(OH)24 attenuated hydrogen peroxide-induced apoptotic cell death in A549 cells, and knockdown of Nrf2 by small interfering ribonucleic acid diminished C60(OH)24-mediated cytoprotection. Taken together, these findings demonstrate that C60(OH)24 may attenuate oxidative stress-induced apoptosis via augmentation of Nrf2-regulated cellular antioxidant capacity, thus providing insights into the mechanisms of the antioxidant properties of C60(OH)24.Keywords: fullerenol, Nrf2, oxidative stress, cytoprotection, A549 cells

Xin-Hua Tian,1,2 Zhi-Gang Wang,1,2 Han Meng,1,2 Yu-Hua Wang,2 Wei Feng,2 Feng Wei,2 Zhi-Chun Huang,2 Xiao-Ning Lin,2 Lei Ren3,41Xiehe Clinical College of Medicine, Fujian Medical University, Fuzhou, 2Department of Neurosurgery, Zhongshan Hospital, 3Research Center of Biomedical Engineering, Department of Biomaterials, College of Materials, 4State Key Laboratory for Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen, People's Republic of ChinaBackground: Gene transfer using a nanoparticle vector is a promising new approach for the safe delivery of therapeutic genes in human disease. The Tat peptide-decorated gelatin-siloxane (Tat-GS) nanoparticle has been demonstrated to be biocompatible as a vector, and to have enhanced gene transfection efficiency compared with the commercial reagent. This study investigated whether intracisternal administration of Tat-GS nanoparticles carrying the calcitonin gene-related peptide (CGRP) gene can attenuate cerebral vasospasm and improve neurological outcomes in a rat model of subarachnoid hemorrhage.Method: A series of gelatin-siloxane nanoparticles with controlled size and surface charge was synthesized by a two-step sol-gel process, and then modified with the Tat peptide. The efficiency of Tat-GS nanoparticle-mediated gene transfer of pLXSN-CGRP was investigated in vitro using brain capillary endothelial cells and in vivo using a double-hemorrhage rat model. For in vivo analysis, we delivered Tat-GS nanoparticles encapsulating pLXSN-CGRP intracisternally using a double-hemorrhage rat model.Results: In vitro, Tat-GS nanoparticles encapsulating pLXSN-CGRP showed 1.71 times higher sustained CGRP expression in endothelial cells than gelatin-siloxane nanoparticles encapsulating pLXSN-CGRP, and 6.92 times higher CGRP expression than naked pLXSN-CGRP. However, there were no significant differences in pLXSN-CGRP entrapment efficiency and cellular uptake between the Tat-GS nanoparticles and gelatin-siloxane nanoparticles. On day 7 of the in vivo experiment, the data indicated better neurological outcomes and reduced vasospasm in the subarachnoid hemorrhage group that received Tat-GS nanoparticles encapsulating pLXSN-CGRP than in the group receiving Tat-GS nanoparticles encapsulating pLXSN alone because of enhanced vasodilatory CGRP expression in cerebrospinal fluid.Conclusion: Overexpression of CGRP attenuated vasospasm and improved neurological outcomes in an experimental rat model of subarachnoid hemorrhage. Tat-GS nanoparticle-mediated CGRP gene delivery could be an innovative strategy for treatment of cerebral vasospasm after subarachnoid hemorrhage.Keywords: gene transfer, nanoparticles, calcitonin gene-related peptide, cerebral vasospasm

Xin-hua Tian1, Feng Wei1, Tian-xiao Wang2, Peng Wang1, Xiao-ning Lin1, Jun Wang2, Dong Wang2, Lei Ren2,31Neurosurgical Department of Affiliated Zhongshan Hospital, 2Research Center of Biomedical Engineering, Department of Biomaterials, College of Materials, 3State Key Laboratory for Physical Chemistry of Solid Surfaces, Xiamen University, Xiamen, People’s Republic of ChinaBackground: Nanobiotechnology can provide more efficient tools for diagnosis, targeted and personalized therapy, and increase the chances of brain tumor treatment being successful. Use of nanoparticles is a promising strategy for overcoming the blood–brain barrier and delivering drugs to the brain. Gelatin-siloxane (GS) nanoparticles modified with Tat peptide can enhance plasmid DNA transfection efficiency compared with a commercial reagent.Methods: SynB-PEG-GS nanoparticles are membrane-penetrable, and can cross the blood–brain barrier and deliver a drug to its target site in the brain. The efficiency of delivery was investigated in vivo and in vitro using brain capillary endothelial cells, a cocultured blood–brain barrier model, and a normal mouse model.Results: Our study demonstrated that both SynB-PEG-GS and PEG-GS nanoparticles had a spherical shape and an average diameter of 150–200 nm. It was shown by MTT assay that SynB-PEG-GS nanoparticles had good biocompatibility with brain capillary endothelial cells. Cellular uptake by SynB-PEG-GS nanoparticles was higher than that for PEG-GS nanoparticles for all incubation periods. The amount of SynB-PEG-GS nanoparticles crossing the cocultured blood–brain barrier model was significantly higher than that of PEG-GS nanoparticles at all time points measured (P

Yanzhao Bi, Yifan Zhang, Chunying Cui, Lulu Ren, Xueyun Jiang School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, People’s Republic of China Abstract: Nanodiamond (ND) is a renowned material in nonviral small interfering RNA (siRNA) carrier field due to its unique physical, chemical, and biological properties. In our previous work, it was proven that ND could deliver siRNA into cells efficiently and downregulate the expression of desired protein. However, synthesizing a high-efficient tumor-targeting carrier using ND is still a challenge. In this study, a novel carrier, NDCONH(CH2)2NH-VDGR, was synthesized for siRNA delivery, and its properties were characterized with methods including Fourier transform infrared spectrometry, transmission electron microscopy, scanning electron microscopy, gel retardation assay, differential scanning calorimetry, confocal microscopy, releasing test, real-time polymerase chain reaction (PCR) assay, enzyme-linked immunosorbent assay (ELISA), flow cytometry, cytotoxicity assay, and gene-silencing efficacy assay in vitro and in vivo. The mechanism of NDCONH(CH2)2NH-VDGR/survivin-siRNA-induced tumor apoptosis was evaluated via flow cytometer assay using Annexin V–fluorescein isothiocyanate/propidium iodide staining method. The NDCONH(CH2)2NH-VDGR/survivin-siRNA nanoparticle with 60–110 nm diameter and 35.65±3.90 mV zeta potential was prepared. For real-time PCR assay, the results showed that the expression of survivin mRNA was reduced to 46.77%±6.3%. The expression of survivin protein was downregulated to 48.49%±2.25%, as evaluated by ELISA assay. MTT assay showed that NDCONH(CH2)2NH-VDGR/survivin-siRNA had an inhibitory effect on MCF-7 cell proliferation. According to these results, the survivin-siRNA could be delivered, transported, and released stably, which benefits in increasing the gene-silencing effect. Therefore, as an siRNA carrier, NDCONH(CH2)2NH-VDGR was suggested to be used in siRNA delivery system and in cancer treatments. Keywords: NDCONH(CH2)2NH-VDGR, siRNA carrier, RNAi, survivin

Guangya Xu,1,* Huashan Shi,2,* Laibin Ren,1 Hongfeng Gou,1 Daoyin Gong,1 Xiang Gao,1–3 Ning Huang11Department of Pathophysiology, West China College of Preclinical Medicine and Forensic Medicine, Sichuan University, Chengdu, Sichuan, People’s Republic of China; 2State Key Laboratory of Biotherapy and Cancer Center, 3Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, People’s Republic of China*These authors contributed equally to this workAbstract: Colorectal cancer, a type of malignant neoplasm originating from the epithelial cells lining the colon and/or rectum, has been the third most frequent malignancy and one of the leading causes of cancer-related deaths in the US. As a bioflavonoid with high anticancer potential, quercetin (Qu) has been proved to have a prospective applicability in chemotherapy for a series of cancers. However, quercetin is a hydrophobic drug, the poor hydrophilicity of which hinders its clinical usage in cancer therapy. Therefore, a strategy to improve the solubility of quercetin in water and/or enhance the bioavailability is desired. Encapsulating the poorly water-soluble, hydrophobic agents into polymer micelles could facilitate the dissolution of drugs in water. In our study, nanotechnology was employed, and quercetin was encapsulated into the biodegradable nanosized amphiphilic block copolymers of monomethoxy poly(ethylene glycol)–poly(ε-caprolactone) (MPEG–PCL), attempting to present positive evidences that this drug delivery system of polymeric micelles is effective. The quercetin-loaded MPEG–PCL nanomicelles (Qu-M), with a high drug loading of 6.85% and a minor particle size of 34.8 nm, completely dispersed in the water and released quercetin in a prolonged period in vitro and in vivo. At the same time, compared with free quercetin, Qu-M exhibited improved apoptosis induction and cell growth inhibition effects in CT26 cells in vitro. Moreover, the mice subcutaneous CT26 colon cancer model was established to evaluate the therapy efficiency of Qu-M in detail, in which enhanced anti-colon cancer effect was proved in vivo: Qu-M were more efficacious in repressing the growth of colon tumor than free quercetin. In addition, better effects of Qu-M on inducing cell apoptosis, inhibiting tumor angiogenesis, and restraining cell proliferation were observed by immunofluorescence analysis. Our study indicated that Qu-M were a novel nanoagent of quercetin with an enhanced antitumor activity, which could serve as a promising potential candidate for colon cancer chemotherapy.Keywords: quercetin, nanoformulation, colon cancer, cell apoptosis, angiogenesis

Background: Polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate-80 have been extensively proposed for delivering drugs into the animal brain and have shown great potential for therapeutic applications. In this study, we made an attempt to deliver the chemotherapeutic drug, temozolomide, into the brain by using PBCA nanoparticles. The physicochemical characteristics, in vitro release, and brain targeting ability of the drug-loaded nanoparticles were investigated., Results: Our results show that a significantly higher concentration of temozolomide in the form of polysorbate-80-coated PBCA nanoparticles was observed in the brain (P < 0.05) in comparison with the free drug., Conclusion: This study indicates that polysorbate-80 coated PBCA nanoparticles could be a feasible carrier for temozolomide delivery to the brain. It is anticipated that the developed formulation may improve on targeted therapy for malignant brain tumors in the future.