A Polysaccharide-based Hydrogel and PLGA Microspheres for Sustained P24 Peptide Delivery: An In vitro and In vivo Study Based on Osteogenic Capability

A bone morphogenetic protein-2(BMP-2) derived synthetic oligopeptide, S [PO4]KIPKASSVPTELSAISTLYLDDD(P24), has shown great potential for facilitating bone regeneration. However, P24 cannot be directly used onto bone defects, while a continuous sustained delivery of P24 may lead to a better formation of bone tissue. Based on this issue, we have developed a sustained delivery system incorporating P24-loaded poly(lactide-co-glycolide)(PLGA) microspheres and nano-hydroxyapatite(n-HA) into the composite hydrogel. The P24-contained compound material was characterized with NMR, FTIR and SEM to demonstrate the fomiation of compound structure containing P24, PLGA and n-HA. A continuous drug release of P24 was observed for over 60 d that evidently enhanced the efficiency in promoting the proliferation of MC3T3-E1 cells and the secrete of alkaline phosphatase(ALP) in vitro. Moreover, the osteoinduction eflect of the hydrogel system with P24 peptide niicrospheres was demonstrated in vivo and manifested by the result of immunohistochemistry. This novel injectable composite hydrogel is expected to be applied to improving the bone defect treatment in bone tissue engineering.     

Effects of Molecular Crowding on G-Quadruplex-hemin Mediated Peroxidase Activity

The concentration of macromolecules in cells can reach up to 50-400 mg/mL.They occupy 40%(volume fraction)of the whole cellar space,known as molecular crowding.The diluted solution condition in vitro is different from the crowded physiological condition in vivo.Therefore,the simulation of the physiological condition is necessary for obtaining the reliable results.It has been reported that G-quadruplex can bind to hemin to enhance its catalytic function for generating oxygen radicals,which can oxidize the lipids,proteins and DNA,thus leading to the damage of cells and tissues.In this paper,we chose PEG400 as molecular crowding reagent to simulate the molecular crowding environment in vivo.The catalytic characteristics of G-quadruplex-hemin complex in H202-ABTS system have been investigated[ABTS=2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate)].The results showed that the binding affinity of G-quadruplex and hemin was decreased with the increasing of PEG400 concentration.They even lose their binding affinity in the presence of 40%PEG400.As a result,the peroxidase activity of G-quadruplex-hemin also reduced.Therefore,in physiological condition,hemin might not bind to G-quadruplex and it might not be the main reason to cause the damages of cells and tissues.     

Engineered Hsp Protein Nanocages for siRNA Delivery

The efficient delivery of small interfering RNA (siRNA) to tumor cells still remains a great challenge. Of the various nanocarriers, protein nanocages have attracted extensive interest due to their unique structure and suitable characteristics derived from their proteinaceous nature. However, most reported protein nanocages that are developed are based on virus capsid proteins, which may raise safety concerns, including those related to gene mutation and carcinogenesis. The development of nonviral protein‐based systems for siRNA delivery is greatly needed. In this study, a novel siRNA delivery system based on heat shock protein (Hsp) nanocages is developed by a genetic engineering method. The delivery system could condense siRNA into stable complexes and protect the condensed siRNA from degradation. A cellular uptake analysis shows that siRNA is introduced into tumor cells mediated by Hsp‐R9 nanocages. Green fluorescent protein (GFP) expression in HeLa‐EGFP cells is significantly downregulated by Hsp‐R9/siRNA complexes. The results indicate that Hsp nanocages may be a good platform for siRNA delivery into tumor cells.     

基于原位聚合技术构建细胞内微环境响应型DNA递送系统北大核心CSCD

传统的非病毒载体基于分子间静电自组装作用与核酸结合,组装的复合物在体内复杂的环境中容易发生结构解离,共价结合的交联聚合物载体有望成为解决传统非病毒载体结构稳定性差的有效方案。选择N-(3-氨丙基)甲基丙烯酰胺盐酸盐、1-乙烯基咪唑、2-甲基丙烯酰氧乙基磷酸胆碱与N,N′-双(丙稀酰)胱胺作为多功能性单体,采用原位聚合方法制备包载质粒DNA(pDNA)的交联聚合物-pDNA复合物。其中,共价键为载体提供优异的结构稳定性;1-乙烯基咪唑能够响应胞内溶酶体酸性微环境,触发质子海绵效应便于复合物的溶酶体逃逸;N,N′-双(丙稀酰)胱胺的二硫键可以响应胞内高水平的谷胱甘肽(GSH),实现复合物在细胞内部选择性解聚,释放内含pDNA。研究表明,该复合物平均水合半径约135 nm,ζ电势约−6.5 mV,形貌近似球形。该复合物可在10 mg/mL肝素环境中保持结构稳定性,具有响应细胞内GSH,触发释放包载核酸分子的功能。细胞实验证明该复合物细胞毒性低。细胞摄取、转染能力强。综上所述,基于原位聚合技术制备交联聚合物载体在基因递送领域具有重要应用前景,本研究为新型基因递送载体的开发提供了新思路。     

Selenocystine-Derived Label-Free Fluorescent Schiff Base Nanocomplex for siRNA Delivery Synergistically Kills Cancer Cells

Chemo and siRNA synergic treatments for tumors is a promising new therapeutic trend. Selenocystine, a selenium analog of cysteine, has been considered a potential antitumor agent due to its redox perturbing role. In this study, we developed a nanocarrier for siRNA based on a selenocystine analog engineered polyetherimide and achieved traceable siRNA delivery and the synergic killing of tumor cells. Notably, we applied the label-free Schiff base fluorescence mechanism, which enabled us to trace the siRNA delivery and to monitor the selenocystine analogs’ local performance. A novel selenocystine-derived fluorescent Schiff base linker was used to crosslink the polyetherimide, thereby generating a traceable siRNA delivery vehicle with green fluorescence. Moreover, we found that this compound induced tumor cells to undergo senescence. Together with the delivery of a siRNA targeting the anti-apoptotic BCL-xl/w genes in senescent cells, it achieved a synergistic inhibition function by inducing both senescence and apoptosis of tumor cells. Therefore, this study provides insights into the development of label-free probes, prodrugs, and materials towards the synergic strategies for cancer therapy.     

Bacteriochlorophyll-a as photosensitizer for photodynamic treatment of transplantable murine tumors.

Bacteriochlorophyll-a (bChla), which absorbs light of 780 nm wavelength, was tested for in vivo photodynamic activity in the SMT-F and RIF transplantable mouse tumor systems. High performance liquid chromatography (HPLC) analysis of tissue extracts showed that bChla was rapidly degraded in vivo to bacteriopheophytin-a (bPheoa) and other breakdown products. These were also photodynamically active, and tumor response could be achieved over a wavelength range of 660 to 780 nm, while tumor cure was restricted to wavelengths of 755 (bPheoa) to 780 nm. A photosensitizing product absorbing at 660 nm was also present in isolated tumor cells. Photodynamic cell kill of tumor cells isolated from tumors after bChla accumulation in vivo, using 755 or 780 nm light _vitro, was exponential up to 20–40 J cm⁻². Above this light dose little or no further damage could be achieved, which is an indication of the rapid photobleaching of these sensitizers. In vivo, vascular occlusion occurred readily if light treatment was delivered shortly after sensitizer administration, but was delayed if light treatment was carried out 24 h after injection. Although up to 70% of tumor cells were lethally damaged after completion of in vivo light treatment, concurrent severe vascular destruction seemed necessary for tumor cure. Normal tissue photosensitivity totally subsided within 5 days after sensitizer administration.     

Mitochondria-targeted fluorescent probe based on vibration-induced emission for real-time monitoring mitophagy-specific viscosity dynamic

Directly monitoring mitophagy-specific viscosity dynamic in living cells is of great significance but remains challenging. Herein, this study reported a novel mitochondria-targeted fluorescent probe DPAC-DY based on vibration-induced emission (VIE) for monitoring viscosity changes during mitochondrial autophagy. This probe contained N,N'-diphenyl- dihydrodibenzo[a,c]phenazine (DPAC) as the VIE core and two positively charged pyridinium moieties for mitochondria anchoring. As the ambient viscosity increased, the vibration of DPAC-DY could be hindered, and subsequently resulting in the enhancement of fluorescence emission. In vitro and intracellular experiments indicated that the probe DPAC-DY showed highly sensitive response to viscosity due to VIE mechanism. Importantly, by virtue of this probe, in situ and real-time visualization of the specific viscosity dynamics during the mitochondrial autophagy process was achieved. Thus, this work provides a novel strategy for VIE-based viscosity response sensors applied to specific organelles and offers a platform for in-depth study of mitochondrial viscosity-related diseases.     

磷脂管的体外组装及其在生物与化学领域的研究进展

由磷脂组成的微管结构在有机体组织及器官中均有存在，在细胞间的物质传递及信息交流等方面起着重要作用。实现磷脂管在体外的简单快速组装是解析生命信息系统的良好途径。由于良好的生物相容性及支架结构特性，对磷脂管进行材料或结构修饰，是扩大其潜在应用的极佳手段。以磷脂管本身为载体以及以磷脂管为模板修饰构建的微纳米材料，在生物、材料及化学等方面的应用也取得了一系列成果。对磷脂管的体外组装及其在多个领域的应用研究进行了梳理归纳，重点对磷脂管的体外组装方式以及磷脂管在生物及化学等领域的应用等做了分类阐述与总结，并提出磷脂管在多功能纳米复合材料方面的未来发展及应用方向。     

Delivery of Survivin siRNA Using Cationic Diphenylalanine Vesicles

Small interfering RNA(siRNA) has been proved to be a powerful tool for silencing target gene in cells, raising the possibility that siRNA can be employed as a therapy for treating cancers and other genetic diseases. However, siRNA transfection has the limitation due to the difficulty in the delivery of siRNA to target cells and tissues. To explore an efficient biocompatible siRNA delivery system, cationic diphenylalanine vesicles(CDPVs) were constructed to transfer survivin siRNA to human ovarian cancer cells. The morphology of CDPVs was characterized by scanning electron microscopy(SEM) and the distribution of survivin siRNA was characterized by confocal laser scanning microscopy, which reveal that diphenylalanine and the survivin siRNA were successfully co-delivered. After co-incubation for 48 h, the CDPVs/siRNA exhibited enhanced tumor cell growth inhabitation and apoptosis inducted in human SK-OV-3 ovarian carcinoma cells. Overall, CDPVs is an efficient siRNA delivery system and has a promising prospect for cancer therapy.     

Multivalent Aptamer-modified DNA Origami as Drug Delivery System for Targeted Cancer Therapy

In spite of great development in nanoparticle-based drug delivery systems(DDSs)for improved therapeutic efficacy,it remains challenging for effective delivery of chemotherapeutic drugs to targeted tumor cells.In this work,we report a triangle DNA origami as targeted DDS for cancer therapy.DNA origami shows excellent biocompatibility and stability in cell culture medium for 24 h.In addition,the DNA origami structures conjugated with multivalent aptamers enable for efficient delivery of anticancer drug doxorubicin(Dox)into targeted cancer cell due to their targeting function,reducing side effects associated with nonspecific distribution.Moreover,we also demonstrated that the multivalent aptamer-modified DNA origami loading Dox exhibits prominent therapeutic efficacy in vitro.Accordingly,this work provides a good paradigm for the development of DNA origami nanostructure-based targeted DDS for cancer therapy.     

Polymeric micelle systems of hydroxycamptothecin based on amphiphilic N-alkyl-N-trimethyl chitosan derivatives

This research investigated the possible utilization of amphiphilic N-octyl-N-trimethyl chitosan (OTMCS) derivatives in solublization and controlled release of 10-hydroxycamptothecin (10-HCPT), a hydrophobic anticancer drug. The release behavior of the 10-HCPT-OTMCS micelles was measured and compared to that of a commercial 10-HCPT lyophilized powder in vitro and in vivo. This research also examined the effects of chemical structure of the chitosan derivatives and the micellar preparation conditions on the encapsulation efficiency, drug loading content, and particle size of the polymeric micelles. The results showed that these chitosan derivatives were able to self-assemble and form spherical shape polymeric micelles with an average particle size range of 24–280 nm and a drug loading content of 4.1–32.5%, depending on the modified structures and loading procedures. The solubility of 10-HCPT in aqueous fluid was increased about 80,000-fold from 2 ng/ml in water to 1.9 mg/ml in OTMCS micellar (degree of octyl and trimethyl substitution is 8% and 54%, respectively) solution. In addition, OTMCS was able to modulate the in vitro release of 10-HCPT and improve its pharmacokinetic properties and lactone ring stability in vivo. These data suggested the possible utilization of the amphiphilic micellar chitosan derivatives as carriers for hydrophobic drugs for improving their delivery and release properties.     

Prostatic Zn determination for prostate cancer diagnosis

We present our studies of prostatic Zn concentration measurements, carried out in the light of a novel prostate cancer (CAP) diagnosis method proposed by us. The method is based on in vivo prostatic Zn mapping by XRF trans-rectal probe.

We report on the extensive clinical studies, intended to assess the validity of the novel proposed diagnostic method. Zn content was measured in vitro in needle-biopsy samples from several hundreds of patients, and was correlated with histological findings and other patient parameters. For this purpose, a technique of absolute Zn content determination in 1 mm³ fresh tissue samples by XRF was developed. The experimental details and the main clinical-evaluation results are presented.

We further outline the suggested design of the XRF trans-rectal probe for an efficient in vivo detection and mapping of the Zn fluorescence radiation from the prostate through the rectal wall. Laboratory phantom studies, a preliminary design concept and its expected performance are also reported.     

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles

In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of −13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.     

Design and Synthesis of Novel 3,4-Dihydro-2H-1,2,4-benzothiadiazine 1,1-Dioxides-based Strobilurins as Potent Fungicide Candidates

To discover novel strobilurins analogues with good and broad spectrum activity, a series of novel 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxides-based strobilurins was designed, synthesized, and tested against various phytopathogenic fungi. Compounds 7b, 7c, and 7k exhibited substantial and broad spectrum antifungal activities against the tested phytopathogenic fungi, especially compound 7b, which showed 100%, 80%, 90%, and 90% antifungal activity(in virto) against Erysiphe graminis(E. graminis), Puccinia sorghi Schw.(P. sorghi Schw.), Colletotrichum lagenarium(C. lagenarium), and Pseudoperonospora cubensis(P. cubensis) at 300 μg/mL, respectively, better or comparable to the positive control azoxystrobin. Moreover, compound 7b exhibited 85% greenhouse inhibition activity(in vivo) against E. graminis even at 0.2 μg/mL, equal to azoxystrobin(90%) and trifloxystrobin(90%). Meanwhile, compound 7b against P. cubensis displayed 70% and 55% greenhouse inhibition activity(in vivo) at 1.56 and 0.2 μg/mL, respectively, much better than those of azoxystrobin and trifloxystrobin(both 0% at 1.56 and 0.2 μg/mL). Therefore, compound 7b could be considered as the most promising fungicidal candidate for further study. Furthermore, based on the effective concentration(EC₅₀) against C. arachidicola, the built CoMSIA model provided the useful reference for the further structural optimization design.     

L-Arginine/nanofish bone nanocomplex enhances bone regeneration via antioxidant activities and osteoimmunomodulatory properties

In this study,a promising strategy has been developed to promote bone regeneration by combining antioxidant activities and osteoimmunomodulatory properties.Herein,an L-arginine/nanofish bone(Arg/NFB) nanocomplex has been prepared and evaluated in vitro and in vivo.The Arg/NFB nanocomplex possesses good antioxidant activities and could modulate the polarization of non-activated macrophage into different types and induce the secretion of pre-inflammato ry,anti-inflammatory,osteogenic as well as angiogenic cytokines.Additionally,the regulated immune microenvironment can enhance the osteogenic differentiation of mouse embryo osteoblast precursor cells(MC3 T3-E1) and angiogenic capacity of human umbilical vein endothelial cells(HUVECs),leading to the improved formation of mineralized nodules,alkaline phosphatase activity and angiogenic effects.In vivo results with cranial defect models reveal that the treatment of Arg/NFB nanocomplex exhibited significant improvement of new bone formation and angiogenesis.All the results demonstrate Arg/NFB nanocomplex with antioxidant activities and osteoimmunomodulatory properties could be a new idea for developing the next generation of bone regeneration biomaterials.     

Solid lipid nanoparticles prepared by solvent diffusion method in a nanoreactor system

In this study, water-in-oil (W/O) miniemulsion was used as nanoreactor to prepare solid lipid nanoparticles (SLN) by solvent diffusion method. n-Hexane, Tween 80 and Span 80 were used as the oil phase and surfactant combination for preparation of W/O miniemulsion, respectively. The stable miniemulsion with the particle size of 27.1 ± 7.6 nm was obtained when the composition of water/Tween 80/Span 80/n-hexane was 1 ml/18 mg/200 mg/10 ml. Clobetasol propionate (CP) was used as a model drug. The physicochemical properties of the SLN, such as particle size, zeta potential, surface morphology, drug entrapment efficiency, drug loading capacity and in vitro drug release behaviors were investigated, comparing with those of SLN prepared by conventional aqueoethod. The SLN prepared by the novel method displayed smaller particles size and higher dus solvent diffusion mrug entrapment efficiency than those of SLN prepared by the conventional method. The drug entrapment efficiency decreased with increasing of charged amount of drug, and 15.9% of drug loading was achieved as the charged amount of drug was 20%. The in vitro drug release tests indicated that the drug release rate was faster than that of SLN prepared by the conventional method, and the drug content in SLN did not affect the in vitro drug release profile.     

Single siRNA Nanocapsules for Enhanced RNAi Delivery

Synthetic siRNA has been considered as a highly promising therapeutic agent for human diseases. However, clinical use of siRNA has been hampered by instability in the body and inability to deliver sufficient RNA interference compounds to the tissues or cells. To address this challenge, we present here a single siRNA nanocapsule delivery technology, which is achieved by encapsulating a single siRNA molecule within a degradable polymer nanocapsule with a diameter around 20 nm and positive surface charge. As proof-of-concept, since CCR5 is considered a major silencing target of HIV therapy, CCR5-siRNA nanocapsules were delivered into 293T cells and successfully downregulated the CCR5 RNA fused with mCherry reporter RNA. In the absence of human serum, nanocapsules and lipofectamine silenced expression of CCR5-mCherry expression to 8% and 15%, respectively. Such nanocapsules maintain the integrity of siRNA inside even after incubation with ribonuclease and serum for 1 h; under the same conditions, siRNA is degraded in the native form or when formulated with lipofectamine. In the presence of serum, CCR5-siRNA nanocapsules knocked down CCR5-mCherry expression to less than 15% while siRNAs delivered through lipofectamine slightly knocked down the expression to 55%. In summary, this work provides a novel platform for siRNA delivery that can be developed for therapeutic purposes.     

胺解改性聚L-谷氨酸苄酯引导骨再生膜的制备

以聚L-谷氨酸苄酯(PBLG)为原料, 通过溶剂浇铸与粒子沥滤法分别构建PBLG单层致密和PBLG单层多孔膜, 利用乙醇胺对薄膜表面改性, 构筑双层引导骨再生膜. 研究了不同胺解改性时间对PBLG-s-PHEG双层膜亲水性和力学性能的影响, 结果表明, 随着PBLG分子量的增大, 薄膜的力学性能增强而降解速率减缓. 延长胺解改性时间可提高薄膜亲水性和体内外降解速率. 细胞实验结果表明, 双层薄膜的致密结构能够有效阻隔成纤维细胞的侵入, 多孔结构能够支持细胞贴壁黏附和铺展. 体外生物活性评价结果表明, 表面改性的PBLG基材料可用于体内骨缺损修复. 本文所构建的双层引导骨再生膜在体外具有良好的力学性能和降解性能, 与组织具有一定的贴合性, 同时可有效阻碍成纤维细胞侵入, 具有潜在应用价值.     

ICG and Sunitinib-loaded NH2-MOFs for Folate-mediated Hepatocellular Carcinoma Dual-modal Therapy

This research investigated a novel folic acid(FA)-modified zirconium core metal-organic framework(MOF) Uio-66 as a nanocarrier to deliver indocyanine green(ICG) and Sunitinib to cancer cells for combination therapy. Platinum-loaded Uio-66 nanoparticles(Pu) were synthesized via a one-pot method, followed by the modification with FA on their surfaces. This afforded FPu that enabled subsequent loading of ICG and Sunitinib to achieve dual-modal cancer therapy. Drug loading/release test and singlet oxygen detection were also conducted in vitro, and the nanoparticles showed considerable drug loading efficiency for both ICG and Sunitinib, coupled with a high singlet oxygen generation rate. Specifically, drug loading and encapsulation efficiency of Sunitinib were 2.30% and 72.67%, while those for ICG were 2.87% and 90.28%, respectively. Additionally, cytotoxicity test on HepG2 human hepatocellular carcinoma cancer cell line revealed that the fully functional nanoparticles possess excellent biocompatibility and as such could be further investigated as a potential drug delivery system for effectual carcinoma cancer treatment.     

Adaptive Amphiphilic Dendrimer‐Based Nanoassemblies as Robust and Versatile siRNA Delivery Systems

siRNA delivery remains a major challenge in RNAi‐based therapy. Here, we report for the first time that an amphiphilic dendrimer is able to self‐assemble into adaptive supramolecular assemblies upon interaction with siRNA, and effectively delivers siRNAs to various cell lines, including human primary and stem cells, thereby outperforming the currently available nonviral vectors. In addition, this amphiphilic dendrimer is able to harness the advantageous features of both polymer and lipid vectors and hence promotes effective siRNA delivery. Our study demonstrates for the first time that dendrimer‐based adaptive supramolecular assemblies represent novel and versatile means for functional siRNA delivery, heralding a new age of dendrimer‐based self‐assembled drug delivery in biomedical applications.