羧甲基壳聚糖复合纳米银、纳米氧化铜的制备及抑菌性研究

本研究在水热条件下利用羧甲基壳聚糖分别还原硝酸银和硫酸铜,得到稳定的羧甲基壳聚糖复合纳米银和纳米氧化铜,并测试其抑菌能力.表征测试显示,所制的纳米银为20～30 nm的球状结构,纳米氧化铜为80～100 nm的花瓣状结构,二者都均匀稳定地分布于羧甲基壳聚糖中.抑菌试验显示,不同浓度的羧甲基壳聚糖复合纳米银(A组2 mg...     

Influence of chitosan coating on magnetic nanoparticles in endothelial cells and acute tissue biodistribution

Chitosan coating on magnetic nanoparticles (MNPs) was studied on biological systems as a first step toward the application in the biomedical field as drug-targeted nanosystems. Composition of MNPs consists of magnetite functionalized with oleic acid and coated with the biopolymer chitosan or glutaraldehyde-cross-linked chitosan. The influence of the biopolymeric coating has been evaluated by in vitro and in vivo assays on the effects of these MNPs on rat aortic endothelial cells (ECs) viability and on the random tissue distribution in mice. Results were correlated with the physicochemical properties of the nanoparticles. Nitric oxide (NO) production by ECs was determined, considering that endothelial NO represents one of the major markers of ECs function. Cell viability was studied by MTT assay. Different doses of the MNPs (1, 10 and 100?μg/mL) were assayed, revealing that MNPs coated with non-cross-linked chitosan for 6 and 24 h did not affect neither NO production nor cell viability. However, a significant decrease in cell viability was observed after 36 h treatment with the highest dose of this nanocarrier. It was also revealed that the presence and dose of glutaraldehyde in the MNPs structureimpact on the cytotoxicity. The study of the acute tissue distribution was performed acutely in mice after 24 h of an intraperitoneal injection of the MNPs and sub acutely, after 28 days of weekly administration. Both formulations greatly avoided the initial clearance by the reticuloendothelial system (RES) in liver. Biological properties found for N1 and N2 in the performed assays reveal that chitosan coating improves biocompatibility of MNPs turning these magnetic nanosystems as promising devices for targeted drug delivery.     

反相高效液相色谱法测定壳聚糖纳米粒中丙酮酸乙酯的含量

背景：有关丙酮酸乙酯含量检测方法的研究较少,采用反相高效液相色谱法检测丙酮酸乙酯含量的文献更少。 目的：建立测定壳聚糖纳米粒中丙酮酸乙酯含量的反相高效液相色谱法。 方法：采用Agilent1200系列高效液相色谱仪检测丙酮酸乙酯-壳聚糖纳米粒中丙酮酸乙酯的含量。色谱柱：ZORBAX Eclipse XDB-C18(4.6 mm×150 mm,5 μm),柱温25 ℃,流动相为乙腈-水(体积比为40∶60),流速1 mL/min,检测波长210 nm,进样量20 μL。 结果与结论：丙酮酸乙酯峰与辅料及溶剂峰分离良好,丙酮酸乙酯质量浓度在1-100 mg/L内于峰面积线性关系良好(r =0.999 6),低、中、高浓度丙酮酸乙酯对照品溶液日内、日间精密度的相对标准偏差均小于3%,重复性实验的相对标准偏差为1.25%,稳定性实验的相对标准偏差为1.3%。3批样品的加样回收率分别为(91.5±1.0)%,(93.5±0.2)%,(94.4±0.4)%;包封率分别为(87.20±0.22)%,(90.50±0.15)%,(91.10±0.17)%。不同批次样品丙酮酸乙酯含量检测结果的相对标准偏差分别为0.9%,0.5%,0.3%。说明反相高效液相色谱法灵敏度高、线性范围宽、专属性强、精密度高、加样回收率好、结果精确可靠,可用于壳聚糖纳米粒中丙酮酸乙酯含量的测定。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

生物活性玻璃与壳聚糖复合的骨修复材料

背景：生物活性玻璃是一种多相复合材料,具有良好的生物活性、骨传导性及生物相容性,但作为骨修复材料仍然存在不能完全降解、机械强度较低等不足。 目的：设计生物活性玻璃/壳聚糖复合材料骨组织工程支架,并检测其理化性能。 方法：将2.0%壳聚糖盐酸溶液与β-甘油磷酸钠以7∶1的体积比混合制备壳聚糖溶液。称取0.5,1.0,1.5 g生物活性玻璃分别加入上述壳聚糖溶液中,使得壳聚糖与生物活性玻璃的质量比为2∶1,1∶1及1∶1.5。将复合材料浸泡于模拟生理体液中7 d进行体外矿化。 结果与结论：扫描电镜见复合支架具有相互贯通的多孔结构,孔隙率最高可达89%,孔径大小合适,为100-  300 µm,生物活性玻璃以针状形式分散在壳聚糖支架之间,均匀排列,被壳聚糖支架充分包裹结合紧密。随生物活性玻璃含量的增加,复合材料的孔隙率逐渐下降,断裂强度逐渐升高,他们之间呈正相关性。X射线衍射图及傅里叶变换红外光谱证实复合支架中的单一材料未发生性质改变,示差扫描量热法分析显示正常体温情况下材料无质量丢失。矿化3 d后材料表面形成的羟基磷灰石逐渐长大为绒毛状,数量也明显增多;矿化7 d后绒毛状的羟基磷灰石长成为针状,数量进一步增多,且众多的矿化物结成球状。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

Self-aggregated nanoparticles of linoleic acid-modified glycol chitosan conjugate as delivery vehicles for paclitaxel: preparation,characterization and evaluation

A series of linoleic acid-modified glycol chitosan (LAGC) conjugates were synthesized and characterized by FTIR and ¹H NMR. The effect of the amount of linoleic acid (LA) on the physicochemical properties of LAGC conjugates was investigated. The mean diameters of three LAGC nanoparticles determined by dynamic light scattering ranged from 204 to 289 nm. The critical aggregation concentration values of LAGC conjugates in aqueous solution were 0.0148, 0.0348, and 0.0807 mg/ml, respectively. Paclitaxel (PTX) was physically loaded into the LAGC nanoparticles by a dialysis method. The drug loading content and encapsulation efficiency of PTX-loaded LAGC (PTX-LAGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates. PTX-LAGC nanoparticles were almost spherical in shape observed by transmission electron microscopy. In vitro release revealed that PTX release from the nanoparticles was reduced as the LA substitution degree of LAGC conjugates increased. Compared with the commercial formulation Taxol, PTX-LAGC-1 nanoparticles exhibited comparable cellular uptake and cytotoxicity against HepG2 cells in vitro. Importantly, PTX-LAGC-1 nanoparticles demonstrated the stronger antitumor efficacy against hepatic H22 tumor-bearing mice than Taxol (p < 0.05). Therefore, glycolipid-like LAGC nanoparticles had a potential as delivery vehicles for tumor therapy.     

Development of chitosan/gelatin hydrogels incorporation of biphasic calcium phosphate nanoparticles for bone tissue engineering

Abstract

The chitosan/gelatin hydrogel incorporated with biphasic calcium phosphate nanoparticles (BCP-NPs) as scaffold (CGB) for bone tissue engineering was reported in this article. Such nanocomposite hydrogels were fabricated by using cycled freeze-thawing method, of which physicochemical and biological properties were regulated by adjusting the weight ratio of chitosan/gelatin/BCP-NPs. The needle-like BCP-NPs were dispersed into composites uniformly, and physically cross-linked with chitosan and gelatin, which were identified via Scanning Electron Microscope (SEM) images and Fourier Transform Infrared Spectroscopy (FT-IR) analysis. The porosity, equilibrium swelling ratio, and compressive strength of CGB scaffolds were mainly influenced by the BCP-NPs concentration. In vitro degradation analysis in simulated body fluids (SBF) displayed that CGB scaffolds were degraded up to at least 30?wt% in one month. Also, CCK-8 analysis confirmed that the prepared scaffolds had a good cytocompatibility through in culturing with bone marrow mesenchymal stem cells (BMSCs). Finally, In vivo animal experiments revealed that new bone tissue was observed inside the scaffolds, and gradually increased with increasing months, when implanted CGB scaffolds into large necrotic lesions of rabbit femoral head. The above results suggested that prepared CGB nanocomposites had the potential to be applied in bone tissue engineering.     

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.     

Preparation and characterization of polyelectrolyte complex nanoparticles based on poly (malic acid), chitosan. A pH-dependent delivery system

The main objective of this work was to develop polyelectrolyte complex (PEC) nanoparticles based on poly (malic acid), chitosan (PMLA/CS) as pH-dependent delivery systems. The results indicated that the PMLA/CS Nps were successfully prepared. The prepared PMLA/CS Nps showed spherical morphology with a mean diameter of 212.81 nm and negative surface charge of ?24.60 mV, and revealing significant pH-sensitive properties as the mass ratio of PMLA to CS was 5:5. The prepared PMLA/CS Nps were characterized by FT-IR, TEM and DLS. The prepared PMLA/CS Nps remained stable over a temperature range of 4–53 °C. Doxorubicin (Dox) as a model drug was loaded on the nanoparticles through the physical adsorption method. The high drug loading efficiency (16.9%) and the sustained release patterns in acidic media were observed, and the release accelerated in alkaline solutions. MTT based cytotoxic analysis also depicted the non-toxic nature of PMLA/CS Nps, while Dox-PMLA/CS Nps showed dose-dependent cytotoxicity towards MDA-MB-231 cells. Hence, the nanoparticles could be potentially applied as pH sensitive drug vehicles for controlled release.     

精氨酸修饰壳聚糖基因纳米粒子对血液补体活性和溶血作用的影响

目的 考察精氨酸修饰壳聚糖基因纳米粒子（ACGN）对血液补体活性和溶血的影响.方法 将精氨酸接枝到壳聚糖上制备精氨酸修饰的壳聚糖,用共沉降的方法制备精氨酸修饰壳聚糖基因纳米粒子,用光子相关光谱检测精氨酸修饰壳聚糖基因纳米粒子的粒径,用凝胶电泳阻滞实验对精氨酸修饰壳聚糖与DNA的相互作用进行研究,并考察壳聚糖基因纳米粒子（CGN）和ACGN在体内外对血液补体活性和溶血作用的影响.结果 精氨酸修饰壳聚糖在电荷比为2∶1时能有效地完全包覆DNA,并形成粒径大约为120～180nm的纳米粒子,ACGN和CGN在体内外对红细胞没有明显的破坏作用,在体外对补体系统均没有明显的影响作用,但在体内引起补体的轻微升高.结论 ACGN在体内外没有引起明显的补体激活和溶血作用,有望成为一种新型的、安全的非病毒基因载体.     

精氨酸修饰壳聚糖基因纳米粒子对血小板GMP-140表达的影响

目的 考察精氨酸修饰壳聚糖基因纳米粒子（ACGN）对血小板GMP-140表达的影响.方法 制备精氨酸修饰的壳聚精（ACS）并朋红外光谱对其进行表征;用共沉降的方法制备精氨酸修饰壳聚糖基因纳米粒子,用凝胶电泳阻滞实验对精氨酸修饰壳聚精与DNA的相互作用进行研究;用酶联免疫双抗夹心法测定血小板α颗粒膜蛋白-140（GMP-140）表达来考察精氨酸修饰壳聚糖基因纳米粒子对血小板激活的影响.结果 红外光谱结果显示精氨酸成功地接枝到壳聚糖上,在正负电荷比≥2：1时,ACS能完全阻滞DNA的迁移,表明所有的DNA均已被ACS完全包覆.壳聚糖基因纳米粒子（CGN）和ACGN在体外均不引起血小板的激活,但在体内则都引起轻微血小板激活.结论 精氨酸修饰壳聚糖纳米粒子对血小板GMP-140的表达没有引起明显的变化,有望成为一种新型的、安全的非病毒基因载体.     

A bilayer composite composed of TiO2-incorporated electrospun chitosan membrane and human extracellular matrix sheet as a wound dressing

We designed bilayer composites composed of an upper layer of titanium dioxide (TiO₂)-incorporated chitosan membrane and a sub-layer of human adipose-derived extracellular matrix (ECM) sheet as a wound dressing for full-thickness wound healing. The dense and fibrous top layer, which aims to protect the wound from bacterial infection, was prepared by electrospinning of chitosan solution followed by immersion in TiO₂ solution. The sponge-like sub-layer, which aims to promote new tissue regeneration, was prepared with acellular ECM derived from human adipose tissue. Using a modified drop plate method, there was a 33.9 and 69.6% reduction in viable Escherichia coli and Staphylococcus aureus on the bilayer composite, respectively. In an in vivo experiment using rats, the bilayer composites exhibited good biocompatibility and provided proper physicochemical and compositional cues at the wound site. Changes in wound size and histological examination of full-thickness wounds showed that the bilayer composites induced faster regeneration of granulation tissue and epidermis with less scar formation, than control wounds. Overall results suggest that the TiO₂-incorporated chitosan/ECM bilayer composite can be a suitable candidate as a wound dressing, with an excellent inhibition of bacterial penetration and wound healing acceleration effects.     

Development of shell cross-linked nanoparticles based on boronic acid-related reactions for self-regulated insulin delivery

Shell cross-linked nanoparticles were fabricated by the complexation of poly(3-methacrylamido phenylboronic acid) (PMAPBA) and thiolated chitosan (chitosan-SH) via boronic acid-related reactions. The formation of PMAPBA/chitosan-SH nanoparticles was confirmed by transmission electron microscopy, dynamic light scattering, and UV spectroscopy. The nanoparticles had a narrow size distribution with a relatively high positive charge density, and the size and zeta potential of the nanoparticles correlated with the chitosan-SH concentration. Furthermore, owing to the cross-linking of the nanoparticle shell, insulin was encapsulated in the nanoparticles with a loading capacity of up to 18%. The release of insulin from the nanoparticles slowed down because of the presence of disulfide bonds and increased with increasing glucose level in the medium. The structure of the released insulin was not distorted. More importantly, the nanoparticles had good cytocompatibility, as demonstrated by in vitro experiments. The simplicity of this strategy along with a high loading capacity, glucose sensitivity, and cytocompatibility of the produced nanoparticles should significantly boost their application in self-regulated insulin delivery.     

Single-step electrochemical deposition of antimicrobial orthopaedic coatings based on a bioactive glass/chitosan/nano-silver composite system

Composite orthopaedic coatings with antibacterial capability containing chitosan, Bioglass® particles (9.8 μm) and silver nanoparticles (Ag-np) were fabricated using a single-step electrophoretic deposition (EPD) technique, and their structural and preliminary in vitro bactericidal and cellular properties were investigated. Stainless steel 316 was used as a standard metallic orthopaedic substrate. The coatings were compared with EPD coatings of chitosan and chitosan/Bioglass®. The ability of chitosan as both a complexing and stabilizing agent was utilized to form uniformly deposited Ag-np. Due to the presence of Bioglass® particles, the coatings were bioactive in terms of forming carbonated hydroxyapatite in simulated body fluid (SBF). Less than 7 wt.% of the incorporated silver was released over the course of 28 days in SBF and the possibility of manipulating the release rate by varying the deposition order of coating layers was shown. The low released concentration of Ag ions (<2.5 ppm) was efficiently antibacterial against Staphyloccocus aureus up to 10 days. Although chitosan and chitosan/Bioglass® coating supported proliferation of MG-63 osteoblast-like cells up to 7 days of culture, chitosan/Bioglass®/Ag-np coatings containing 342 μg of Ag-np showed cytotoxic effects. This was attributed to the relatively high concentration of Ag-np incorporated in the coatings.     

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles

Abstract

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor.

Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo.

Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system).

Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.     

Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport

The present study was to demonstrate the effects of goblet cell-targeting nanoparticles on the oral absorption of insulin in vitro, ex vivo and in vivo, and identify the targeting mechanism as well as the influence of mucus. The insulin loaded nanoparticles were prepared using trimethyl chitosan chloride (TMC) modified with a CSKSSDYQC (CSK) targeting peptide. Compared with unmodified nanoparticles, the CSK peptide modification could facilitate the uptake of nanoparticles in villi, enhance the permeation of drugs across the epithelium, meanwhile, induce a significantly higher internalization of drugs via clathrin and caveolae mediated endocytosis on goblet cell-like HT29-MTX cells. In transport studies across Caco-2/HT29-MTX co-cultured cell monolayer (simulating intestinal epithelium), the CSK peptide modification also showed enhanced transport ability, even if the targeting recognition was partially affected by mucus. Moreover, it was found the existence of mucus was propitious to the transport of insulin from both modified and unmodified nanoparticles. In the pharmacological and pharmacokinetic studies in diabetic rats, the orally administrated CSK peptide modified nanoparticles produced a better hypoglycemic effect with a 1.5-fold higher relative bioavailability compared with unmodified ones. In conclusion, CSK peptide modified TMC nanoparticles showed sufficient effectiveness as goblet cell-targeting nanocarriers for oral delivery of insulin.     

Facile synthesis of magnetic-/pH-responsive hydrogel beads based on Fe3O4 nanoparticles and chitosan hydrogel as MTX carriers for controlled drug release

In the present study, methotrexate (MTX)-encapsulated magnetic-/pH-responsive hydrogel beads based on Fe₃O₄ nanoparticles and chitosan were successfully prepared through a one-step gelation process, which is a very facile, economic and environmentally friendly route. The developed hydrogel beads exhibited homogeneous porous structure and super-paramagnetic responsibility. MTX can be successfully encapsulated into magnetic chitosan hydrogel beads, and the drug encapsulation efficiency (%) and encapsulation content (%) were 93.8 and 6.28%, respectively. In addition, the drug release studies in vitro indicated that the MTX-encapsulated magnetic chitosan hydrogel beads had excellent pH-sensitivity, 90.6% MTX was released from the magnetic chitosan hydrogel beads within 48 h at pH 4.0. WST-1 assays in human liver hepatocellular carcinoma cells (HepG2) demonstrated that the MTX-encapsulated magnetic chitosan hydrogel beads had good cytocompatibility and high anti-tumor activity. Therefore, our results revealed that the MTX-encapsulated magnetic chitosan hydrogel beads would be a competitive candidate for controlled drug release in the area of targeted cancer therapy in the near future.     

Effects of silver nanoparticles on oxidative DNA damage–repair as a function of p38 MAPK status: A comparative approach using human Jurkat T cells and the nematode Caenorhabditis elegans

The large‐scale use of silver nanoparticles (AgNPs) has raised concerns over potential impacts on the environment and human health. We previously reported that AgNP exposure causes an increase in reactive oxygen species, DNA damage, and induction of p38 MAPK and PMK‐1 in Jurkat T cells and in Caenorhabditis elegans. To elucidate the underlying mechanisms of AgNP toxicity, here we evaluate the effects of AgNPs on oxidative DNA damage–repair (in human and C. elegans DNA glycosylases hOGG1, hNTH1, NTH‐1, and 8‐oxo‐GTPases—hMTH1, NDX‐4) and explore the role of p38 MAPK and PMK‐1 in this process. Our comparative approach examined viability, gene expression, and enzyme activities in wild type (WT) and p38 MAPK knock‐down (KD) Jurkat T cells (in vitro) and in WT and pmk‐1 loss‐of‐function mutant strains of C. elegans (in vivo). The results suggest that p38 MAPK/PMK‐1 plays protective role against AgNP‐mediated toxicity, reduced viability and greater accumulation of 8OHdG was observed in AgNP‐treated KD cells, and in pmk‐1 mutant worms compared with their WT counterparts, respectively. Furthermore, dose‐dependent alterations in hOGG1, hMTH1, and NDX‐4 expression and enzyme activity, and survival in ndx‐4 mutant worms occurred following AgNP exposure. Interestingly, the absence or depletion of p38 MAPK/PMK‐1 caused impaired and additive effects in AgNP‐induced ndx‐4(ok1003); pmk‐1(RNAi) mutant survival, and hOGG1 and NDX‐4 expression and enzyme activity, which may lead to higher accumulation of 8OHdG. Together, the results indicate that p38 MAPK/PMK‐1 plays an important protective role in AgNP‐induced oxidative DNA damage–repair which is conserved from C. elegans to humans. Environ. Mol. Mutagen. 55:122–133, 2014. © 2013 Wiley Periodicals, Inc.