Search

Yu-Guo Yuan,1,2,* Ya-Xin Zhang,1,* Song-Zi Liu,1,* Abu Musa Md Talimur Reza,3 Jia-Lin Wang,1,2 Ling Li,1,2 He-Qing Cai,1,2 Ping Zhong,1 Il-Keun Kong4 1College of Veterinary Medicine, Yangzhou University, Yangzhou, People’s Republic of China; 2Jiangsu Co-Innovation Center of Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, People’s Republic of China; 3Department of Molecular Biology and Genetics Faculty of Basic Sciences, Gebze Technical University, Kocaeli, Republic of Turkiye; 4Division of Applied Life Science (BK21 Four), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam Province, Republic of Korea*These authors contributed equally to this workCorrespondence: Yu-Guo Yuan; Il-Keun Kong, Email yyg9776430@163.com; ikong7900@gmail.comIntroduction: The increasing industrial and biomedical utilization of graphene oxide silver nanoparticles (GO-AgNPs) raises the concern of nanosafety: exposure to the AgNPs or GO-AgNPs increases the generation of reactive oxygen species (ROS), causes DNA damage and alters the expression of whole transcriptome including mRNA, miRNA, tRNA, lncRNA, circRNA and others. Although the roles of different RNAs in epigenetic toxicity are being studied during the last decade, but still we have little knowledge about the role of circle RNAs (circRNAs) in epigenetic toxicity.Methods: Rabbit fetal fibroblast cells (RFFCs) were treated with 0, 8, 16, 24, 32 and 48 μg/mL GO-AgNPs to test the cell viability and 24 μg/mL GO-AgNPs was selected as the experimental dose. After 24 h treatment with 24 μg/mL GO-AgNPs, the level of ROS, malondialdehyde (MDA), superoxide dismutase (SOD), intracellular ATP, glutathione peroxidase (GPx), and glutathione reductase (Gr) were measured in the RFFCs. High-throughput whole transcriptome sequencing was performed to compare the expression of circRNAs, long non-coding RNAs (lncRNA) and mRNA between 24 μg/mL GO-AgNPs-treated RFFCs and control cells. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to validate the accuracy of circRNA sequencing data. Bioinformatics analyses were performed to reveal the potential functional roles and related pathways of differentially expressed circRNAs, lncRNA and mRNA and to construct a circRNA–miRNA–mRNA interaction network.Results: We found that 57 circRNAs, 75 lncRNAs, and 444 mRNAs were upregulated while 35 circRNAs, 21 lncRNAs, and 186 mRNAs were downregulated. These differentially expressed genes are mainly involved in the transcriptional mis-regulation of cancer through several pathways: MAPK signaling pathway (circRNAs), non-homologous end-joining (lncRNAs), as well as PPAR and TGF-beta signaling pathways (mRNAs).Conclusion: These data revealed the potential roles of circRNAs in the GO-AgNPs induced toxicity through oxidative damage, which would be the basis for further research to determine their roles in the regulation of different biological processes.Keywords: circRNAs, GO-AgNPs, oxidative stress, epigenetic toxicity

Fengjun Xia,1,2,* Xiaoyan Tao,2,3,* Haichen Wang,1,2 Jian Shui,4 Changhang Min,1,2 Yubing Xia,1,2 Jun Li,1,2 Mengli Tang,1,2 ZhaoJun Liu,1,2 Yongmei Hu,1,2 Huidan Luo,1,2 Mingxiang Zou1,2 1National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 2Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 3Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China; 4Department of Clinical Laboratory, Changsha Central Hospital, Changsha, 410008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Mingxiang Zou, National Clinical Research Center for Geriatric Disorders, Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, Hunan Province, 410008, People’s Republic of China, Tel/Fax +86-7384327440, Email zoumingxiang@csu.edu.cnPurpose: As an under-explored biomaterial, bacterial biofilms have a wide range of applications in the green synthesis of nanomaterials. The biofilm supernatant of Pseudomonas aeruginosa PA75 was used to synthesize novel silver nanoparticles (AgNPs). BF75-AgNPs were found to possess several biological properties.Methods: In this study, we biosynthesized BF75-AgNPs using biofilm supernatant as the reducing agent, stabilizer, and dispersant and investigated their biopotential in terms of antibacterial, antibiofilm, and antitumor activities.Results: The synthesized BF75-AgNPs demonstrated a typical face-centered cubic crystal structure; they were well dispersed; and they were spherical with a size of 13.899 ± 4.036 nm. The average zeta potential of the BF75-AgNPs was − 31.0 ± 8.1 mV. The BF75-AgNPs exhibited strong antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase Escherichia coli (ESBL-EC), extensively drug-resistant Klebsiella pneumoniae (XDR-KP), and carbapenem-resistant Pseudomonas aeruginosa (CR-PA). Moreover, the BF75-AgNPs had a strong bactericidal effect on XDR-KP at 1/2 × MIC, and the expression level of reactive oxygen species (ROS) in bacteria was significantly increased. A synergistic effect was observed when the BF75-AgNPs and colistin were used for the co-treatment of two colistin-resistant XDR-KP strains, with fractional inhibitory concentration index (FICI) values of 0.281 and 0.187, respectively. Furthermore, the BF75-AgNPs demonstrated a strong biofilm inhibition activity and mature biofilm bactericidal activity against XDR-KP. The BF75-AgNPs also exhibited a strong antitumor activity against melanoma cells and low cytotoxicity against normal epidermal cells. In addition, the BF75-AgNPs increased the proportion of apoptotic cells in two melanoma cell lines, and the proportion of late apoptotic cells increased with BF75-AgNP concentration.Conclusion: This study suggests that BF75-AgNPs synthesized from biofilm supernatant have broad prospects for antibacterial, antibiofilm, and antitumor applications.Graphical Abstract: Keywords: multidrug resistant, green synthesis, colistin, metallic silver, melanoma

Li Lin,1,* Qingyong Chen,1,* Yue Dai,2 Yufeng Xia1 1Department of Pharmacognosy, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People’s Republic of China; 2Department of Traditional Chinese Medicine and Pharmacology, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, 211198, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yufeng Xia, Department of Pharmacognosy, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 639 Longmian Avenue, Jiangning District, Nanjing, 211198, People’s Republic of China, Tel +862583271400, Fax +862585301528, Email yfxiacpu@126.comPurpose: Madecassic acid (MCA) is a natural triterpenoid isolated from centellae herba that has diverse biological effects, such as anti-inflammatory, antioxidant, and anticancer activities. However, the efficacy of MCA is limited by low oral bioavailability caused by its extremely poor aqueous solubility. This study aimed to develop a self-nanoemulsifying drug delivery system (SNEDDS) for MCA to improve its oral absorption.Methods: The utilized oil phases, surfactants, and co-surfactants for SNEDDS were selected based on the solubility of MCA and emulsification efficiency. The optimized formulation was characterized for pharmaceutical properties and its pharmacokinetic behavior was examined in rats. Besides, the intestinal absorption property of MCA was investigated using in situ single-pass intestinal perfusion and intestinal lymphatic transport.Results: The optimized nanoemulsion formula consists of Capryol 90:Labrasol:Kolliphor ELP:Transcutol HP in a weight ratio of 1:2.7:2.7:3.6 (w/w/w/w). MCA-loaded SNEDDS presented a small droplet size (21.52 ± 0.23 nm), with a zeta potential value of − 3.05 ± 0.3 mV. Compared with pure MCA, SNEDDS had a higher effective permeability coefficient and showed 8.47-fold and 4.01-fold of maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC), respectively. Cycloheximide was pretreated before the experiment to evaluate the degree of lymphatic uptake. The results showed that cycloheximide greatly influenced the absorption of SNEDDS, resulting in 82.26% and 76.98% reduction in Cmax and AUC, respectively.Conclusion: This study reports the MCA-loaded SNEDDS with distinctly enhanced in vitro and in vivo performance compared with pure MCA and concludes that the SNEDDS formulation could be a viable and effective strategy for improving the dissolution rate and bioavailability of poor aqueous-soluble ingredients.Keywords: madecassic acid, self-nanoemulsifying drug delivery system, pharmacokinetics, intestinal lymphatic transport

Haoyu Zhao,1,* Yachao Jia,1,* Feng Wang,1 Yimin Chai,1 Chunfu Zhang,2 Jia Xu,1 Qinglin Kang1 1Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Department of Orthopedic Surgery, Shanghai, People’s Republic of China; 2Shanghai Jiao Tong University, School of Biomedical Engineering, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jia Xu; Qinglin Kang, Email xujia0117@126.com; orthokang@163.comIntroduction: Large bone abnormalities are commonly treated using distraction osteogenesis (DO), but it is not suitable for a long-term application; therefore, there is an urgent need for adjuvant therapy that can accelerate bone repair.Methods: We have synthesized mesoporous silica-coated magnetic nanoparticles doped with cobalt ions (Co-MMSNs) and assessed their capacity to quicken bone regrowth in a mouse model of DO. Furthermore, local injection of the Co-MMSNs significantly accelerated bone healing in DO, as demonstrated by X-ray imaging, micro-CT, mechanical tests, histological evaluation, and immunochemical analysis.Results: In vitro, the Co-MMSNs exhibited good biocompatibility and induced angiogenic gene expression and osteogenic development in bone mesenchymal stem cells. And the Co-MMSNs can promote bone regeneration in a rat DO model.Discussion: This study demonstrated the significant potential of Co-MMSNs to shorten the DO treatment duration and effectively reduce the incidence of complications.Keywords: mesoporous silica-coated magnetic nanoparticles, mesenchymal stem cells, large bone defects, distraction osteogenesis, bone regeneration

Jing Wang,1,* Weijian Chen,1,2,* Wenxiang Du,2 Hongjie Zhang,2 Matthias Ilmer,3 Lei Song,2 Yuan Hu,2 Xiaopeng Ma1 1Department of General Surgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, People’s Republic of China; 2State Key Laboratory of Fire Science, University of Science and Technology of China, Hefei, Anhui, 230026, People’s Republic of China; 3Department of General, Visceral, and Transplantation Surgery, Ludwig-Maximilians-University (LMU), Campus Grosshadern, Munich, 81377, Germany*These authors contributed equally to this workCorrespondence: Yuan Hu; Xiaopeng Ma, Email yuanhu@ustc.edu.cn; XiaopengMa@fsyy.ustc.edu.cnIntroduction: Tamoxifen (TAM) has proven to be a therapeutic breakthrough to reduce mortality and recurrence in estrogen receptor-positive (ER+) breast cancer patients. However, the application of TAM exhibits low bioavailability, off-target toxicity, instinct and acquired TAM resistance.Methods: We utilized black phosphorus (BP) as a drug carrier and sonosensitizer, integrated with TAM and tumor-targeting ligand folic acid (FA) to construct TAM@BP-FA for synergistic endocrine and sonodynamic therapy (SDT) of breast cancer. The exfoliated BP nanosheets were modified through in situ polymerization of dopamine, followed by electrostatic adsorption of TAM and FA. The anticancer effect of TAM@BP-FA was evaluated through in vitro cytotoxicity and in vivo antitumor model. RNA-sequencing (RNA-seq), quantitative real-time PCR, Western blot analysis, flow cytometry analysis and peripheral blood mononuclear cells (PBMCs) analysis were performed for mechanism investigation.Results: TAM@BP-FA had satisfactory drug loading capacity, the TAM release behavior can be controlled through pH microenvironment and ultrasonic stimulation. An amount of hydroxyl radical (∙OH) and singlet oxygen (1O2) were as expected generated under ultrasound stimulation. TAM@BP-FA nanoplatform showed excellent internalization in both TAM-sensitive MCF7 and TAM-resistant (TMR) cells. Using TMR cells, TAM@BP-FA displayed significantly enhanced antitumor ability in comparison with TAM (7.7% vs 69.6% viability at 5μg/mL), the additional SDT further caused 15% more cell death. RNA-seq unraveled the TAM@BP-FA antitumor mechanisms including effects on cell cycle, apoptosis and cell proliferation. Further analysis showed additional SDT successfully triggering reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) reduction. Moreover, PBMCs exposed to TAM@BP-FA induced an antitumor immune response by natural killer (NK) cell upregulation and immunosuppression macrophage reduction.Conclusion: The novel BP-based strategy not only delivers TAM specifically to tumor cells but also exhibits satisfactory antitumor effects through targeted therapy, SDT, and immune cell modulation. The nanoplatform may provide a superior synergistic strategy for breast cancer therapy.Keywords: black phosphorus, breast cancer, sonodynamic therapy, tamoxifen, PBMC, combination therapy

Yuexing Gu,1,2,* Yan Li,3,* Shengjie Ge,1,2 Wenbo Lu,4 Yu Mao,1,2 Miao Chen,1,2 Yayun Qian1,2 1Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, People’s Republic of China; 2Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases, Yangzhou University, Yangzhou, People’s Republic of China; 3Department of Obstetrics and Gynecology, The Second People’s Hospital of Taizhou City, Taizhou, People’s Republic of China; 4Shanxi Normal University, College of Chemistry and Material Science, Linfen, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yayun Qian, Email yyqian@yzu.edu.cnPurpose: This paper aims to construct a surface-enhanced Raman spectroscopy (SERS) biosensor based on functionalized Au-Si nanocone arrays (Au-SiNCA) using a dual signal amplification strategy (SDA-CHA) to evaluate telomerase activity during epithelial-mesenchymal transition (EMT) in laryngeal carcinoma (LC).Methods: A SERS biosensor based on functionalized Au-SiNCA was designed with an integrated dual-signal amplification strategy to achieve ultrasensitive detection of telomerase activity during EMT in LC patients.Results: Labeled probes (Au-AgNRs@4-MBA@H1) and capture substrates (Au-SiNCA@H2) were prepared by modifying hairpin DNA and Raman signal molecules. Using this scheme, telomerase activity in peripheral mononuclear cells (PMNC) could be successfully detected with a limit of detection (LOD) as low as 10− 6 IU/mL. In addition, biological experiments using BLM treatment of TU686 effectively mimicked the EMT process. The results of this scheme were highly consistent with the ELISA scheme, confirming its accuracy.Conclusion: This scheme provides a reproducible, selective, and ultrasensitive assay for telomerase activity, which is expected to be a potential tool for the early screening of LC in future clinical applications.Keywords: surface-enhanced Raman scattering, telomerase, laryngeal carcinoma, epithelial-mesenchymal transition, Au-SiNCA

Wen Ting Zhu,1,* Xiang Feng Zeng,2,* Hua Yang,2 Meng Lei Jia,1 Wei Zhang,2 Wei Liu,2 Sheng Yao Liu3 1Department of Pharmacy, Biomedicine Research Center, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, People’s Republic of China; 2Department of Orthopedics, The Affiliated Nanhua Hospital, Hengyang Medical School, University of South China, Hengyang, 421001, People’s Republic of China; 3Department of Orthopedics, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, People’s Republic of China*These authors contributed equally to this workCorrespondence: Wei Liu, Department of Orthopedics, The Affiliated Nanhua Hospital, Hengyang Medical School, University of South China, Hengyang, 421001, People’s Republic of China, Email lwsurgery@163.com Sheng Yao Liu, Department of Orthopedics, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, People’s Republic of China, Email liushengyao12@163.comBackground: Osteosarcoma is a malignant bone tumor with a high rate of lung metastasis and mortality. It has been demonstrated that resveratrol can inhibit tumor proliferation and metastasis, but its application is limited due to poor water solubility and low bioavailability. In this study, we proposed to prepare folate-modified liposomes loaded with resveratrol to investigate its anti-osteosarcoma effect in vitro and in vivo.Methods: We prepared and characterized resveratrol liposomes modified with folate (denoted as, FA-Res/Lps). The effects of FA-Res/Lps on human osteosarcoma cell 143B proliferation, apoptosis, and migration were investigated by MTT, cell cloning, wound-healing assay, transwell, and flow cytometry. A xenograft tumor and lung metastasis model of osteosarcoma was constructed to study the therapeutic effects of FA-Res/Lps on the growth and metastasis of osteosarcoma in vivo.Results: The FA-Res/Lps were prepared with a particle size of 118.5 ± 0.71 and a small dispersion coefficient of 0.154 ± 0.005. We found that FA-modified liposomes significantly increased resveratrol uptake by osteosarcoma cells 143B in flow cytometric assay, resulting in FA-Res/Lps, which inhibit tumor proliferation, migration and induce apoptosis more effectively than free Res and Res/Lps. The mechanism of action may be associated with the inhibition of JAK2/STAT3 signaling. In vivo imaging demonstrated that FA-modified DiR-modified liposomes significantly increased the distribution of drugs at the tumor site, leading to significant inhibition of osteosarcoma growth and metastasis by FA-Res/Lps. Furthermore, we found that FA-Res/Lps did not cause any adverse effects on mice body weight, liver, or kidney tissues.Conclusion: Taken together, the anti-osteosarcoma effect of resveratrol is significantly enhanced when it is loaded into FA-modified liposomes. FA-Res/Lps is a promising strategy for the treatment of osteosarcoma.Keywords: resveratrol, liposomes, osteosarcoma, lung metastasis, JAK2/STAT3 pathway

Chunjiao Lu,* Yi Liu,* Yao Liu, Guanhua Kou, Yang Chen, Xuewei Wu, Yuhang Lv, Jiahao Cai, Renyuan Chen, Juanjuan Luo, Xiaojun Yang Guangdong Provincial Key Laboratory of Infectious Disease and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People’s Republic of China*These authors contributed equally to this workCorrespondence: Juanjuan Luo; Xiaojun Yang, Guangdong Provincial Key Laboratory of Infectious Disease and Molecular Immunopathology, Shantou University Medical College, Shantou, 515041, People’s Republic of China, Tel +86-754-88900236, Fax +86-754-88900276, Email lujj770@163.com; yangx@stu.edu.cnIntroduction: Silver nanoparticles (AgNP) are widely used as coating materials. However, the potential risks of AgNP to human health, especially for neural and vascular systems, are still poorly understood.Methods: The vascular and neurotoxicity of various concentrations of AgNP in zebrafish were examined using fluorescence microscopy. In addition, Illumina high-throughput global transcriptome analysis was performed to explore the transcriptome profiles of zebrafish embryos after exposure to AgNP. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to elucidate the top 3000 differentially expressed genes (DEGs) between AgNP-exposed and control groups.Results: We systematically investigated the neural and vascular developmental toxicities of AgNP exposure in zebrafish. The results demonstrated that AgNP exposure could cause neurodevelopmental anomalies, including a small-eye phenotype, neuronal morphology defects, and inhibition of athletic abilities. In addition, we found that AgNP exposure induces angiogenesis malformation in zebrafish embryos. Further RNA-seq revealed that DEGs were mainly enriched in the neuroactive ligand-receptor interaction and vascular endothelial growth factor (Vegf) signaling pathways in AgNP-treated zebrafish embryos. Specifically, the mRNA levels of the neuroactive ligand-receptor interaction pathway and Vegf signaling pathway-related genes, including si:ch73-55i23.1, nfatc2a, prkcg, si:ch211-132p1.2, lepa, mchr1b, pla2g4aa, rac1b, p2ry6, adrb2, chrnb1, and chrm1b, were significantly regulated in AgNP-treated zebrafish embryos.Conclusion: Our findings indicate that AgNP exposure transcriptionally induces developmental toxicity in neural and vascular development by disturbing neuroactive ligand-receptor interactions and the Vegf signaling pathway in zebrafish embryos.Keywords: silver nanoparticles, neurological development, vascular development, developmental toxicity, zebrafish

Jianhua Yan,1 Qifei Wang,1,2 Junlin Yang,2 Paige Rutter,1 Malcolm Xing,3 Bingyun Li1 1Department of Orthopaedics, School of Medicine, West Virginia University, Morgantown, WV, 26506, USA; 2Spine Center, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 3Department of Mechanical Engineering, University of Manitoba, Winnipeg, R3T2N2, CanadaCorrespondence: Bingyun Li, Department of Orthopaedics, School of Medicine, West Virginia University, 64 Medical Center Drive, Morgantown, WV, 26506, USA, Tel +1 681-285-5956 ; +1 304-293-1075, Fax +1 304-293-7070, Email bili@hsc.wvu.eduBackground: The wide use of antibiotics has created challenges related to antibiotic-resistant bacteria, which have been increasingly found in recent decades. Antibiotic resistance has led to limited choices of antibiotics. Multiple old antimicrobial agents have high antimicrobial properties toward bacteria, but they unfortunately also possess high toxicity toward humans. For instance, silver (Ag) compounds were frequently used to treat tetanus and rheumatism in the 19th century and to treat colds and gonorrhea in the early 20th century. However, the high toxicity of Ag has limited its clinical use.Purpose: We aimed to reformulate Ag to reduce its toxicity toward human cells like osteoblasts and to optimize its antimicrobial properties.Results: Ag, an old antimicrobial agent, was reformulated by hybriding nanomaterials of different dimensions, and silver nanoparticles (AgNPs) of controllable sizes (95– 200 nm) and varying shapes (cube, snowflake, and sphere) were synthesized on carbon nanotubes (CNTs). The obtained AgNP-CNT nanohybrids presented significantly higher killing efficacy against Staphylococcus aureus (S. aureus) compared to AgNPs at the same molar concentration and showed synergism in killing S. aureus at 0.2 and 0.4 mM. AgNPs presented significant osteoblast toxicity; in contrast, AgNP-CNT nanohybrids demonstrated significantly enhanced osteoblast viability at 0.04– 0.8 mM. The killing of S. aureus by AgNP-CNT nanohybrids was fast, occurring within 15 min.Conclusion: Ag was successfully reformulated and Ag nanohybrids with various AgNP shapes on CNTs were synthesized. The nanohybrids presented significantly enhanced antimicrobial properties and significantly higher osteoblast cell viability compared to AgNPs, showing promise as an innovative antimicrobial nanomaterial for a broad range of biomedical applications.Keywords: silver nanoparticle, nanohybrid, antimicrobial, cytotoxicity, reformulation, antibiotic resistance

Tzu-Hao Huang,1 Chih-Jung Chen,2– 4 Hsin-Chia Angela Lin,1 Chun-Han Chen,1,* Jia-You Fang5– 7,* 1Division of General Surgery, Department of Surgery, Chang Gung Memorial Hospital, Chiayi, Taiwan; 2Department of Pathology and Laboratory Medicine, Taichung Veterans General Hospital, Taichung, Taiwan; 3School of Medicine, Chung Shan Medical University, Taichung, Taiwan; 4Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung, Taiwan; 5Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan; 6Research Center for Food and Cosmetic Safety, Research Center for Chinese Herbal Medicine, and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Kweishan, Taoyuan, Taiwan; 7Department of Anesthesiology, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan*These authors contributed equally to this workCorrespondence: Chun-Han Chen, Division of General Surgery, Department of Surgery, Chang Gung Memorial Hospital, Chiayi, 6, W. Sec., Jiapu Road, Puzih City, Chiayi County, 61363, Taiwan, Tel +886-5-3621000 ext. 3821, Fax +886-5-3623002, Email chen5746@cgmh.org.tw Jia-You Fang, Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan, Taoyuan, 333, Taiwan, Tel +886-3-2118800, Fax +886-3-2118236, Email fajy@mail.cgu.edu.twPurpose: Morbid obesity and its related metabolic syndrome are an important health issue. Recently, sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB) have accounted for the most popular bariatric surgeries. Valsartan (VST) is a common hypertension drug, and nano-carriers can increase its solubility and bioavailability. This study aims to explore the nano-VST formula in bariatric surgery subjects.Methods: High-fat fed animals were used as obese models. Operations were performed according to a standardized protocol. The drug was administrated by gavage, and blood samples were taken by serial tail vein sampling. Caco-2 cells were used for examining cell viability and drug uptake. A self-nano-emusifying drug delivery system (SNEDDS) formula was composed of sefsol-218, RH-40 and propylene glycol by a specified ratio, while high-performance liquid chromatography (HPLC) was used for determining drug concentrations.Results: Post-operatively, subjects that underwent RYGB lost more body weight compared to the SG group. The SNEDDS did not exhibit cytotoxicity after adequate dilution, and the cytotoxicity was not related to VST dose. A better cellular uptake of SNEDDS was observed in vitro. The SNEDDS formula achieved a diameter of 84 nm in distilled water and 140 nm in simulated gastric fluid. In obese animals, the maximum serum concentration (Cmax) of VST was increased 1.68-folds by SNEDDS. In RYGB with SUS, the Cmax was reduced to less than 50% of the obese group. SNEDDS increased the Cmax to 3.5 folds higher than SUS and resulted in 3.28-folds higher AUC0-24 in the RYGB group. Fluorescence imaging also confirmed a stronger signal of SNEDDS in the gastrointestinal mucosa. SNEDDS accumulated a higher drug concentration than suspension alone in the liver of the obese group.Conclusion: SNEDDS could reverse the VST malabsorption in RYGB. Further studies are mandatory to clarify post-SG change of drug absorption.Keywords: gastric bypass, sleeve gastrectomy, valsartan, nano-drug delivery system

Chengjie Zhong,1,* Jiajiu Chen,2,* Yi Ling,3 Deng Liu,3 Jing Xu,2 Luofu Wang,2 Chengguo Ge,4 Qing Jiang4 1The Second Clinical Medical College, Chongqing Medical University, Chongqing, 400016, People’s Republic of China; 2Department of Urology, Daping Hospital, Army Medical University, Chongqing, 400038, People’s Republic of China; 3Department of Ultrasound, Southwest Hospital, Army Medical University, Chongqing, 400042, People’s Republic of China; 4Department of Urology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chengguo Ge; Qing Jiang, Department of Urology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, People’s Republic of China, Tel +86 13883510963 ; +86 13508346066, Email gcg2022@hospital.cqmu.edu.cn; 300899@hospital.cqmu.edu.cnBackground and Purpose: The early diagnosis and differential diagnosis of renal cell carcinoma (RCC) has always been a clinical difficulty and a research focus. Carbonic anhydrase IX (CA IX) is highly expressed on the cell membrane of RCC but is not expressed in normal renal tissues. In this study, nanobubbles (NBs) targeting CA IX with ultrasound and photoacoustic multimodal imaging capabilities were prepared to explore a new method for the diagnosis and differential diagnosis of RCC.Methods: Indocyanine green (ICG)-loaded lipid NBs (ICG-NBs) were prepared by using the filming rehydration method, and anti-CA IX polypeptides (ACPs) were attached to their surfaces to prepare CA IX-targeted NBs (ACP/ICG-NBs). The particle size, zeta potential and ICG encapsulation efficiency of these nanobubbles were measured, and their specific targeting and binding abilities to RCC cells were determined. The in vitro and in vivo ultrasound, photoacoustic and fluorescence imaging characteristics of these nanobubbles were also assessed.Results: The particle size of the ACP/ICG-NBs was 475.9 nm in diameter, and their zeta potential was − 2.65 mV. Laser confocal microscopy and flow cytometry both confirmed that ACP/ICG-NBs had specific binding activity and ideal affinity to CA IX-positive RCC cells (786-O) but not to CA IX-negative RCC cells (ACHN). The intensities of the in vitro ultrasound, photoacoustic and fluorescence imaging were positively correlated with the concentrations of ACP/ICG-NBs. In in vivo ultrasound and photoacoustic imaging experiments, ACP/ICG-NBs exhibited specific enhanced ultrasound and photoacoustic imaging effects in 786-O xenograft tumors.Conclusion: The ICG- and ACP-loaded targeted nanobubbles that we prepared had the capability of ultrasound, photoacoustic and fluorescence multimodal imaging and could specifically enhance the ultrasound and photoacoustic imaging of RCC xenograft tumors. This outcome has potential clinical application value for the diagnosis of RCC at the early stage and the differential diagnosis of benign and malignant kidney tumors.Keywords: contrast-enhanced ultrasound imaging, photoacoustic imaging, renal cell carcinoma, carbonic anhydrase IX, targeted nanobubble, fluorescence imaging

Qi Lou,1,* Lu Pan,1,* Shengjin Xiang,1,2,* Yueting Li,1 Jiahui Jin,1 Jingyang Tan,1 Baoshan Huang,1,2 Kaihui Nan,1,2 Sen Lin1,2 1National Engineering Research Center of Ophthalmology and Optometry, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, 325027, People’s Republic of China; 2State Key Laboratory of Ophthalmology, Optometry and Vision Science, School of Ophthalmology & Optometry, Wenzhou Medical University, Wenzhou, 325027, People’s Republic of China*These authors contributed equally to this workCorrespondence: Kaihui Nan; Sen Lin, School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, 325027, People’s Republic of China, Tel +86-577-88067962, Email lin_sen@wmu.edu.cn; nankh@l63.comIntroduction: Benzalkonium chloride (BAC) is widely employed as a preservative in eye drops, which will cause the death of corneal epithelial cells due to ROS production, DNA strand breakage, and mitochondrial dysfunction, resulting in dry eye disease (DED)-like changes in ocular surface tissues. In this study, Melatonin (MT) liposomes (TAT-MT-LIPs) designed by loading MT into TAT-modified liposomes have been developed, characterized, and used for inhibiting BAC-induced DED (BAC-DED).Methods: The TAT was chemically grafted onto the Mal-PEG2000-DSPE by Michael’s addition between the sulfhydryl group in TAT and the maleimide group in Mal-PEG2000-DSPE. TAT-MT-LIPs were prepared using film dispersion followed by the extrusion method and topically treated in rats once a day. BAC-DED was induced in rats by topical administration with 0.2% BAC twice daily. Defects, edema, and inflammation of the corneas, as well as IOP, were examined. Histologic analyses of corneas were performed to assess the change of mitochondrial DNA oxidation and NLRP3/Caspase-1/GSDMD signaling transduction.Results: After topical administration, TAT-MT-LIPs significantly alleviated DED-clinical symptoms of experimental animals by inhibiting tissue inflammation and preventing the loss of the corneal epithelium and conjunctival goblet cells. Our data suggested continuous ocular surface exposure of BAC-induced NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis, which was not reported before. BAC caused substantial mt-DNA oxidation, which promoted the transduction of NLRP3/Caspase-1/GSDMD and consequent corneal epithelium pyroptosis. TAT-MT-LIPs could efficiently suppress the BAC-induced corneal epithelium pyroptosis and inflammation by inhibiting mt-DNA oxidation and the subsequent signal transmission.Conclusion: NLRP3/Caspase-1/GSDMD mediated corneal epithelium pyroptosis is involved in the development of BAC-DED. The present study provided new insights into the adverse effects of BAC, which can serve as a new target for protecting corneal epithelium when applying BAC as a preservative in eye drops. The developed TAT-MT-LIPs can efficiently inhibit BAC-DED and give great potential to be developed as a new DED treatment.Keywords: melatonin liposome, dry eye disease, benzalkonium chloride, pyroptosis, NLRP3/Caspase-1/GSDMD signaling axis

Fei Tong,1– 4 Pei Wang,1,3,4 Ziqiang Chen,1,3,4 Yifan Liu,1,3,4 Lianguo Wang,1,3,4 Jun Guo,1,3,4 Zhihua Li,1,3,4 Hu Cai,2,4 Junchao Wei1– 4 1School of Stomatology, Nanchang University, Nanchang, 330006, People’s Republic of China; 2School of Chemistry and Chemical Engineering, Nanchang University, Nanchang, Jiangxi Province, 330031, People’s Republic of China; 3The Key Laboratory of Oral Biomedicine, Nanchang, Jiangxi Province, 330006, People’s Republic of China; 4Jiangxi Province Clinical Research Center for Oral Diseases, Nanchang, 330006, People’s Republic of ChinaCorrespondence: Hu Cai, School of Chemistry and Chemical Engineering, Nanchang University, 999# Xuefu Road, Honggutan District, Nanchang, Jiangxi, 330031, People’s Republic of China, Tel +86 791 83969514, Email caihu@ncu.edu.cn Junchao Wei, School of Stomatology, Nanchang University, 49# Fuzhou Road, Donghu District, Nanchang, Jiangxi, 330006, People’s Republic of China, Tel +86 791 86236950, +86 791 6361141, Email weijunchao@ncu.edu.cnIntroduction: The critical challenge for periodontitis therapy is thoroughly eliminating the dental plaque biofilm, particularly penetrating the deep periodontal tissue. Regular therapeutic strategies are insufficient to penetrate the plaque without disturbing the commensal microflora of the oral cavity. Here, we constructed a Fe3O4 magnetic nanoparticle loading minocycline (FPM NPs) to penetrate the biofilm physically and effectively eliminate periodontal biofilm.Methods: In order to penetrate and remove the biofilm effectively, Fe3O4 magnetic nanoparticles were modified with minocycline using a co-precipitation method. The particle size and dispersion of the nanoparticles were characterized by transmission electron microscopy, scanning electron microscopy, and dynamic light scattering. The antibacterial effects were examined to verify the magnetic targeting of FPM NPs. Confocal laser scanning microscopy was employed to check the effect of FPM + MF and develop the best FPM NPs treatment strategy. Additionally, the therapeutic effect of FPM NPs was investigated in periodontitis rat models. The expression of IL-1β, IL-6, and TNF-α in periodontal tissues was measured by qRT-PCR and Western blot.Results: The multifunctional nanoparticles exhibited intense anti-biofilm activity and good biocompatibility. The magnetic forces could pull FMP NPs against the biofilm mass and kill bacteria deep in the biofilms both in vivo and in vitro. The integrity of the bacterial biofilm is disrupted under the motivation of the magnetic field, allowing for improved drug penetration and antibacterial performance. The periodontal inflammation recovered well after FPM NPs treatment in rat models. Furthermore, FPM NPs could be monitored in real-time and have magnetic targeting potentials.Conclusion: FPM NPs exhibit good chemical stability and biocompatibility. The novel nanoparticle presents a new approach for treating periodontitis and provides experimental support for using magnetic-targeted nanoparticles in clinic applications.Keywords: ferromagnetic nanoparticle, periodontitis, biofilm, magnetic targeting

Mateusz Wierzbicki,1 Katarzyna Zawadzka,1 Barbara Wójcik,1 Sławomir Jaworski,1 Barbara Strojny,1 Agnieszka Ostrowska,1 Artur Małolepszy,2 Marta Mazurkiewicz-Pawlicka,2 Ewa Sawosz1 1Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, 02-786, Poland; 2Faculty of Chemical and Process Engineering, Warsaw University of Technology, Warsaw, 00-654, PolandCorrespondence: Mateusz Wierzbicki, Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw, 02-786, Poland, Tel +48 22 5936676, Email mateusz_wierzbicki@sggw.edu.plIntroduction: Diamond nanoparticles are considered to be one of the most cytocompatible carbon nanomaterials; however, their toxicity varies significantly depending on the analysed cell types. The aim was to investigate the specific sensitivity of endothelial cells to diamond nanoparticles dependent on exposure to nanoparticles.Methods: Diamond nanoparticles were characterized with Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS). Toxicity of diamond nanoparticles was assessed for endothelial cells (HUVEC), human mammary epithelial cells (HMEC) and HS-5 cell line. The effect of diamond nanoparticles on the level of ROS, NO, NADPH and protein synthesis of angiogenesis-related proteins of endothelial cells was evaluated.Results and Discussion: Our studies demonstrated severe cell type-specific toxicity of diamond nanoparticles to endothelial cells (HUVEC) depending on nanoparticle surface interaction with cells. Furthermore, we have assessed the effect on cytotoxicity of the bioconjugation of nanoparticles with a peptide containing the RGD motive and a serum protein corona. Our study suggests that the mechanical interaction of diamond nanoparticles with the endothelial cell membranes and the endocytosis of nanoparticles lead to the depletion of NADPH, resulting in an intensive synthesis of ROS and a decrease in the availability of NO. This leads to severe endothelial toxicity and a change in the protein profile, with changes in major angiogenesis-related proteins, including VEGF, bFGF, ANPT2/TIE-2, and MMP, and the production of stress-related proteins, such as IL-6 and IL-8.Conclusion: We confirmed the presence of a relationship between the toxicity of diamond nanoparticles and the level of cell exposure to nanoparticles and the nanoparticle surface. The results of the study give new insights into the conditioned toxicity of nanomaterials and their use in biomedical applications.Keywords: diamond nanoparticles, endothelial cells, nanotoxicity, oxidative toxicity

Lan Sun,1 Hong-Juan Yao,2 Jing-Cao Li,1 Bao-Quan Zhao,1 Yong-An Wang,1 Ying-Ge Zhang1 1Key Laboratory of Nanopharmacology and Nanotoxicology, Beijing Institute of Pharmacology and Toxicology, Beijing, People’s Republic of China; 2Key Laboratory of Antibiotic Bioengineering of National Health and Family Planning Commission (NHFPC), Institute of Medicinal Biotechnology (IMB), Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC), Beijing, People’s Republic of ChinaCorrespondence: Ying-Ge Zhang; Yong-An Wang, Beijing Institute of Pharmacology and Toxicology, Beijing, People’s Republic of China, Tel +86 10-66930654 ; +86 10-66874607, Fax +86 10-68211656, Email Zhangyg58@126.com; yonganw@126.comIntroduction: Hepatocellular cancer stem cells (CSCs) play crucial roles in hepatocellular cancer initiation, development, relapse, and metastasis. Therefore, eradication of this cell population is a primary objective in hepatocellular cancer therapy. We prepared a nanodrug delivery system with activated carbon nanoparticles (ACNP) as carriers and metformin (MET) as drug (ACNP-MET), which was able to selectively eliminate hepatocellular CSCs and thereby increase the effects of MET on hepatocellular cancers.Methods: ACNP were prepared by ball milling and deposition in distilled water. Suspension of ACNP and MET was mixed and the best ratio of ACNP and MET was determined based on the isothermal adsorption formula. Hepatocellular CSCs were identified as CD133+ cells and cultured in serum-free medium. We investigated the effects of ACNP-MET on hepatocellular CSCs, including the inhibitory effects, the targeting efficiency, self-renewal capacity, and the sphere-forming capacity of hepatocellular CSCs. Next, we evaluated the therapeutic efficacy of ACNP-MET by using in vivo relapsed tumor models of hepatocellular CSCs.Results: The ACNP have a similar size, a regular spherical shape and a smooth surface. The optimal ratio for adsorption was MET: ACNP=1:4. ACNP-MET could target and inhibit the proliferation of CD133+ population and decrease mammosphere formation and renewal of CD133+ population in vitro and in vivo.Conclusion: These results not only suggest that nanodrug delivery system increased the effects of MET, but also shed light on the mechanisms of the therapeutic effects of MET and ACNP-MET on hepatocellular cancers. ACNP, as a good nano-carrier, could strengthen the effect of MET by carrying drugs to the micro-environment of hepatocellular CSCs.Keywords: nanodrug delivery system, metformin, CD133, hepatocellular cancer stem cells

Yu Been Shin,1,* Ju-Yeon Choi,2,* Dae Hwan Shin,1 Jeong-Won Lee2,3 1College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea; 2Research Institute for Future Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 3Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea*These authors contributed equally to this workCorrespondence: Dae Hwan Shin, College of Pharmacy, Chungbuk National University, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju, 28160, Republic of Korea, Tel +82 43 261 2820, Fax +82 43 268 2732, Email dshin@chungbuk.ac.kr Jeong-Won Lee, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81, Irwon-ro, Gangnam-gu, Seoul, 06351, South Korea, Tel +82-2-3410-1382, Fax +82-2-3410-0630, Email garden.lee@samsung.comPurpose: We aimed to inhibit ovarian cancer (OC) development by interfering with microtubule polymerization and inhibiting mTOR signaling. To achieve this, previously developed micelles containing fenbendazole and rapamycin were applied.Methods: Herein, we prepared micelles for drug delivery using fenbendazole and rapamycin at a 1:2 molar ratio and methoxy poly(ethylene glycol)-b-poly(caprolactone)(mPEG-b-PCL) via freeze-drying. We revealed their long-term storage capacity of up to 120 days. Furthermore, a cytotoxicity test was performed on the OC cell line HeyA8, and an orthotopic model was established for evaluating in vivo antitumor efficacy.Results: Fenbendazole/rapamycin-loaded mPEG-b-PCL micelle (M-FR) had an average particle size of 37.2 ± 1.10 nm, a zeta potential of − 0.07 ± 0.09 mV, and a polydispersity index of 0.20 ± 0.02. Additionally, the average encapsulation efficiency of fenbendazole was 75.7 ± 4.61% and that of rapamycin was 98.0 ± 1.97%. In the clonogenic assay, M-FR was 6.9 times more effective than that free fenbendazole/rapamycin. The in vitro drug release profile showed slower release in the combination formulation than in the single formulation.Conclusion: There was no toxicity, and tumor growth was suppressed substantially by our formulation compared with that seen with the control. The findings of our study lay a foundation for using fenbendazole and rapamycin for OC treatment.Keywords: ovarian cancer treatment, combination therapy, drug delivery, nanoformulation

Wenjing Ta, Xingyue Li, Jihong Song, Ruochen Hua, Yuting Zheng, Wen Lu School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, People’s Republic of ChinaCorrespondence: Wen Lu, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, 710061, People’s Republic of China, Email lvlu2004@xjtu.edu.cnPurpose: Nanotechnology-based drug delivery systems (nano-DDS) have been developed to be a promising strategy to improve the efficacy, safety, physicochemical and pharmacokinetic/pharmacodynamics properties of drugs. It is very necessary to elucidate the delivery process in vivo or in cells for the rational design and accurate preparation of nano-DDS. The aim of this study was to construct a nano-DDS to visualize and quantify the intracellular behavior of the loaded cargo and carrier in such a system.Methods: A carboxyl-terminal end of poly(lactic-co-glycolic acid) polymer was fluorescently labeled with rhodamine B by conjugation of ethylenediamine. Dual-fluorescent nanoparticles (DFPs) were prepared from this fluorescently labeled polymer to encapsulate a fluorescent cargo, coumarin 6. The carrier and cargo of DFPs were monitored by confocal fluorescence microscopy during cellular uptake. Furthermore, the transcellular transportation of DFPs was evaluated quantitatively by measuring the fluorescence intensity.Results: The obtained fluorescent polymer showed stable and quantifiable characteristics. DFPs could be customized in terms of coumarin 6 content (97.7± 1.0%), size (367.3± 1.7 nm) and dual-emission fluorescence (green cargo and red carrier). DFPs did not significantly affect cell viability, the integrity of cell monolayers and the microscopic morphology at concentrations below 0.7 mg/mL within 3 h of co-incubation with Caco-2 cells. Multichannel fluorescence monitoring revealed that the fluorescence intensity of the carrier and cargo increased with time, but not synchronously. By calculating the residual, intracellular, and transport amounts of DFPs, the material balance between the total amount of cellular transport and the dose administered was obtained.Conclusion: Based on the advantages of dual fluorescent labeling, the differential behavior of cell trafficking can be visualized and quantitatively analyzed for the cargo and carrier of DFPs. These results provide insights into the cellular transport process of holistic nanoparticles and complement our understanding of the biological behaviors of nano-DDS.Keywords: dual-fluorescent nanoparticles, tracking cellular uptake, fluorescence imaging, quantitative cell transport, drug delivery

Mohammed F Aldawsari,1 Ahmad H Alhowail,2 Md Khalid Anwer,1 Mohammed Muqtader Ahmed1 1Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-kharj, 11942, Saudi Arabia; 2Department of Pharmacology and Toxicology, College of Pharmacy, Qassim University, Buraydah, Al-Qassim, 51452, Saudi ArabiaCorrespondence: Mohammed F Aldawsari, Department of Pharmaceutics, College of Pharmacy, Prince Sattam Bin Abdulaziz University, Al-kharj, 11942, Saudi Arabia, Tel +966-555101369, Email moh.aldawsari@psau.edu.saBackground: The aim of the present investigation is to prepare baricitinib (BAR)-loaded diphenyl carbonate (DPC) β-cyclodextrin (βCD) based nanosponges (NSs) to improve the oral bioavailability.Methods: BAR-loaded DPC-crosslinked βCD NSs (B-DCNs) were prepared prepared by varying the molar ratio of βCD: DPC (1:1.5 to 1:6). The developed B-DCNs loaded with BAR were characterized for particle size, polydispersity index (PDI), zeta potential (ZP), % yield and percent entrapment efficiency (%EE).Results: Based on the above evaluations, BAR-loaded DPC βCD NSs (B-CDN3) was optimized with mean size (345.8± 4.7 nm), PDI (0.335± 0.005), Yield (91.46± 7.4%) and EE (79.1± 1.6%). The optimized NSs (B-CDN3) was further confirmed by SEM, spectral analysis, BET analysis, in vitro release and pharmacokinetic studies. The optimized NSs (B-CDN3) showed 2.13 times enhancement in bioavailability in comparison to pure BAR suspension.Conclusion: It could be anticipated that NSs loaded with BAR as a promising tool for release and bioavailability for the treatment of rheumatic arthritis and Covid-19.Keywords: baricitinib, cyclodextrin, diphenyl carbonate, crosslinker, bioavailability, BAR, DPC

Peiru Si,1 Jiaxing Lei,1 Chen Yang,1 Peipei Zhang,1 Xiaojiao Li,2 Shaohua Zheng,3 Qingqing Li,1 Jiye Zhang1 1School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, People’s Republic of China; 2Biobank, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People’s Republic of China; 3Department of Anesthesiology, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, People’s Republic of ChinaCorrespondence: Qingqing Li; Jiye Zhang, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76 Yanta Westroad, Xi’an, People’s Republic of China, Email liqingqing0217@xjtu.edu.cn; zjy2011@xjtu.edu.cnIntroduction: Hepatic ischemia-reperfusion injury (HIRI) is the main reason for liver dysfunction or failure after liver resection and liver transplantation. As excess accumulation of reactive oxygen species (ROS) is the leading factor, ceria nanoparticle, a cyclic reversible antioxidant, is an excellent candidate for HIRI.Methods: Manganese doped mesoporous hollow ceria nanoparticles (MnOx-CeO2 NPs) were prepared, and the physicochemical characteristics, such as particle size, morphology, microstructure, etc. were elucidated. The in vivo safety and liver targeting effect were examined after i.v. injection. The anti-HIRI was determined by a mouse HIRI model.Results: MnOx-CeO2 NPs with 0.40% Mn doped exhibited the strongest ROS-scavenging capability, which may due to the increased specific surface area and surface oxygen concentration. The nanoparticles accumulated in the liver after i.v. injection and exhibited good biocompatibility. In the HIRI mice model, MnOx-CeO2 NPs significantly reduced the serum ALT and AST level, decreased the MDA level and increased the SOD level in the liver, prevent pathological damages in the liver.Conclusion: MnOx-CeO2 NPs were successfully prepared and it could significantly inhibit the HIRI after i.v. injection.Graphical Abstract: Keywords: hepatic ischemia reperfusion injury, Mn-doped mesoporous hollow cerium oxide nanoparticles, oxidative stress

Michal Gorzkiewicz,1,2 Monika Marcinkowska,1 Maciej Studzian,3,4 Iwona Karwaciak,4,5 Lukasz Pulaski,3,4 Barbara Klajnert-Maculewicz1 1Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 2Department of Molecular Medicine II, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany; 3Department of Oncobiology and Epigenetics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 4Laboratory of Transcriptional Regulation, Institute of Medical Biology PAS, Lodz, Poland; 5Laboratory of Epigenetics, Institute of Medical Biology PAS, Lodz, PolandCorrespondence: Michal Gorzkiewicz, Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 141/143 Pomorska Street, Lodz, 90-236, Poland, Tel +48 42 635 41 47, Email michal.gorzkiewicz@biol.uni.lodz.plBackground: Mesalazine is one of the main drugs used to treat inflammatory bowel diseases. However, its applicability is limited by its rapid inactivation and removal from the organism, as well as the need for its membrane transporter-dependent cellular uptake to exert therapeutic effect. The present study involved the development of an innovative nanocarrier, based on poly(amidoamine) (PAMAM) dendrimer of the 4th generation, to obtain higher concentrations of the drug in the intestinal epithelial cells, thus increasing its anti-inflammatory potential. The work involved synthesis and in vitro characterization of covalent PAMAM-mesalazine conjugate with succinic linker.Results: PAMAM-mesalazine conjugate was synthesized and characterized by 1H NMR, 13C NMR, FTIR and MALDI-TOF MS. This allowed to confirm the purity of the obtained compound and intermediates. Based on the analyses, it was found that ~45 drug molecules were successfully attached to one molecule of PAMAM dendrimer. The conjugate was then characterized in terms of hydrodynamic diameter, zeta potential, spectral properties, drug release from the carrier, as well as cellular uptake and cytotoxicity in two in vitro models of gastrointestinal epithelium (CaCo-2 and HT-29 human cell lines). Analyzing cellular parameters related to the specific mechanism of action of mesalazine (inhibition of NF-κB signaling, decrease in interleukin and prostaglandin synthesis, and ROS scavenging), we showed that such a dendrimer-based carrier may enhance cellular uptake of the drug, which translated into its improved anti-inflammatory efficacy.Conclusion: The use of PAMAM macromolecule as a carrier for mesalazine increases the bioavailability of the drug, ensuring enhanced cellular uptake and bypassing the need to utilize mesalazine-specific membrane transporters. All these characteristics translate into an improved anti-inflammatory activity of mesalazine in vitro. In conjunction with appropriately designed in vivo studies, such a compound may prove to be a promising alternative to the therapeutics currently used in inflammatory bowel diseases.Graphical Abstract: Keywords: PAMAM dendrimer, mesalazine, inflammatory bowel disease, drug delivery

Tao Rui,1,2 Kai Wang,1,2 Aizhai Xiang,1 Jufeng Guo,1 Ning Tang,1 Xin Jin,1 Yimou Lin,3 Jian Liu,1 Xiaobing Zhang1,2 1Department of Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, People’s Republic of China; 2Key Laboratory of Integrated Oncology and Intelligent Medicine of ZheJiang Province, Zhejiang University School of Medicine, Hangzhou, 310003, People’s Republic of China; 3Department of Surgery, Collaborative Innovation Center for the Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University Hangzhou, Hangzhou, 310003, People’s Republic of ChinaCorrespondence: Tao Rui; Xiaobing Zhang, Email 11818248@zju.edu.cn; 11818341@zju.edu.cnBackground: Serum exosome-based liquid biopsy has significant advantages for screening and diagnosing hepatocellular carcinoma (HCC). P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are novel small silencing RNAs that have been identified to function in cancer-related signalling pathways. However, studies on the presence of piRNAs in serum exosomes from HCC patients and their diagnostic values in HCC are not well reported. Our aim is to validate serum exosome-derived piRNAs as the valuable component of liquid biopsy for diagnosing HCC.Methods: We used small RNA (sRNA) sequencing to profile piRNAs from serum exosomes and describe the base distribution characteristics of serum exosome-derived piRNAs. Serum exosomes from 125 HCC patients and 44 nontumor donors were included in this study.Results: We found that piRNAs were components of serum exosomes from HCC patients. A total of 253 differentially expressed serum exosome-derived piRNAs were screened from HCC compared with the piRNAs from nontumor donors. Serum exosome-derived piRNAs from HCC displayed a distinctive base distribution. To further confirm the potential diagnostic value of serum exosome-derived piRNAs in HCC, we detected the levels of the top 5 upregulated piRNAs in our Chinese cohort. The training set and validation set both showed that all 5 piRNAs were dramatically increased in the serum exosomes from HCC compared with the piRNAs from non-tumour donors. The piRNAs could strongly identify HCC patients from non-tumour donors according to the area under the receiver operating characteristic (AUROC) model. Additionally, the piRNAs could also present significant values for the diagnosis of HCC with low tumour burden.Conclusion: piRNAs enriched the components of serum exosomes from HCC and could serve as promising biomarkers for HCC diagnosis.Keywords: biomarker, exosome, sRNA-seq, HCC, piRNA

Hao Deng,1,* Yumei Wang,1,* Yue Zhou,1,* Dongliang Zhai,1 Jie Chen,1 Shilei Hao,2 Xiaoliang Chen1 1Department of Nuclear Medicine, Chongqing University Cancer Hospital, Chongqing, 400030, People’s Republic of China; 2Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400030, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoliang Chen, Department of Nuclear Medicine, Chongqing University Cancer Hospital, No. 181 HanYu St, Shapingba District, Chongqing, 400030, People’s Republic of China, Tel/Fax +86 023-65079156, Email chenxiaoliang26@163.com Shilei Hao, Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, 174 Shazhengjie, Shapingba District, Chongqing, 400030, People’s Republic of China, Tel +86023-135 9463 5765, Email Shilei_hao@cqu.edu.cnBackground: Prostate cancer (PCa) ranks second in the incidence of all malignancies in male worldwide. The presence of multi-organ metastases and tumor heterogeneity often leads to unsatisfactory outcomes of conventional radiotherapy treatments. This study aimed to develop a novel folate-targeted nanohydroxyapatite (nHA) coupling to deliver adriamycin (Doxorubicin, DOX), 32P, and 99mTc simultaneously for the diagnosis and treatment of prostate-specific membrane antigen (PSMA) positive prostate cancer.Methods: The spherical nHA was prepared by the biomimetic method and characterized. Folic acid (FA) was coupled to nHA with polyethylene glycol (PEG), and the grafting ratio of PEG-nHA and FA-PEG-nHA was determined by the thermogravimetric analysis (TGA) method. In addition, 32P, 99mTc, and DOX were loaded on nHA by physisorption. And the labeling rate and stability of radionuclides were measured by a γ-counter. The loading and release of DOX at different pH were determined by the dialysis method. Targeting of FA-PEG-nHA loaded with 99mTc was verified by in vivo SPECT imaging. In vitro anti-tumor effect of 32P/DOX-FA-PEG-nHA was assessed with apoptosis assay. The safety of the nano-drugs was verified by histopathological analysis.Results: The SEM images showed that the synthesized nHA was spherical with uniform particle size (average diameter of about 100nm). The grafting ratio is about 10% for PEG and about 20% for FA. The drug loading and the delayed release of DOX at different pH confirmed its long-term therapeutic ability. The labeling of 32P and 99mTc was stable and the labeling rate was great. SPECT showed that FA-PEG-nHA showed well in vivo tumor targeting and less damage to normal tissues.Conclusion: FA-targeted nHA loaded with 32P, 99mTc, and DOX may be a new diagnostic and therapeutic strategy for targeting PSMA-positive prostate cancer tumors, which may achieve better therapeutic results while circumventing the severe toxic side effects of conventional chemotherapeutic agents.Keywords: prostate cancer, folic acid targeting, nanohydroxyapatite, 99mTc/ 32P, adriamycin

Noha Khalifa Abo Aasy,1 Sarah A El-Lakany,1 Perusi M Masanga,2 Elbadawy A Kamoun,3,4 Shahira H EL-Moslamy,5 Marwa Abu-Serie,6 Rania G Aly,7 Nazik A Elgindy1 1Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt; 2St John’s University of Tanzania, Dodoma, Tanzania; 3Polymeric Materials Research Department, Advanced Technology and New Materials Research Institute (ATNMRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, Egypt; 4Nanotechnology Research Center (NTRC), The British University in Egypt (BUE), El-Sherouk City, Cairo, 11837, Egypt; 5Bioprocess Development Department (BID), Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab City, Alexandria, 21934, Egypt; 6Medical Biotechnology Department (MBD), Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-city), New Borg El-Arab City, Alexandria, 21934, Egypt; 7Department of Surgical Pathology, Faculty of Medicine, Alexandria University, Alexandria, EgyptCorrespondence: Sarah A El-Lakany, Department of Industrial Pharmacy Faculty of Pharmacy, Alexandria University, 1 El Khartoum Square, PO Box 21521, Alexandria, Egypt, Tel +201002828663, Fax +203 4871668, Email sarah.ellakany@alexu.edu.eg; sarah.abdalla89@gmail.comPurpose: Diabetes mellitus is among the disrupting factors of orchestrated events in wound healing. This necessitates the urge for tailored medications, which are continually offered by nano-sized materials. Herein, we present greenly synthesized copper oxide nanoparticles (CuO NPs), obtained from either Punica granatum L. (PG) or Pisidium guajava L. (GV) extract, to function as potent bactericidal and fungicidal materials that promote regeneration and healing of the targeted diabetic wounded tissues.Methods: PG or GV plant extracts were compared as source of reducing agents for CuO NPs synthesis process. The yield and photocatalytic degradation potential were compared. NPs obtained from the superior extract, PG, were characterized using particles size, zeta potential, XRD, TEM, SEM, and EDX. The antimicrobial effects were evaluated on multidrug-resistant human pathogens and then the percentage biofilm inhibitory concentration was determined. The cytotoxicity and wound scratch study were conducted on a normal human skin cell line. In-vivo wound healing activity in diabetic rats was assessed along with histopathological and immunohistochemical examination of CD45 and α-SMA.Results: The greenly synthesized CuO NPs are spherical in shape having a diameter of 233nm. CuO NPs (250μg/mL) acted as promising biocontrol agent against a variety of multidrug-resistant human pathogens. They significantly exhibited 29.460± 0.811% healing of the scratched wound compared to only 2.001± 0.155% for the control. Wound healing experiments revealed the safety of a low CuO NPs concentration in a diabetic animal model as well as on human normal skin fibroblast cell line. The treated group with a dose of 2mg/cm2 showed superior results with a WC50 value of 7.2 days, and 92% wound contraction after 13-days. Immunohistochemical investigation of the same group demonstrated well-established fibrous tissue (5.7± 3.7/HPF), and an amplified granulation tissue of recently developed blood vessels (70± 1.5/HPF).Conclusion: Green synthesized CuO NPs could overcome drug resistance and promote wound healing process effectively.Keywords: plant extraction, metal ion, cell compatibility, wound contraction, biofilm inhibitory concentration

Qing Xia,* Yue Tang,* Wang Li, Tingting Liang, Yue Zhou, Jun Liu, Feila Liu School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Feila Liu, School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing, People’s Republic of China, Tel +86-15123002638, Fax +86 2362563190, Email liufeila@cqut.edu.cnIntroduction: The immunosuppressive tumor microenvironment (TME) of solid tumors inhibits most drug delivery system-based nanomaterials from achieving deep penetration in tumor tissue and interferes with T cell activity in terms of differentiation and exhaustion, which is becoming a critical therapy hurdle for solid tumors. Therefore, developing a therapeutic strategy with abilities of rapid establishment of tumor-targeted cells, elimination of immune obstacles, and enhanced active immunization is very important, while is still a big challenge.Methods: A new strategy was explored to enhance immune therapy via the conjugation of microRNA155 (miR) to the surface of therapeutic monocyte with graphene quantum dots (GQDs).Results: TME was reversed using surface-engineered monocyte immunotherapy via reprogramming pro-tumoral M2 TAMs into antitumor M1, and thus tumor elimination was dramatically enhanced.Conclusion: Such a surface-engineered monocyte immunotherapy has been demonstrated to be well tolerated to intravenous administration and bio-compatible, showing the potential to be extended for the solid tumor treatment.Graphical Abstract: Keywords: solid tumor, surface-engineered monocyte, GQDs, miR155, immunotherapy

Lingzhou Zhao,* Jiali Gong,* Qinli Qi,* Changcun Liu, Hongxing Su, Yan Xing, Jinhua Zhao Department of Nuclear Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jinhua Zhao; Yan Xing, Department of Nuclear Medicine, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, No. 100, Haining Road, Shanghai, 200080, People’s Republic of China, Tel/Fax +86 21 3779 8352, Email zhaojinhua1963@126.com; xy.1@163.comPurpose: The unique structure of nanobodies is advantageous for the development of radiopharmaceuticals for nuclear medicine. Nanobodies targeted to human epidermal growth factor receptor 2 (HER2) can be used as tools for the imaging and therapy of HER2-overexpressing tumors. In this study, we aimed to describe the generation of a 131I-labeled anti-HER2 nanobody as a targeted radionuclide therapy (TRNT) agent for HER2-positive breast cancer.Methods: The anti-HER2 nanobody NM-02 was labeled with 131I using the iodogen method, and its radiochemical purity and stability in vitro were assessed. The pharmacokinetic profile of 131I-NM-02 was investigated in normal mice. Tumor accumulation, biodistribution, and therapeutic potential of 131I-NM-02 were evaluated in HER2-positive SKBR3 xenografts; HER2-negative MB-MDA-231 xenografts were used as the control group.Results: 131I-NM-02 could be readily prepared with satisfactory radiochemical purity and stability in vitro. Apparent tumor uptake was observed in HER2-positive tumor-bearing mice with rapid blood clearance and favorable biodistribution. 131I-NM-02 could significantly inhibit tumor growth and extend the life of these mice with good organ compatibility. Negligible tumor accumulation and inhibitory effects of 131I-NM-02 were observed in the negative control group.Conclusion: 131I-NM-02 has the potential to be explored as a novel tool for TRNT of HER2-positive breast cancer.Keywords: human epidermal growth factor receptor 2, nanobody, 131I, targeted radionuclide therapy

Mei Fu,1 Jun Li,2 Mingchong Liu,1 Chensong Yang,1 Qidong Wang,1 Hongrui Wang,3 Bingdi Chen,2 Qingge Fu,3 Guixin Sun1 1Department of Traumatic Surgery, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 2Institute for Regenerative Medicine, Shanghai East Hospital, The Institute for Biomedical Engineering & Nano Science, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 3Department of Orthopedic Trauma, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai, People’s Republic of ChinaCorrespondence: Guixin Sun; Qingge Fu, Email sunguixin@tongji.edu.cn; fqg7821@163.comBackground: Immune responses and osteogenesis differentiation induced by implants are crucial for bone tissue regeneration. Consideration of only one of those properties is not sufficient. To investigate the synergistic actions, we designed alginate/graphene oxide/sericin/nanohydroxyapatite (Alg/GO/Ser/nHAP) nanocomposite hydrogels with both osteoimmunomodulatory and osteoinductive activities. This study aimed to explore the effect of hydrogel with osteoimmunomodulatory properties on promoting osteogenesis of bone marrow stem cells (BMSCs).Methods: Alg/GO/Ser/nHAP nanocomposite hydrogel was fabricated and was characterized by SEM, FTIR, XRD, stress-strain, rheology, swelling and degradation. After the impact of sericin on M2 macrophage polarization was identified, the BMSCs viability and adhesion were evaluated by CCK8 assay, live/dead staining, cytoskeleton staining. The cell osteogenic differentiation was observed by ALP/ARS staining, immunofluorescence staining, RT-PCR, and Western blotting, respectively. Rat cranial defect model was used to assess osteoimmunomodulatory effects of scaffolds in vivo by micro‑computed tomographic, histological, and immunohistochemical analyses after 8 weeks of healing.Results: In vitro experiments revealed that the hydrogel presented desirable mechanical strength, stability, porosity, and biocompatibility. Significantly, sericin and nHAP appeared to exert synergistic effects on bone regeneration. Sericin was observed to inhibit the immune response by inducing macrophage M2-type polarization to create a positive osteoimmune microenvironment, contributing to improving osseointegration at the bone-implant interface to promote osteogenesis. However, the osteogenic differentiation in rat BMSCs was further enhanced by combining nHAP and sericin in the nanocomposite hydrogel. Eventually, the hydrogel was implanted into the rat cranial defect model, assisting in the reduction of local inflammation and efficient bone regeneration.Conclusion: The nanocomposite hydrogel stimulated bone formation by the synergistic effects of immunomodulation of macrophage polarization by sericin and direct osteogenic induction by nHAP, demonstrating that such a scaffold that modulates the osteoimmune microenvironment to promote osteogenesis is a promising approach for the development of bone tissue engineering implants in the future.Graphical Abstract: Keywords: hydrogel, nano-hydroxyapatite, sericin, osteoimmunomodulation, osteoinduction, bone regeneration

Mingyuan Li,1 Meng Li,1 Xinyi Li,1 Wanhui Shao,1 Xiujuan Pei,2 Ruyue Dong,1 Hongmeng Ren,1 Lin Jia,1 Shiqin Li,1 Wenlin Ma,1 Yi Zeng,1 Yun Liu,1 Hua Sun,1 Peng Yu1 1College of Biotechnology/Tianjin Enterprise Key Laboratory for Application Research of Hyaluronic Acid, Tianjin University of Science & Technology, Tianjin, 300457, People’s Republic of China; 2Tianjin Shiji Kangtai Biomedical Engineering Co.,Ltd, Tianjin, 300462, People’s Republic of ChinaCorrespondence: Hua Sun; Peng Yu, Email sunhua@tust.edu.cn; yupeng@tust.edu.cnPurpose: In the present study, we prepared collagen liposomes with the addition of polyol, which is expected to not only increase the solubility of collagen but also improve skin penetration.Methods: Collagen liposomes were prepared by the film dispersion method, and their characteristics, integrity and biosafety were evaluated by Fourier transform infrared spectroscopy (FTIR), UV-VIS spectroscopy, polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS) and transmission electron microscope (TEM). The transdermal absorption of collagen and collagen liposomes were tested by an ex vivo horizontal Valia-Chien diffusion cell system.Results: We first demonstrated that collagen extracted from bovine Achilles tendon was type I collagen. The results of DLS measurement and TEM observation showed that the collagen liposomes were spherical in shape with average diameter (75.34± 0.93 nm) and maintained high stability at low temperature (4°C) for at least 42 days without toxicity. The encapsulation rate of collagen liposomes was 57.80 ± 0.51%, and SDS-PAGE analysis showed that collagen was intact in liposomes. Finally, permeability studies indicated that the collagen-loaded liposomes more easily penetrated the skin compared to collagen itself.Conclusion: This study proposed a new method to improve the bioavailability and permeability of bovine type I collagen, which improves the applicability of collagen in biomedicine, cosmeceuticals and pharmaceutical industries.Graphical Abstract: Keywords: collagen, liposome, physicochemical properties of collagen, skin penetration, drug delivery

Wenli Hui, Wenhua Mu, Cong Zhao, Dan Xue, Zihua Zhong, Yani Fang, Ming Gao, Xiao Li, Shihao Gao, Kaiyue Liu, Kunping Yan College of Life Science, Northwest University, Xi’an City, Shaanxi Province, 710069, People’s Republic of ChinaCorrespondence: Kunping Yan, Email kpyan@nwu.edu.cnIntroduction: A limitation of hemoglobin-based oxygen carriers (HBOCs) as oxygen therapeutics is unpolymerized hemoglobin, which induces vasoconstriction leading to hypertension. The removal of unpolymerized hemoglobin from polymerized hemoglobin (PolyHb) is complex, expensive, and time-consuming.Methods: Herein, we developed a method to completely polymerize hemoglobin almost without unpolymerized hemoglobin. Hemoglobin was adsorbed on the anion-exchange resin Q Sepharose Fast Flow or DEAE Sepharose Fast Flow, and acetal, a crosslinker prepared from glutaraldehyde and ethylene glycol, was employed to polymerize the hemoglobin. The polymerization conditions, including reaction time, pH, resin type, and molar ratios of glutaraldehyde to ethylene glycol and hemoglobin to acetal, were optimized. The blood pressure and blood gas of mice injected with PolyHb were monitored as well.Results: The optimal polymerization condition of PolyHb was when the molar ratio of glutaraldehyde to ethylene glycol was 1:20, and the molar ratio of 10 mg/mL hemoglobin adsorbed on anion-exchange resin to glutaraldehyde was 1:300 for 60 min. Under optimized reactive conditions, hemoglobin was almost completely polymerized, with < 1% hemoglobin remaining unpolymerized, and the molecular weight of PolyHb was more centrally distributed. Furthermore, hypertension was not induced in mice by PolyHb, and there were also no pathological changes observed in arterial oxygen, blood gas, electrolytes, and some metabolic indicators.Conclusion: The findings of this study indicate that the use of solid-phase polymerization and acetal is a highly effective and innovative approach to HBOCs, resulting in the almost completely polymerized hemoglobin. These results offer promising implications for the development of new methods for preparing HBOCs.Graphical Abstract: Keywords: polymerized hemoglobin, glutaraldehyde, acetal, solid-phase adsorption, hemoglobin-based oxygen carriers, HBOCs

Yuxun Wei,1– 4,&ast; Yumeng Wei,1– 4,&ast; Lin Sheng,1– 4 Jingwen Ma,1– 4 Zhilian Su,1– 4 Jie Wen,1– 4 Lanmei Li,5 Qiang Jia,2,6 Huiyang Liu,1– 4 Hui Si,2,4 Linjin Xiong,1– 4 Jinglin Chen,1– 4 Ju Cheng,3,4 Ying Zuo,2,7 Hongru Yang,8 Ling Zhao2– 4 1Key Laboratory of Medical Electrophysiology, Ministry of Education, School of Pharmacy of Southwest Medical University, Luzhou, People’s Republic of China; 2Key Laboratory of Medical Electrophysiology, Ministry of Education, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University; Luzhou, Sichuan, People’s Republic of China; 3Central Nervous System Drug Key Laboratory of Sichuan Province, School of Pharmacy of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 4Luzhou Key Laboratory of Traditional Chinese Medicine for Chronic Diseases Jointly Built by Sichuan and Chongqing, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 5Nanchong Key Laboratory of Individualized Drug Therapy, Department of Pharmacy, Nanchong Central Hospital, The Second Clinical Medical College, North Sichuan Medical College, Nanchong, Sichuan, People’s Republic of China; 6Ethics Committee Office, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 7Department of Comprehensive Medicine, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China; 8Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ling Zhao, Key Laboratory of Medical Electrophysiology, Ministry of Education, Luzhou Key Laboratory of Traditional Chinese Medicine for Chronic Diseases Jointly Built by Sichuan and Chongqing, The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China, Tel/Fax +86 830 3160093, Email zhaoling-998@163.com Hongru Yang, Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, People’s Republic of China, Tel/Fax +86 830 8585668, Email yanghongru801@126.comPurpose: The present study aimed to construct a co-loading platform encapsulating curcumin and paclitaxel at ratios of 2:1– 80:1 (w/w) designated “CU-PTX-LNP” and explored the synergistic effects of CU-PTX at different composite proportions on liver cancer cells using the combination index (CI) method.Methods: The CU lipid nanoplatform (CU-LNP) formulation was optimized via single-factor and orthogonal experiments. Various concentrations of PTX were added to the optimal formulation of CU-LNP to generate CU-PTX-LNP and the nanoplatform characterized via differential scanning calorimetry (DSC), transmission electron microscope (TEM), X-ray diffraction (XRD), zeta potential, polydispersity index (PDI), and size analyses. The cumulative release, stability, and cytotoxicity of CU-PTX-LNP in LO2, HepG2, and SMMC-7221 cells were assessed in vitro, followed by safety investigation and pharmacokinetic studies in vivo. The anti-tumor activity of CU-PTX-LNP was also evaluated using nude mice.Results: CU-PTX-LNP formulations containing CU:PTX at a range of proportions (2:1– 80:1; w/w) appeared as uniformly dispersed nanosized spherical particles with high entrapment efficiency (EE> 90%), sustained release and long-lasting stability. Data from in vitro cytotoxicity assays showed a decrease in the IC50 value of PTX of CU-PTX-LNP (by 5.47– 332.7 times in HepG2 and 4.29– 143.21 times in SMMC-7221 cells) compared to free PTX. In vivo, CU-PTX-LNP displayed excellent biosafety, significant anti-tumor benefits and enhanced pharmacokinetic behavior with longer mean residence time (MRT(0-t); CU: 4.31-fold, PTX: 4.61-fold) and half-life (t1/2z; CU: 1.83-fold, PTX: 2.28-fold) relative to free drugs.Conclusion: The newly designed CU-PTX-LNP platform may serve as a viable technological support system for the successful production of CU-PTX composite preparations.Keywords: curcumin, paclitaxel, lipid nano platform, synergistic effect

Afrah E Mohammed,1,&ast; Sahar S Alghamdi,2,3 Ashwag Shami,1,&ast; Rasha Saad Suliman,4 Kawther Aabed,1 Modhi O Alotaibi,1 Ishrat Rahman5 1Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, 11671, Saudi Arabia; 2Department of Pharmaceutical Sciences, College of Pharmacy, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 3King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia; 4Department of Pharmacy, Fatima College of Health Sciences, Abu Dhabi, 3798, United Arab Emirates; 5Department of Basic Dental Sciences, College of Dentistry, Princess Nourah bint Abdulrahman University, Riyadh, 11671, Saudi Arabia&ast;These authors contributed equally to this workCorrespondence: Ishrat Rahman, Department of Basic Dental Sciences, College of Dentistry, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671, Saudi Arabia, Email imrahman@pnu.edu.saIntroduction: Biogenic silver nanoparticles (AgNPs) may be a feasible therapeutic option in the research and development towards selectively targeting specific cancers and microbial infections, lending a role in precision medicine. In-silico methods are a viable strategy to aid in drug discovery by identifying lead plant bioactive molecules for further wet lab and animal experiments.Methods: Green synthesis of M-AgNPs was performed using the aqueous extract from the Malvaviscus arboreus leaves, characterized using UV spectroscopy, FTIR, TEM, DLS, and EDS. In addition, Ampicillin conjugated M-AgNPs were also synthesized. The cytotoxic potential of the M-AgNPs was evaluated using the MTT assay on MDA-MB 231, MCF10A, and HCT116 cancer cell lines. The antimicrobial effects were determined using the agar well diffusion assay on methicillin-resistant S. aureus (MRSA) and S. mutans, E. coli, and Klebsiella pneumoniae. Additionally, LC-MS was used to identify the phytometabolites, and in silico techniques were applied to determine the pharmacodynamic and pharmacokinetic profiles of the identified metabolites.Results: Spherical M-AgNPs were successfully biosynthesized with a mean diameter of 21.8 nm and were active on all tested bacteria. Conjugation with ampicillin increased the susceptibility of the bacteria. These antibacterial effects were most predominant in Staphylococcus aureus (p < 0.0001). M-AgNPs had potent cytotoxic activity against the colon cancer cell line (IC50=29.5 μg/mL). In addition, four secondary metabolites were identified, Astragalin, 4-hydroxyphenyl acetic acid, Caffeic acid, and Vernolic acid. In silico studies identified Astragalin as the most active antibacterial and anti-cancer metabolite, binding strongly to the carbonic anhydrase IX enzyme with a comparatively higher number of residual interactions.Discussion: Synthesis of green AgNPs presents a new opportunity in the field of precision medicine, the concept centered on the biochemical properties and biological effects of the functional groups present in the plant metabolites used for reduction and capping. M-AgNPs may be useful in treating colon carcinoma and MRSA infections. Astragalin appears to be the optimal and safe lead for further anti-cancer and anti-microbial drug development.Keywords: biogenic AgNPs, LC-MS, Astragalin, biological activity

Noran M Tawfik,1,2 Mohammed S Teiama,3,4 Sameh Samir Iskandar,5 Ahmed Osman,1,6 Sherif F Hammad7,8 1Biotechnology Program, Basic and Applied Sciences Institute, Egypt-Japan University of Science and Technology, Alexandria, Egypt; 2Department of Zoology, Faculty of Science, Suez Canal University, Ismailia, Egypt; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Cairo, Egypt; 4Department of Pharmaceutics, Faculty of Pharmacy, Galala University, Suez, Egypt; 5Fellow and Head of Surgical Oncology Department, Ismailia Teaching Oncology Hospital (GOTHI), Ismailia, Egypt; 6Department of Biochemistry, Faculty of Science, Ain Shams University, Cairo, Egypt; 7PharmD Programs, Egypt-Japan University of Science and Technology, Alexandria, Egypt; 8Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Cairo, EgyptCorrespondence: Noran M Tawfik; Ahmed Osman, Biotechnology Program, Basic and Applied Sciences Institute, Egypt-Japan University of Science and Technology, New Borg El-Arab, Alexandria, 21934, Egypt, Email noran.tawfik@ejust.edu.eg; ahmed.osman@ejust.edu.egBackground: Thalidomide (THD) and its analogues were recently reported as a promising treatment for different types of solid tumors due to their antiangiogenic effect.Methods: In this work, we synthesized a novel THD analogue (TA), and its chemistry was confirmed with different techniques such as IR, mass spectroscopy, elemental analysis as well as 1H and 13C NMR. To increase solubility and anticancer efficacy, a new oil in water (O/W) nanoemulsion (NE) was used in the formulation of the analogue. The novel formula’s surface charge, size, stability, FTIR, FE-TEM, in vitro drug release and physical characteristics were investigated. Furthermore, molecular docking studies were conducted to predict the possible binding modes and molecular interactions behind the inhibitory activities of the THD and TA.Results: TA showed a significant cytotoxic activity with IC50 ranging from 0.326 to 43.26 μmol/mL when evaluated against cancerous cells such as MCF-7, HepG2, Caco-2, LNCaP and RKO cell lines. The loaded analogue showed more potential cytotoxicity against MDA-MB-231 and MCF-7-ADR cell lines with IC50 values of 0.0293 and 0.0208 nmol/mL, respectively. Moreover, flow cytometry of cell cycle analysis and apoptosis were performed showing a suppression in the expression levels of TGF-β, MCL-1, VEGF, TNF-α, STAT3 and IL-6 in the MDA-MB-231 cell line.Conclusion: The novel NE formula dramatically reduced the anticancer dosage of TA from micromolar efficiency to nanomolar efficiency. This indicates that the synthesized analogue exhibited high potency in the NE formulation and proved its efficacy against triple-negative breast cancer cell line.Graphical Abstract: Keywords: thalidomide analogue, antiangiogenic, nanoemulsion, cytotoxicity, triple-negative breast cancer, docking study

Abeer S Hassan,1 Amal Hofni,2 Mohammed AS Abourehab,3 Iman AM Abdel-Rahman4 1Department of Pharmaceutics, Faculty of Pharmacy, South Valley University, Qena, Egypt; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, South Valley University, Qena, Egypt; 3Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt; 4Department of Pharmacognosy, Faculty of Pharmacy, South Valley University, Qena, EgyptCorrespondence: Abeer S Hassan, Department of Pharmaceutics, Faculty of Pharmacy, South Valley University, Qena, Egypt, Tel +201012060262, Email abeer.saad@svu.edu.egIntroduction: Ginger extract (GE) has sparked great interest due to its numerous biological benefits. However, it suffers from limited skin permeability, which challenges its transdermal application. The target of the current work was to develop transethosomes as a potential nanovehicle to achieve enhanced transdermal delivery of GE through the skin.Methods: GE–loaded transethosomes were prepared by cold injection using different edge activators. The fabricated nanovesicles were evaluated for particle size, ζ-potential, encapsulation efficiency, and in vitro drug release. The selected formulation was then laden into the hydrogel system and evaluated for ex vivo permeability and in vivo anti-inflammatory activity in a carrageenan-induced rat-paw edema model.Results: The selected formulation comprised of sodium deoxycholate exhibited particle size of 188.3± 7.66 nm, ζ-potential of − 38.6± 0.08 mV, and encapsulation efficiency of 91.0%± 0.24%. The developed transethosomal hydrogel containing hydroxypropyl methylcellulose was homogeneous, pseudoplastic, and demonstrated sustained drug release. Furthermore, it exhibited improved flux (12.61± 0.45 μg.cm2/second), apparent skin permeability (2.43± 0.008× 10− 6 cm/second), and skin deposition compared to free GE hydrogel. In vivo testing and histopathological examination revealed that the GE transethosomal hydrogel exhibited significant inhibition of edema swelling compared to free GE hydrogel and ketoprofen gel. The animals that were treated with ginger transethosome hydrogel showed a significant decrement in reactive oxygen species and prostaglandin E2 compared to untreated animals.Conclusion: Transethosomes might be a promising new vehicle for GE for effective skin permeation and anti-inflammation. To the best of our knowledge, this work is the first utilization of transethosomes laden into hydrogel as a novel transdermal delivery system of GE.Graphical Abstract: Keywords: nanovesicles, transethosomes, ginger, ex vivo permeation, transdermal delivery, inflammation

Woo-Jin Jeon,1,&ast; Hong-Ki Lee,2,3,&ast; Young-Guk Na,1 Minwoo Jung,1 Su-Cheol Han,2 Jeong Ho Hwang,2 Eunhye Jung,4 Dasom Hwang,4 Jin Soo Shin,4 Cheong-Weon Cho1 1College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, 34134, Republic of Korea; 2Center for Companion Animal New Drug Development, Jeonbuk Branch, Korea Institute of Toxicology (KIT), Jeongeup, Jeollabuk-do, 53212, Republic of Korea; 3Human Health Risk Assessment Center, Jeonbuk Branch, Korea Institute of Toxicology (KIT), Jeongeup, Jeollabuk-do, 53212, Republic of Korea; 4Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea&ast;These authors contributed equally to this workCorrespondence: Jin Soo Shin, Infectious Diseases Therapeutic Research Center, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea, Email jsshin@krict.re.kr Cheong-Weon Cho, College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea, Email chocw@cnu.ac.krIntroduction: The ongoing SARS-CoV-2 pandemic has affected public health, the economy, and society. This study reported a nanotechnology-based strategy to enhance the antiviral efficacy of the antiviral agent remdesivir (RDS).Results: We developed a nanosized spherical RDS-NLC in which the RDS was encapsulated in an amorphous form. The RDS-NLC significantly potentiated the antiviral efficacy of RDS against SARS-CoV-2 and its variants (alpha, beta, and delta). Our study revealed that NLC technology improved the antiviral effect of RDS against SARS-CoV-2 by enhancing the cellular uptake of RDS and reducing SARS-CoV-2 entry in cells. These improvements resulted in a 211% increase in the bioavailability of RDS.Conclusion: Thus, the application of NLC against SARS-CoV-2 may be a beneficial strategy to improve the antiviral effects of antiviral agents.Keywords: remdesivir, nanostructured lipid carrier, antiviral effect, SARS-CoV-2, virus entry

Madalitso M Mlozen,1,2 Jacqueline Van Marwijk,1 Brendan Shane Wilhelmi,1 Chris Whiteley1 1Department of Biochemistry and Microbiology, Rhodes University, Makhanda (Grahamstown), South Africa; 2Malawi Adventist University, Malamulo Campus, Department of Biomedical Sciences, Makwasa, MalawiCorrespondence: Madalitso M Mlozen, Malawi Adventist University, Malamulo campus, Department of Biomedical Sciences, P.O.Box 55, Makwasa, Tel +265 884628334, Email mlozenim@mchs.advenist.orgBackground: Regardless of the efforts to ease cases of human African trypanosomiasis (HAT), an increased number of cases get reported annually. This is because of drug resistant Trypanosoma brucei (Tb), the causative agent of the illness. This has renewed the need for creative methods to find new anti-trypanosomal drugs. The blood stream form (BSF) of the parasite depends exclusively on the glycolytic pathway for energy production while it is in the human host. Interruptions in this pathway efficiently kills the parasite. Trypanosoma brucei hexokinase (TbHK) is the first enzyme in glycolysis, and any effectors or inhibitors of TbHK would have potential as anti-trypanosomal agents.Methods: TbHK and human glucokinase (hGCK) were over-expressed with a 6 histidine-tag in E. coli BL21(DE3) cells having the pRARE2 plasmid.Results: TbHK had thermal and pH stability between 30°C and 55°C and 7.5 and 8.5, respectively, while hGCK exhibited thermal and pH stability between 30°C and 40°C and 7.0 and 8.0, respectively. Kinetically, TbHK had a Km of 39.3 μM, Vmax of 0.066 μmol.min− 1.mL− 1, kcat of 2.05 min− 1 and kcat/Km of 0.0526 min− 1.μmol− 1. hGCK exhibited a Km of 4.5 μM, Vmax of 0.032 μnmol.min− 1.mL− 1, kcat of 11.25 min− 1, and kcat/Km of 2.5 min− 1.μmol− 1. Interaction kinetic studies of silver nanoparticles (AgNPs) (0.1 μM) of average size of 6 nm with TbHK and hGCK were conducted. AgNPs selectively inhibited TbHK over hGCK. TbHK showed a non-competitive inhibition with a 50% and 28% decrease in Vmax, and kcat/km, respectively. HsGCK showed a 33% increase in affinity, 9% decrease in Vmax, and a 50% increase in enzyme efficiency.Conclusion: The observed pattern of hGCK and AgNPs falls under the uncompetitive inhibition. The observed highly selective inhibitory effects of AgNPs between TbHK and hGCK may be used in development of new anti-trypanosomal drugs.Keywords: nanoparticles, hexokinase, trypanosomiasis, nanomedicine

Meng Chen,1,2 Yang Li,1,2 Wen-Xiu Hou,3 Da-Yong Peng,1,2 Jing-Kun Li,1,2 Hao-Xuan Zhang1,2 1Department of Orthopedic Surgery, The First Affiliated Hospital of Shandong First Medical University, Jinan, Shandong Province, 250014, People’s Republic of China; 2Department of Orthopedic Surgery, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan, Shandong Province, 250014, People’s Republic of China; 3Department of Spine Surgery, Shandong University Qilu Hospital, Jinan, Shandong, 250000, People’s Republic of ChinaCorrespondence: Hao-Xuan Zhang, Department of Orthopedics, The First Affiliated Hospital of Shandong First Medical University, No. 16766, Lixia District, Jingshi Road, Jinan, Shandong Province, 250014, People’s Republic of China, Tel/Fax +86531-89268540, Email hoho0605@126.comPurpose: The repair and treatment of infected bone defects (IBD) is a common challenge faced by orthopedic clinics, medical materials science, and tissue engineering.Methods: Based on the treatment requirements of IBD, we utilized multidisciplinary knowledge from clinical medicine, medical materials science, and tissue engineering to construct a high-efficiency vancomycin sustained-release system with nanodiamond (ND) and prepare a composite scaffold. Its effect on IBD treatment was assessed from materials, cytology, bacteriology, and zoology perspectives.Results: The results demonstrated that the Van-ND-45S5 scaffold exhibited an excellent antibacterial effect, biocompatibility, and osteogenesis in vitro. Moreover, an efficient animal model of IBD was established, and a Van-ND-45S5 scaffold was implanted into the IBD. Radiographic and histological analyses and bone repair-related protein expression, confirmed that the Van-ND-45S5 scaffold had good biocompatibility and osteogenic and anti-infective activities in vivo.Conclusion: Collectively, our findings support that the Van-ND-45S5 scaffold is a promising new material and approach for treating IBD with good antibacterial effects, biocompatibility, and osteogenesis.Graphical Abstract: Keywords: antibacterial effect, nanodiamond, scaffold, infected bone defects

Xiao Lei,1,&ast; Guodong Tan,2,&ast; Yiming Wang,3,&ast; Li Chen,4,&ast; Yuan Cao,1 Bingxin Si,2 Zhen Zhen,2 Bei Li,5 Yan Jin,5 Wei Wang,6 Fang Jin1 1Shaanxi Clinical Research Center for Oral Disease & Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 2Air Force Medical Center, Fourth Military Medical University, Beijing, People’s Republic of China; 3Shaanxi Clinical Research Center for Oral Diseases & Department of Oral Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 4Shaanxi Key Laboratory of Energy Chemical Process Intensification & Institute of Polymer Science in Chemical Engineering, School of Chemical Engineering and Technology, Xi’an Jiao Tong University, Xi’an, Shaanxi, People’s Republic of China; 5State Key Laboratory of Military Stomatology & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 6State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Fang Jin, Shaanxi Clinical Research Center for Oral Disease & Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China, Email fangjin191@163.com Wei Wang, State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China, Email weiwang_0510@163.comIntroduction: Osteoarthritis (OA) is a chronic degenerative joint disease accompanied by an elevated macrophage proinflammatory phenotype, which is triggered by persistent pathologically elevated calcium ion levels in mitochondria. However, existing pharmacological compounds targeting the inhibition of mitochondrial calcium ion (m[Ca2+]) influx are currently limited in terms of plasma membrane permeability and low specificity for ion channels and transporters. In the present study, we synthesized mesoporous silica nanoparticle-amidated (MSN)-ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)/triphenylphosphine (TPP)-polyethylene glycol (PEG) [METP] nanoparticles (NPs), which specifically target mitochondria and block excess calcium ion influx.Methods: m[Ca2+] overload in OA mouse bone marrow-derived macrophages (BMDMs) was detected by a fluorescence probe. A tissue in situ fluorescence colocalization assay was used to evaluate METP NP uptake by macrophages. BMDMs from healthy mice were pretreated with a concentration gradient of METP NPs followed by lipopolysaccharide (LPS) stimulation and detection of m[Ca2+] levels in vitro. The optimal METP NP concentration was further applied, and the endoplasmic reticulum (ER) and cytoplasm calcium levels were detected. The inflammatory phenotype was measured by surface markers, cytokine secretion and intracellular inflammatory gene/protein expression. A Seahorse cell energy metabolism assay was performed to elucidate the mechanism by which METP NPs reverse the BMDM proinflammatory phenotype.Results: The present study identified calcium overload in BMDM mitochondria of OA mice. We demonstrated that METP NPs reversed the increased m[Ca2+] levels in mitochondria and the proinflammatory phenotype of BMDMs, with both in vivo and in vitro experiments, via the inhibition of the mitochondrial aspartate-arginosuccinate shunt and ROS production.Conclusion: We demonstrated that METP NPs are effective and highly specific regulators of m[Ca2+] overload. In addition, we demonstrated that these METP NPs reverse the macrophage proinflammatory phenotype by restoring m[Ca2+] homeostasis, thereby inhibiting the tissue inflammatory response and achieving a therapeutic effect for OA.Graphical Abstract: Keywords: nanoparticle, macrophage, mitochondria, calcium homeostasis, osteoarthritis

Ya-Chun Chu,1,2,* Hsu-Wei Fang,2,3,* Yu-Yi Wu,1 Yu-Jun Tang,1 Erh-Hsuan Hsieh,1 YiZhou She,4 Che-Yi Chang,1 I-Chan Lin,5,6 Yin-Ju Chen,1,7– 9 Guei-Sheung Liu,1,10– 12 Ching-Li Tseng1,8,9,13 1Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan; 2Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei City, Taiwan; 3Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli County, Taiwan; 4School of Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan; 5Department of Ophthalmology, Wan Fang Hospital, Taipei Medical University, Taipei City, Taiwan; 6Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan; 7Department of Radiation Oncology, Taipei Medical University Hospital, Taipei City, Taiwan; 8International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan; 9Center for Precision Medicine and Translational Cancer Research, Taipei Medical University Hospital, Taipei City, Taiwan; 10Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; 11Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; 12Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia; 13Research Center of Biomedical Device, College of Biomedical Engineering, Taipei Medical University, Taipei City, Taiwan*These authors contributed equally to this workCorrespondence: Ching-Li Tseng, Tel +886 2 2736 1661 (ext. 5214), Email chingli@tmu.edu.twBackground: Corneal neovascularization (NV) is a process of abnormal vessel growth into the transparent cornea from the limbus and can disturb the light passing through the cornea, resulting in vision loss or even blindness. The use of nanomedicine as an effective therapeutic formulation in ophthalmology has led to higher drug bioavailability and a slow drug release rate. In this research, we designed and explored the feasibility of a new nanomedicine, gp91 ds-tat (gp91) peptide-encapsulated gelatin nanoparticles (GNP-gp91), for inhibiting corneal angiogenesis.Methods: GNP-gp91 were prepared by a two-step desolvation method. The characterization and cytocompatibility of GNP-gp91 were analyzed. The inhibition effect of GNP-gp91 on HUVEC cell migration and tube formation was observed by an inverted microscope. The drug retention test in mouse cornea was observed by in vivo imaging system, fluorescence microscope, and DAPI/TAMRA staining. Finally, the therapeutic efficacy and evaluation of neovascularization-related factors were conducted through the in vivo corneal NV mice model via topical delivery.Results: The prepared GNP-gp91 had a nano-scale diameter (550.6 nm) with positive charge (21.7 mV) slow-release behavior (25%, 240hr). In vitro test revealed that GNP-gp91 enhanced the inhibition of cell migration and tube formation capacity via higher internalization of HUVEC. Topical administration (eyedrops) of the GNP-gp91 significantly prolongs the retention time (46%, 20 min) in the mouse cornea. In chemically burned corneal neovascularization models, corneal vessel area with a significant reduction in GNP-gp91 group (7.89%) was revealed when compared with PBS (33.99%) and gp91 (19.67%) treated groups via every two days dosing. Moreover, GNP-gp91 significantly reduced the concentration of Nox2, VEGF and MMP9 in NV’s cornea.Conclusion: The nanomedicine, GNP-gp91, was successfully synthesized for ophthalmological application. These data suggest that GNP-gp91 contained eyedrops that not only have a longer retention time on the cornea but also can treat mice corneal NV effectively delivered in a low dosing frequency, GNP-gp91 eyedrops provides an alternative strategy for clinical ocular disease treatment in the culture.Keywords: gp91 peptide, gelatin, nanoparticles, ocular retention, control release, corneal neovascularization, eye drops

Fatma M Elsharkawy,1 Maha M Amin,2 Hesham A Shamsel-Din,3 Walaa Ibrahim,3 Ahmed B Ibrahim,3 Sinar Sayed2 1Regulatory Affairs Department, Al Andalous for Pharmaceutical Industries, Giza, Egypt; 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt; 3Labeled Compounds Department, Hot Labs Center, Egyptian Atomic Energy Authority, Cairo, 13759, EgyptCorrespondence: Sinar Sayed, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo, 11562, Egypt, Tel +2 01010421543, Email sinar.fouad@pharma.cu.edu.egPurpose: The research objective is to design intranasal brain targeted CLZ loaded lecithin based polymeric micelles (CLZ- LbPM) aiming to improve central systemic CLZ bioavailability.Methods: In our study, intranasal CLZ loaded lecithin based polymeric micelles (CLZ- LbPM) were formulated using soya phosphatidyl choline (SPC) and sodium deoxycholate (SDC) with different CLZ:SPC:SDC ratios via thin film hydration technique aiming to enhance drug solubility, bioavailability and nose to brain targeting efficiency. Optimization of the prepared CLZ-LbPM using Design-Expert® software was achieved showing that M6 which composed of (CLZ:SPC: SDC) in respective ratios of 1:3:10 was selected as the optimized formula. The optimized formula was subjected to further evaluation tests as, Differential Scanning Calorimetry (DSC), TEM, in vitro release profile, ex vivo intranasal permeation and in vivo biodistribution.Results: The optimized formula with the highest desirability exhibiting (0.845), small particle size (12.23± 4.76 nm), Zeta potential of (− 38 mV), percent entrapment efficiency of > 90% and percent drug loading of 6.47%. Ex vivo permeation test showed flux value of 27 μg/cm².h and the enhancement ratio was about 3 when compared to the drug suspension, without any histological alteration. The radioiodinated clozapine ([131I] iodo-CLZ) and radioiodinated optimized formula ([131I] iodo-CLZ-LbPM) were formulated in an excellent radioiodination yield more than 95%. In vivo biodistribution studies of [131I] iodo-CLZ-LbPM showed higher brain uptake (7.8%± 0.1%ID/g) for intranasal administration with rapid onset of action (at 0.25 h) than the intravenous formula. Its pharmacokinetic behavior showed relative bioavailability, direct transport percentage from nose to brain and drug targeting efficiency of 170.59%, 83.42% and 117% respectively.Conclusion: The intranasal self-assembling lecithin based mixed polymeric micelles could be an encouraging way for CLZ brain targeting.Graphical Abstract: Keywords: clozapine, soy lecithin, self-assembling polymeric micelles, intranasal route, brain targeting, radioiodinated clozapine, 131I

Yongai Xiong,1,2 Tingting Chen,1,2 Lei Chen,1,2 Rongshan Cai1,2 1Key Laboratory of Basic Pharmacology of Guizhou Province and School of Pharmacy, Zunyi Medical University, Zunyi, Guizhou, 563000, People’s Republic of China; 2Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou, 563000, People’s Republic of ChinaCorrespondence: Yongai Xiong, Email yaxiong@zmu.edu.cnIntroduction: Radiotherapy and chemotherapy are the fundamental causes of myelosuppression in cancer patients, which usually induce a serious hematopoietic system toxicity, causing the hemocytes and immunity decline of patients. Ziyuglycoside I (ZgI), an active ingredient isolated from traditional Chinese medicine Sanguisorba officinalis L, has been demonstrated to increase the leucocytes and protect hematopoietic stem cells, which is related to its promotion of autophagy in hematopoietic stem cells.Methods: In the present study, we formulated the SH-PEG-NH2-coated gold nanoparticles loading ZgI (ZgI-AuNPs) with a enhanced autophagy promotion in hematopoietic stem cells. ZgI-AuNPs were prepared by HAuCl4-sodium citrate reduction method, and the synthesis of ZgI-AuNPs was validated by XRD, FT-IR, DSC, and TEM findings. Furthermore, the drug loading rate and the release of ZgI were evaluated, and the ZgI-AuNPs’ effects on autophagy and immunofluorescence staining for LC3B were tested. Finally, the effect of ZgI-AuNPs on the autophagy and hematopoietic ability of HSCs in vivo was also carried out.Results: The prepared ZgI-AuNPs have an irregular cubic crystal structure by TEM observation, and the average particle size was 340 ± 16.5 nm determined by DLS. The XRD, FT-IR and DSC detection showed that the ZgI had been well loaded in AuNPs, and the AuNPs can load the ZgI at a content of 160.63 ± 1.35 μg·mg− 1. Meanwhile, the AuNPs can reduce the drug release rate of ZgI. Importantly, the ZgI-AuNPs enhanced autophagy of HSCs both in vitro and in vivo. At the same time, the gold nanoparticles enhance the hematopoietic effect of ZgI on mice HSCs.Conclusion: Our research suggests that SH-PEG-NH2-coated gold nanoparticles loading ZgI has potential application in myelosuppression therapy.Graphical Abstract: Keywords: myelosuppression, HSCS, autophagy, gold nanoparticles, Ziyuglycoside I

Piktel E, Markiewicz KH, Wilczewska AZ, et al. Int J Nanomedicine. 2020;15:4573–4589. It has been brought to the authors attention that Figure 6E showing nanosystem-mediated DNA fragmentation in DLD-1 colorectal cancer cells is a duplicate of part of Figure 3C that was previously published in the review article: Wnorowska U, Fiedoruk K, Piktel E, et al. J Nanobiotechnology. 2020;18(1):3 (https://doi.org/10.1186/s12951-019-0566-z). In addition, both Figures 5E and 6E were described with incorrect treatment conditions. Data obtained during corresponding experiments were verified; consequently, Figure 5E and 6E were updated using data from the original study. The correct Figure 5 is as follows. Figure 5 Continued. Figure 5 Anticancer activity of ceragenin CSA-131 and ceragenin-containing nanoformulations against lung carcinoma A549 cells. Increase of intracellular levels of reduced thiols in A549 cells treated with CSA-131 (grey bar), MNP@CSA-131 (red bar), CSA-131+MNP (blue bar) and MNP (yellow bar) when compared to untreated control (0 μg/mL; black bar) (A). The percentages of dead cells (black columns), PI-negative cells with low viability (dark grey columns) and healthy cells (light grey columns) in lung carcinoma cells treated with CSA-131, MNP@CSA-131, CSA-131 and MNP or naked MNPs (B). The proliferation of cancer cells treated with CSA-131 (grey squares), MNP@CSA-131 (red circles), CSA-131 + MNP (blue diamonds) and MNP (yellow inverted triangles) when compared to untreated control (black squares) estimated using resazurin-based fluorimetric method (C). Induction of apoptosis in A549 cells by CSA-131 and its magnetic derivatives (D). Percentage of early apoptotic (black columns), late apoptotic/dead cells (dark grey columns) and dead cells (light grey columns). For the purpose of the clarity of the presented data, live cells (Annexin V-negative and 7-AAD-negative) were not presented in the provided figures. Morphological alternations in nuclei of A549 cells upon treatment with CSA-131, MNP@CSA-131 and CSA-131 + MNP when compared to uncreated cells (E). White arrows indicate treatment-induced morphological changes in nuclei of treated cells. All experiments were performed using agents at a concentration of 10 μg/mL for 24 h. (A–D) demonstrate results from 3 to 6 individual experiments ± SD, for panel E results from one representative experiment are shown. * and ^ indicate statistical significance (p-value

Walaa Ebrahim Abd El Hady,1 Ghada Ahmed El-Emam,1 Nora E Saleh,2 Marwa M Hamouda,2 Amira Motawea1 1Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; 2Department of Medical Parasitology, Faculty of Medicine, Mansoura University, Mansoura, EgyptCorrespondence: Amira Motawea, Email a_metawea@mans.edu.egBackground: Schistosomiasis is a chronic debilitating parasitic disease accompanied with severe mortality rates. Although praziquantel (PZQ) acts as the sole drug for the management of this disease, it has many limitations that restrict the use of this treatment approach. Repurposing of spironolactone (SPL) and nanomedicine represents a promising approach to improve anti-schistosomal therapy. We have developed SPL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to enhance the solubility, efficacy, and drug delivery and hence decrease the frequency of administration, which is of great clinical value.Methods: The physico-chemical assessment was performed starting with particle size analysis and confirmed using TEM, FT-IR, DSC, and XRD. The antischistosomal effect of the SPL-loaded PLGA NPs against Schistosoma mansoni (S. mansoni)-induced infection in mice was also estimated.Results: Our results manifested that the optimized prepared NPs had particle size of 238.00 ± 7.21 nm, and the zeta potential was − 19.66 ± 0.98 nm, effective encapsulation 90.43± 8.81%. Other physico-chemical features emphasized that nanoparticles were completely encapsulated inside the polymer matrix. The in vitro dissolution studies revealed that SPL-loaded PLGA NPs showed sustained biphasic release pattern and followed Korsmeyer–Peppas kinetics corresponding to Fickian diffusion (n< 0.45). The used regimen was efficient against S. mansoni infection and induced significant reduction in spleen, liver indices, and total worm count (ρ< 0.05). Besides, when targeting the adult stages, it induced decline in the hepatic egg load and the small intestinal egg load by 57.75% and 54.17%, respectively, when compared to the control group. SPL-loaded PLGA NPs caused extensive damage to adult worms on tegument and suckers, leading to the death of the parasites in less time, plus marked improvement in liver pathology.Conclusion: Collectively, these findings provided proof-of-evidence that the developed SPL-loaded PLGA NPs could be potentially used as a promising candidate for new antischistosomal drug development.Keywords: spironolactone, poly (lactic-co-glycolic acid), nanoparticles, Schistosoma mansoni, egg load, oogram

Liwen Kuang,1,&ast; Chenchen Zhang,1,&ast; Binghui Li,1 Haibo Deng,1 Ran Chen,1 Gongchi Li2 1Department of Wound Repair Surgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430062, People’s Republic of China; 2Department of Hand Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Gongchi Li, Department of Hand Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Road, Wuhan, Hubei, 430022, People’s Republic of China, Tel +8613618615209, Email ligongchi@foxmail.comPurpose: Diabetic wound is a highly prevalent and refractory disease. Extensive studies have confirmed that keratinocytes and macrophages play an important role in the process of wound healing. Additionally, exosomes are regarded as a vital intercellular communication tool. This study aimed to investigate the role of human keratinocyte-derived exosomal MALAT1 in the treatment of diabetic wound by influencing the biological function of macrophages.Methods: We mainly assessed the function of MALAT1 on the biological changes of macrophages, and the expression of MALAT1 in the keratinocyte-exosomes analyzed by quantitative real-time polymerase chain reaction (RT-qPCR). The downstream interaction between RNAs or proteins was assessed by mechanistic experiments. Besides, we evaluated the effects of human keratinocyte-derived exosomal MALAT1 on diabetic wound healing in vivo to verify in vitro results.Results: We demonstrated that human keratinocyte-derived exosomal MALAT1 enhanced the biological functions of high glucose-injured macrophages, including phagocytosis, converting to a pro-healing phenotype and reducing apoptosis. Mechanistically, MALAT1 accelerated the expression of MFGE8 by competitively binding to miR-1914-3p, thereby affecting the function of macrophages and the signal axis of TGFB1/SMAD3, and finally promoting the healing of diabetic wounds. Human keratinocyte-derived exosomal MALAT1 might promote collagen deposition, ECM remodeling, and expression of MFGE8, VEGF, and CD31 but reduce the expression of TGFB and SMAD3 in an in vivo model of diabetic mice wounds, which accelerated diabetic wound healing and restored its function.Conclusion: The current study revealed that human keratinocyte-derived exosomal MALAT1 would suppress miR-1914-3p to activate MFGE8 and eventually promote wound healing by enhancing macrophage phagocytosis, converting to a pro-healing phenotype and reducing apoptosis. It proposed that keratinocyte-derived exosomes might have the capacity to serve as a new method for the clinical treatment of diabetic wound.Graphical Abstract: Keywords: exosomes, keratinocytes, macrophages, MALAT1, diabetic wound healing

Fangqing Wang,1,&ast; Nianlu Li,2,&ast; Wenbo Wang,1 Long Ma,3 Yaru Sun,4 Hong Wang,1 Jinhua Zhan,5 Dexin Yu1 1Department of Radiology, Qilu Hospital, Shandong University, Affiliated Hospital of Shandong University, Jinan, 250012, People’s Republic of China; 2Physical and Chemical Laboratory, Shandong Academy of Occupational Health and Occupational Medicine, Affiliated Hospital of Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250002, People’s Republic of China; 3The Testing Center of Shandong Bureau of China Metallurgical Geology Bureau, Shandong Normal University, Jinan, 250014, People’s Republic of China; 4Department of Nuclear Medicine, The Second Hospital of Shandong University, Affiliated Hospital of Shandong University, Jinan, 250033, People’s Republic of China; 5School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Jinhua Zhan, School of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100, People’s Republic of China, Email jhzhan@sdu.edu.cn Dexin Yu, Department of Radiology, Qilu Hospital, Shandong University, Affiliated Hospital of Shandong University, Jinan, 250012, People’s Republic of China, Tel +86-18560081629, Fax +86-531-86927544, Email yudexin0330@sina.comPurpose: Owing to lack of specific molecular targets, the current clinical therapeutic strategy for triple negative breast cancer (TNBC) is still limited. In recent years, some nanosystems for malignancy treatment have received considerable attention. In this study, we prepared caramelized nanospheres (CNSs) loaded with doxorubicin (DOX) and Fe3O4 to achieve the synergistic effect of combined therapy and real-time magnetic resonance imaging (MRI) monitoring, so as to improve the diagnosis and therapeutic effect of TNBC.Methods: CNSs with biocompatibility and unique optical properties were prepared by hydrothermal method, DOX and Fe3O4 were loaded on it to obtain Fe3O4/DOX@CNSs nanosystem. Characteristics including morphology, hydrodynamic size, zeta potentials and magnetic properties of Fe3O4/DOX@CNSs were evaluated. The DOX release was evaluated by different pH/near-infrared (NIR) light energy. Biosafety, pharmacokinetics, MRI and therapeutic treatment of Fe3O4@CNSs, DOX and Fe3O4/DOX@CNSs were examined in vitro or in vivo.Results: Fe3O4/DOX@CNSs has an average particle size of 160 nm and a zeta potential of 27.5mV, it demonstrated that Fe3O4/DOX@CNSs is a stable and homogeneous dispersed system. The hemolysis experiment of Fe3O4/DOX@CNSs proved that it can be used in vivo. Fe3O4/DOX@CNSs displayed high photothermal conversion efficiency, extensive pH/heat-induced DOX release. 70.3% DOX release is observed under the 808 nm laser in the pH = 5 PBS solution, obviously higher than pH = 5 (50.9%) and pH = 7.4 (less than 10%). Pharmacokinetic experiments indicated the t1/2β, and AUC0–t of Fe3O4/DOX@CNSs were 1.96 and 1.31 -fold higher than those of DOX solution, respectively. Additionally, Fe3O4/DOX@CNSs with NIR had the greatest tumor suppression in vitro and in vivo. Moreover, this nanosystem demonstrated distinct contrast enhancement on T2 MRI to achieve real-time imaging monitoring during treatment.Conclusion: Fe3O4/DOX@CNSs is a highly biocompatible, double-triggering and improved DOX bioavailability nanosystem that combines chemo-PTT and real-time MRI monitoring to achieve integration of diagnosis and treatment of TNBC.Keywords: caramelized nanotheranostic system, combining chemo-photothermal therapy, magnetic resonance imaging monitoring, double-triggering, triple negative breast cancer

Xiaoya Liu,1,2 Haiyang Fan,3 Zhiyun Meng,2 Zhuona Wu,2 Ruolan Gu,2 Xiaoxia Zhu,2 Hui Gan,2 Guifang Dou2 1Department of Pharmacy, Shenzhen Children’s Hospital, Shenzhen, Guangdong Province, 518026, People’s Republic of China; 2Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing, 100850, People’s Republic of China; 3Department of Thoracic Surgery, The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen, Guangdong Province, 518102, People’s Republic of ChinaCorrespondence: Guifang Dou; Hui Gan, Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing, 100850, People’s Republic of China, Tel +86 10 66932951, Fax +86 10 66931993, Email dougf@bmi.ac.cn; ganh2003@163.comIntroduction: Silver sulfadiazine (AgSD) is widely used in burn wound treatment due to its broad-spectrum antibacterial activity. However, its application in wound healing is greatly hindered by the low solubility of AgSD particles and their cellular cytotoxicity. Herein, we studied the safety and in vivo efficacy of nano-sized silver sulfadiazine loaded in poloxamer thermosensitive hydrogel (NS/Gel).Methods: In NS/Gel, silver sulfadiazine was prepared into silver sulfadiazine nanosuspension (NS) to improve the solubility and enhance its antibacterial activity, whereas the poloxamer thermosensitive hydrogel was selected as a drug carrier of NS to achieve slow drug release and reduced cytotoxicity. The acute toxicity of silver sulfadiazine nanosuspension was first evaluated in healthy mice, and its median lethal dose (LD50) was calculated by the modified Karber method. Furthermore, in vivo antibacterial effect and wound healing property of NS/Gel were evaluated on the infected deep second-degree burn wound mice model.Results: The mortality ratio of mice was concentration-dependent, and the LD50 for silver sulfadiazine nanosuspension was estimated to be 252.1 mg/kg (230.8 to 275.4 mg/kg, 95% confidence limit). The in vivo dosages used for burn wound treatment (40– 50 mg/kg) were far below LD50 (252.1 mg/kg). NS/Gel significantly accelerated wound healing in the deep second wound infection mice model, achieving > 85% wound contraction on day 14. Staphylococcus aureus in the wound region was eradicated after 7 days in NS/Gel group, while the bacterial colony count was still measurable in the control group. Histological analysis and cytokines measurement confirmed that the mice treated with NS/Gel exhibited well-organized epithelium and multiple keratinized cell layers compared to control groups with the modulated expression of IL-6, VEGF, and TGF-β.Conclusion: The combination of silver sulfadiazine nanosuspension and thermo-responsive hydrogel has great potential in clinical burn wound treatment.Keywords: silver sulfadiazine, antibacterial agent, thermosensitive hydrogel, acute toxicity, burn wound model, median lethal dose

Xin-Yue Yang,1,&ast; Lina Geng,2,&ast; Ronghui Li,2 Ji-Xian Song,1 Cui-Ling Jia,1 Ji-Ren An,1,3 Meng-Fan Sun,1 Shan Xu,1 Ya-Jing Guo,1 Yashuo Zhao,1 En-Sheng Ji1 1Hebei Technology Innovation Center of TCM Combined Hydrogen Medicine, Hebei University of Chinese Medicine, Shijiazhuang, People’s Republic of China; 2College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang, People’s Republic of China; 3The First Clinical College, Liaoning University of Traditional Chinese Medicine, Shenyang, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yashuo Zhao; En-Sheng Ji, No. 3, Luqian Xingyuan Road, Shijiazhuang, 050200, People’s Republic of China, Email zys870207@126.com; jesphy@126.comBackground: Chronic intermittent hypoxia (CIH) could cause neuronal damage, accelerating the progression of dementia. However, safe and effective therapeutic drugs and delivery are needed for successful CIH therapy.Purpose: To investigate the neuroprotective effect of Huperzine A (HuA) packaged with nanoliposomes (HuA-LIP) on neuronal damage induced by CIH.Methods: The stability and release of HuA-LIP in vitro were identified. Mice were randomly divided into the Control, CIH, HuA-LIP, and HuA groups. The mice in the HuA and HuA-LIP groups received HuA (0.1 mg/kg, i.p.), and HuA-LIP was administered during CIH exposure for 21 days. HuA-LIP contains the equivalent content of HuA.Results: We prepared a novel formulation of HuA-LIP that had good stability and controlled release. First, HuA-LIP significantly ameliorated cognitive dysfunction and neuronal damage in CIH mice. Second, HuA-LIP elevated T-SOD and GSH-Px abilities and decreased MDA content to resist oxidative stress damage induced by CIH. Furthermore, HuA-LIP reduced brain iron levels by downregulating TfR1, hepcidin, and FTL expression. In addition, HuA-LIP activated the PKAα/Erk/CREB/BDNF signaling pathway and elevated MAP2, PSD95, and synaptophysin to improve synaptic plasticity. Most importantly, compared with HuA, HuA-LIP showed a superior performance against neuronal damage induced by CIH.Conclusion: HuA-LIP has a good sustained-release effect and targeting ability and efficiently protects against neural injury caused by CIH.Keywords: huperzine A, nanoliposomes, neuronal damage, chronic intermittent hypoxia, Iron

Zhangsong Peng,1,&ast; Qiang Chang,1,2,&ast; Malcolm Xing,2 Feng Lu1 1Department of Plastic and Reconstruction Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, People’s Republic of China; 2Department of Mechanical Engineering, University of Manitoba, Children’s Hospital Research Institute of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada&ast;These authors contributed equally to this workCorrespondence: Malcolm Xing, Department of Mechanical Engineering, University of Manitoba, Children’s Hospital Research Institute of Manitoba, Winnipeg, Manitoba, R3T 2N2, Canada, Email malcolm.xing@umanitoba.ca Feng Lu, Department of Plastic and Reconstruction Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, People’s Republic of China, Email doctorlufeng@hotmail.comPurpose: Graphene oxide (GO) and its derivatives have recently been identified as promising candidates for early disease diagnosis and therapy. However, the physiological stability and precise launch requirements present limitations on further clinical practices. Adipose-derived stem cells (ADSCs) were employed as an unobstructed biological vehicle to address the validate this ADSC-based tumor-targeting system for highly efficient GO delivery combined with two-stage NIR radiation for superior tumor ablation.Methods: GO was modified with poly-ethylene glycol (PEG) and folic acid (FA). Afterward, the GO nanocomposite was internalized into ADSCs. The GO-PEG-FA-laden ADSCs were injected into the tail veins of the tumor-bearing mice. Subsequently, first-stage NIR radiation was utilized to disrupt the ADSCs for GO-PEG-FA release. After this, the heat generated by secondary-stage NIR radiation destroy the malignant cells and shrink the tumor, and the cascade process could be recycled until complete tumor ablation if necessary.Results: The GO-PEG-FA nanocomposite exhibited negligible cytotoxicity and could be internalized into ADSCs to target specific tumor sites after 32 days of intravenous injection. The nanocomposite was released from the ADSCs and taken up into cancer cells again with the assistance of FA after the first dose of near-infrared radiation. Then, the second radiation dose could directly strike the cancer cell for cancer ablation.Conclusion: In summary, we reported a stem cell-based anticancer system that used GO-PEG-FA-laden ADSCs for breast cancer therapy through NIR treatment in mice potentially opens a new avenue not only to address precise drug targeting in tumor therapy, but also future clinical practice in diverse areas.Keywords: graphene oxide, adipose-derived stem cells, nanocomposite, photothermal therapy, breast cancer

Pengzheng Chen,1 Mengqi Gu,1 Shuting Wan,1 Xiaotong Jiang,1 Fengyuan Zhang,2 Yuchen Li,2 Qian Zhou,2 Yuan Lu,2 Lei Li,1– 3 Xietong Wang1– 4 1Department of Obstetrics and Gynaecology, Shandong Provincial Hospital, Shandong University, Jinan, People’s Republic of China; 2Department of Obstetrics and Gynaecology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, People’s Republic of China; 3Laboratory of Medical Science and Technology Innovation Center (Institute of Translational Medicine), Shandong First Medical University (Shandong Academy of Medical Sciences), Jinan, People’s Republic of China; 4Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Shandong Provincial Maternal and Child Health Care Hospital, Jinan, People’s Republic of ChinaCorrespondence: Lei Li; Xietong Wang, Department of Obstetrics and Gynaecology, Shandong Provincial Hospital, Shandong University, Jinan, People’s Republic of China, Tel +8615168889200 ; +8615168888928, Email lilei@sdfmu.edu.cn; 198657000067@email.sdu.edu.cnBackground: Fetal lung underdevelopment (FLUD) is associated with neonatal and childhood severe respiratory diseases, among which gestational diabetes mellitus (GDM) play crucial roles as revealed by recent prevalence studies, yet mechanism underlying GDM-induced FLUD, especially the role of trophoblasts, is not all known.Methods: From the perspective of trophoblast-derived exosomes, we established in vitro, ex vivo, in vivo and GDM trophoblast models. Utilizing placenta-derived exosomes (NUB-exos and GDMUB-exos) isolated from normal and GDM umbilical cord blood plasma and trophoblast-derived exosomes (NC-exos and HG-exos) isolated from HTR8/SVneo trophoblasts medium with/without high glucose treatment, we examined their effects on fetal lung development and biological functions.Results: We found that, compared with the NUB-exos group, the exosome concentration increased in GDMUB-exos group, and the content of exosomes also changed evidenced by 61 dysregulated miRNAs. After applying these exosomes to A549 alveolar type II epithelial cells, the proliferation and biological functions were suppressed while the proportion of apoptotic cells was increased as compared to the control. In ex vivo studies, we found that GDMUB-exos showed significant suppression on the growth of the fetal lung explants, where the number of terminal buds and the area of explant surface decreased and shrank. Besides, the expression of Fgf10, Vegfa, Flt-1, Kdr and surfactant proteins A, B, C, and D was downregulated in GDMUB-exos group, whilst Sox9 was upregulated. For in vivo studies, we found significant suppression of fetal lung development in GDMUB-exos group. Importantly, we found consistent alterations when we used NC-exos and HG-exos, suggesting a dominant role of trophoblasts in placenta-derived exosome-induced FLUD.Conclusion: In conclusion, GDM can adversely affect trophoblasts and alter exosome contents, causing crosstalk disorder between trophoblasts and fetal lung epithelial cells and finally leading to FLUD. Findings of this study will shine insight into the theoretical explanation for the pathogenesis of FLUD.Keywords: gestational diabetes mellitus, fetal lung underdevelopment, trophoblast, placenta-derived exosome, trophoblast-derived exosomes

Chenyi Zhang,1,&ast; Lai Pan,1,&ast; Haizheng Zhang,1 Ting Ke,1 Yuxuan Yang,1 Lan Zhang,2 Lili Chen,1 Jingyi Tan1 1Department of Periodontology, The Second Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou, People’s Republic of China; 2Stomatology Department, Zhejiang Hospital, Hangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Lili Chen; Jingyi Tan, Email chenlili_1030@zju.edu.cn; tanjingyi@zju.edu.cnObjective: Emerging studies have explained the crucial role of non-coding RNA (lncRNA) in various pathological progressions. The study was designed to examine the role of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miRNA-124 in the differentiation of osteoclasts, to provide new clues or evidences for the pathogenesis of periodontitis.Methods: We constructed an osteoblast-osteoclast Transwell co-culture system and osteoblast-derived exosomes (OB-exo) intervention model. We assessed the osteoclastogenesis as well as the level of lncRNA-MALAT1 and miRNA-124. The mechanism for lncRNA MALAT1 targeting miR-124 modulating the differentiation of osteoclasts was investigated by cell transfection, quantitative real-time reverse transcription PCR (RT-qPCR), Western blot, and Dual-Luciferase reporter assays.Results: Osteoblast-derived exosomes were isolated and identified. Co-culture and OB-exo intervention can promote osteoclastogenesis, also significantly up-regulate the expression of MALAT1, while the level of miR-124 is the opposite. Transfection of cells with small interfering RNA (si-MALAT1) and miR-124 mimic decreased the formation of TRAP+ osteoclasts and inhibited the expression of NFATc1. However, the effect was reversed when transfected with miR-124 inhibitor and si-MALAT1. The Dual-Luciferase reporter assay confirmed the binding sites between MALAT1 and miR-124, and miR-124 and NFATc1.Conclusion: LncRNA MALAT1 functioned as an endogenous sponge by competing for miR-124 binding to regulate NFATc1 expression, accelerating the progression of osteoclastogenesis.Graphical Abstract: Keywords: exosome, lncRNA, miRNA-124, osteoclast differentiation

Jingmou Yu,1,2 Xin Xie,2 Liangliang Wang,3 Wenbo Liu,4 Huifeng Xu,4 Xiangmei Lu,4 Xiaofan Li,4 Jin Ren,2,4 Weidong Li2,5 1Huzhou Key Laboratory of Medical and Environmental Applications Technologies, School of Life Sciences, Huzhou University, Huzhou, 313000, People’s Republic of China; 2Jiangxi Provincial Key Laboratory of System Biomedicine, Jiujiang University, Jiujiang, 332000, People’s Republic of China; 3Affiliated Hospital of Jiujiang University, Jiujiang, 332000, People’s Republic of China; 4School of Pharmacy and Life Sciences, Jiujiang University, Jiujiang, 332000, People’s Republic of China; 5Jiujiang NO.1 People’s Hospital & Water of Life Hospital, Jiujiang, 332000, People’s Republic of ChinaCorrespondence: Jin Ren; Weidong Li, Email yanjiushengrj@126.com; 346880908@qq.comIntroduction: Metastasis is a major challenge in breast cancer therapy. The successful chemotherapy of breast cancer largely depends on the ability to block the metastatic process. Herein, we designed a dual-targeting and stimuli-responsive drug delivery system for targeted drug delivery against breast cancer metastasis.Methods: AS1411 aptamer-modified chondroitin sulfate A-ss-deoxycholic acid (ACSSD) was synthesized, and the unmodified CSSD was used as the control. Chemotherapeutic drug doxorubicin (DOX)-containing ACSSD (D-ACSSD) micelles were prepared by a dialysis method. The ACSSD conjugate was confirmed by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), and transmission electron microscopy (TEM). In vitro cellular uptake and cytotoxicity of D-ACSSD micelles were studied by confocal laser scanning microscopy (CLSM) and MTT assay in breast tumor cells. The inhibition capability of D-ACSSD micelles in cell migration and invasion was carried out in 4T1 cells. In vivo antitumor activity of DOX-containing micelles was investigated in metastatic 4T1-bearing Balb/c mice.Results: D-ACSSD and DOX-loaded CSSD (D-CSSD) micelles exhibited high drug encapsulation content and reduction-responsive characteristics. D-ACSSD micelles were spherical in shape. Compared with D-CSSD, D-ACSSD showed higher cellular uptake and more potent killing activity in 4T1 and MDA-MB-231 cells. Additionally, D-ACSSD exhibited stronger inhibitory effects on the invasion and migration of highly metastatic 4T1 cells than unmodified D-CSSD. Among the DOX-containing formulations, D-ACSSD micelles presented the most effective inhibition of tumor growth and lung metastasis in orthotopic 4T1-bearing mice in vivo. It also revealed that ACSSD micelles did not exhibit obvious systemic toxicity.Conclusion: The smart D-ACSSD micelles could be a promising delivery system for the therapy of metastatic breast cancer.Graphical Abstract: Keywords: metastasis, dual-targeting, breast cancer therapy, reduction-sensitive micelles, drug delivery

Sangseo Kim,1 Sadikalmahdi Abdella,1 Fatima Abid,1 Franklin Afinjuomo,1 Souha H Youssef,1 Amy Holmes,1 Yunmei Song,1 Sachin Vaidya,2 Sanjay Garg1 1Centre for Pharmaceutical Innovation, Clinical and Health Sciences, University of South Australia, Adelaide, SA, 5000, Australia; 2Central Adelaide Local Health Network, The Queen Elizabeth Hospital, Woodville, SA, 5011, AustraliaCorrespondence: Sanjay Garg, Tel +61 8 8302 1575, Email Sanjay.garg@unisa.edu.auBackground: Imiquimod (IMQ) is an immunomodulating drug that is approved for the treatment of superficial basal cell carcinoma, actinic keratosis, external genital warts and perianal warts. However, IMQ cream (Aldara®) has several drawbacks including poor skin permeation, local toxicity, and compromised patient compliance as a topical pharmacological option.Methods: Our research aimed to develop and optimize nanostructured lipid carriers (NLCs) containing IMQ for the first time using a hybrid design of experiments approach. The optimized formulation was then incorporated into a matrix-type topical patch as an alternative dosage form for topical application and evaluated for IMQ deposition across different skin layers in comparison to the performance of the commercial product. Additionally, our work also attempted to highlight the possibility of implementing environment-friendly practices in our IMQ-NLCs formulation development by reviewing our analytical methods and experimental designs and reducing energy and solvent consumption where possible.Results: In this study, stearyl alcohol, oleic acid, Tween® 80 (polysorbate 80), and Gelucire® 50/13 (Stearoyl polyoxyl-32 glycerides) were selected for formulation development. The formulation was optimized using a 2k factorial design and a central composite design. The optimized formulation achieved the average particle size, polydispersity index, and zeta potential of 75.6 nm, 0.235, and – 30.9 mV, respectively. Subsequently, a matrix-type patch containing IMQ-NLCs was developed and achieved a statistically significant improvement in IMQ deposition in the deeper skin layers. The IMQ deposition from the patch into the dermis layer and receptor chamber was 3.3 ± 0.9 μg/cm2 and 12.3 ± 2.2 μg/cm2, while the commercial cream only deposited 1.0 ± 0.8 μg/cm2 and 1.5 ± 0.5 μg/cm2 of IMQ, respectively.Conclusion: In summary, IMQ-NLC-loaded patches represent great potential as a topical treatment option for skin cancer with improved patient compliance.Keywords: imiquimod, nanostructured lipid carrier, design of experiments, topical patches, skin cancer, greenness evaluation