寡胺-葡聚糖类阳离子化合物合成及其在基因转染中的应用

应用氧化葡聚糖与精胺通过还原胺法反应,制备了聚阳离子非病毒基因载体葡聚糖-寡胺化合物;用红外光谱测定仪、核磁共振波谱仪、紫外光谱仪,激光纳米粒度测定仪、透射电子显微镜进行了表征.证实了该载体为葡聚糖-精胺化合物DSP.其平均粒径为170nm,Zeta电位为+31.1mV,镜下呈园球形状.将载体DSP与pCMV-GFP的质粒DNA以8:1(N/P)组成复合物时,对SMMC-7721人肝癌细胞进行体外转染,转染效率高于阳性对照组transfectine 2000,为31.1%.实验表明该材料有望成为临床的非病毒基因载体.     

Synthesis and characterization of thiolated carboxymethyl chitosan-graft-cyclodextrin nanoparticles as a drug delivery vehicle for albendazole

A new strategy for the synthesis of thiolated carboxymethyl chitosan-g-cyclodextrin nanoparticles by an ionic-gelation method is presented. The synthetic approach was based on the utilization of 1,6-hexamethylene diisocyanate during cyclodextrin grafting onto carboxymethyl chitosan. The use of the 1,6-hexamethylene diisocyanate resulted in reactions between cyclodextrin and active sites at the C₆-position of chitosan, and preserved amino groups of chitosan for subsequent reactions with thioglycolic acid, as the thiolating agent, and tripolyphosphate, as the gelling counterion. Various methods such as scanning electron microscopy, rheology and in vitro release studies were employed to exhibit significant features of the nanoparticles for mucosal albendazole delivery applications. It was found that the thiolated carboxymethyl chitosan-g-cyclodextrin nanoparticles prepared using an aqueous solution containing 1 wt% of tripolyphosphate and having 115.65 (μmol/g polymer) of grafted thiol groups show both the highest mucoadhesive properties and the highest albendazole entrapment efficiency. The latter was confirmed theoretically by calculating the enthalpy of mixing of albendazole in the above thiolated chitosan polymer.     

Synthesis and characterization of Ag@polycarbazole nanoparticles and their novel optical behavior

Ag@polycarbazole as a new 3D nanocomposite material was successfully fabricated using microwave polyol reduction method followed by cations assisted oxidative polymerization of carbazole. The material was characterized by transmission electron microscopes (TEM), scanning electron microscope (SEM), FT-IR, and Raman measurements. The results confirmed that Ag nanoparticles are entirely enclosed by 3,6 polycarbazole. The interfacial study was carried out by X-ray photoelectron spectroscopy, which revealed that Ag surface atoms are intact chemically and blue shifted due to polymer composite formation. The surface plasmon resonance (SPR) and photoluminescence (PL) behavior were found quite sensitive to surface composition of Ag nanoparticles, which is greatly influenced by cations dopant and enclosing polymer in contrary way. The polycarbazole played a contributive role to counterbalance the effect of cations dopant in SPR and PL behaviors along with displaying its luminescence in violet region.     

Cationic silica nanoparticles as gene carriers: synthesis, characterization and transfection efficiency in vitro and in vivo

The potential of cationic SiO2 nanoparticles was investigated for in vivo gene transfer in this study. Cationic SiO2 nanoparticles with surface modification were generated using amino-hexyl-amino-propyltri-methoxysilane (AHAPS). The zeta potential of the nanoparticles at pH = 7.4 varied from -31.4 mV (unmodified particles; 10 nm) to +9.6 mV (modified by AHAPS). Complete immobilization of DNA at the nanoparticle surface was achieved at a particle ratio of 80 (w/w nanoparticle/DNA ratio). The surface modified nanoparticle had a size of 42 nm with a distribution from 10-100 nm. The ability of these particles to transfect pCMVbeta reporter gene was tested in Cos-1 cells, and optimum results were obtained in the presence of FCS and chloroquine at a particle ratio of 80. These nanoparticles were tested for their ability to transfer genes in vivo in the mouse lung, and a two-times increase in the expression levels was found with silica particles in comparison to EGFP alone. Very low or no cell toxicity was observed, suggesting silica nanoparticles as potential alternatives for gene transfection.     

Synthesis and characterization of PHV-block-mPEG diblock copolymer and its formation of amphiphilic nanoparticles for drug delivery

Despite the recent research interest in the field of nanoparticles delivery system, their structure modification and transport behavior of various hydrophobic drugs is poorly developed. In this article the synthesis of novel amphiphilic diblock copolymer poly([R]-3-hydroxyvalerate)-block-monomethoxy poly(ethylene glycol) (PHV-block-mPEG) was undertaken by modifying the structure of biodegradable and hydrophobic poly([R]-3-hydroxyvalerate) (PHV) with hydrophilic monomethoxy poly(ethylene glycol) (mPEG). The chemical combination of the two blocks was carried out in the melt using bis(2-ethylhexanoate) tin as transesterification catalyst. The synthesized product was characterized by gel permeation chromatography (GPC), 1H nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC) analysis. The block copolymer self-assembled into amphiphilic nanoparticles with a core of hydrophobic PHV and a shell of hydrophilic mPEG in aqueous solution. Characterization of the nanoparticles showed the formation of discrete, spherically shaped nanoparticles with mean particle size of 200 +/- 1 nm and zeta potential of -14 +/- 1 mV. A hydrophobic drug thymoquinone was efficiently incorporated into the core hydrophobic domain of the nanoparticles and its release kinetics was studied in vitro. The amphiphilic PEGylated nanoparticles showed biocompatibility when checked in neuronal hippocampal cells of prenatal rat. Our results suggest that the amphiphilic nanoparticles with core-shell structures are potentially useful to develop novel drug carriers.     

Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection-rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.     

Structural characterization of novel gemini non-viral DNA delivery systems for cutaneous gene therapy

The structural and physicochemical properties of novel cationic lipid-based DNA complexes have been investigated for the purpose of designing micro/nano-scale self-assembling delivery systems for cutaneous gene therapy. DNA/gemini surfactant (spacer n?=?3–16; chain m?=?12 or 16) complexes (1?:?10 charge ratio), with or without dioleoylphosphatidyl-ethanolamine (DOPE), designed for cellular transfection, were generally in the range of 100–200?nm as demonstrated by atomic force microscopy and particle size analysis. Small-angle X-ray scattering measurements indicated that the DNA/gemini complexes lacked long-range order, whereas DNA/gemini/DOPE complexes exhibited lamellar and polymorphic phases other than hexagonal. Correlation studies using transfection efficiency data in PAM 212 keratinocytes and in vitro skin absorption indicated that formulations containing gemini surfactants having the ability to induce structures other than lamellar in the resulting complexes, generally exhibited greater transfection activity and cutaneous absorption.     

Synthesis and characterization of highly stable dispersions of copper nanoparticles by a novel one-pot method

Highly stable dispersions of copper nanoparticles with an average particle size of about 2 nm were synthesized via a simple and cost-effective one-pot method with CS₂ as a reactant and a solvent as well for synthesizing surface-capping agent and Cu nanoparticles. As-prepared copper nanoparticles were characterized by X-ray powder diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy, and Fourier transform infrared spectrometry. It has been found that surface-capped copper nanoparticles with a narrow size distribution can be readily fabricated with the established method. As-obtained spherical surface-capped Cu nanoparticles can form stable dispersions which do not show any sign of sediment even after storage at ambient conditions for 6 months, showing promising potential as novel lubricant additive.     

Surface-modified superparamagnetic nanoparticles for drug delivery: preparation, characterization, and cytotoxicity studies

Superparamagnetic iron oxide nanoparticles have been used for many years as magnetic resonance imaging (MRI) contrast agents or in drug delivery applications. In this study, a novel approach to prepare magnetic polymeric nanoparticles with magnetic core and polymeric shell using inverse microemulsion polymerization process is reported. Poly(ethyleneglycol) (PEG)-modified superparamagnetic iron oxide nanoparticles with specific shape and size have been prepared inside the aqueous cores of AOT/n-Hexane reverse micelles and characterized by various physicochemical means such as transmission electron microscopy (TEM), infrared spectroscopy, atomic force microscopy (AFM), vibrating sample magnetometry (VSM), and ultraviolet/visible spectroscopy. The inverse microemulsion polymerization of a polymerizable derivative of PEG and a cross-linking agent resulted in a stable hydrophilic polymeric shell of the nanoparticles. The results taken together from TEM and AFM studies showed that the particles are spherical in shape with core-shell structure. The average size of the PEG-modified nanoparticles was found to be around 40-50 nm with narrow size distribution. The magnetic measurement studies revealed the superparamagnetic behavior of the nanoparticles with saturation magnetization values between 45-50 electromagnetic units per gram. The cytotoxicity profile of the nanoparticles on human dermal fibroblasts as measured by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the particles are nontoxic and may be useful for various in vivo and in vitro biomedical applications.     

一种均苯型可溶性聚酰亚胺的研究

采用二步法合成了一种新型的均苯四甲酸二酐型 (均苯型 )聚酰亚胺 ,实验分别考察了聚酰亚胺的溶解性能及其特性粘度 (Iv)与玻璃化温度 (Tg)、热分解温度 (Td)的关系 ,并对其预聚体—聚酰胺酸的亚胺化过程进行了表征。结果表明 :该聚酰亚胺可溶于N ,N′ -二甲基甲酰胺等强极性溶剂 ;Tg和Td随Iv值增大而升高 ,Iv值较低时 ,其对Tg和Td的影响较显著 ;随着热处理时间的延长或温度的提高 ,亚胺化程度增加。     

In vitro comparative evaluation of monolayered multipolymeric films embedded with didanosine-loaded solid lipid nanoparticles: a potential buccal drug delivery system for ARV therapy

Drug delivery via the buccal route has emerged as a promising alternative to oral drug delivery. Didanosine (DDI) undergoes rapid degradation in the gastrointestinal tract, has a short half-life and low oral bioavailability, making DDI a suitable candidate for buccal delivery. Recent developments in buccal drug delivery show an increased interest toward nano-enabled delivery systems. The advantages of buccal drug delivery can be combined with that of nanoparticulate delivery systems to provide a superior delivery system. The aim of this study was to design and evaluate the preparation of novel nano-enabled films for buccal delivery of DDI. Solid lipid nanoparticles (SLNs) were prepared via hot homogenization followed by ultrasonication and were characterized before being incorporated into nano-enabled monolayered multipolymeric films (MMFs). Glyceryl tripalmitate with Poloxamer 188 was identified as most suitable for the preparation of DDI-loaded SLNs. SLNs with desired particle size (PS) (201?nm), polydispersity index (PDI) (0.168) and zeta potential (?18.8?mV) were incorporated into MMFs and characterized. Conventional and nano-enabled MMFs were prepared via solvent casting/evaporation using Eudragit RS100 and hydroxypropyl methylcellulose. Drug release from the nano-enabled films was found to be faster (56% versus 20% in first hour). Conventional MMFs exhibited higher mucoadhesion and mechanical strength than nano-enabled MMFs. SLNs did not adversely affect the steady state flux (71.63?±?13.54?µg/cm²?h versus 74.39?±?15.95?µg/cm²?h) thereby confirming the potential transbuccal delivery of DDI using nano-enabled MMFs. Nano-enabled buccal films for delivery of DDI can be successfully prepared, and these physico-mechanical studies serve as a platform for future formulation optimization work in this emerging field.     

Albumin as a carrier for NO delivery: preparation,physicochemical characterization,and interaction with gold nanoparticles

Background: Nitric oxide (NO) is a gaseous transmitter playing numerous physiological roles and characterized by a short half-life. Its binding to endogenous thiols increases its stability, facilitating its storage and transport. The purpose of this study was to investigate the nitrosated serum albumin (SA-SNO) and to provide a reference for its easy preparation for further use in in vitro studies.

Methods: Serum albumin (SA) was S-nitrosated by reacting with (i) NaNO₂ in acidic medium; (ii) different low-molecular weight S-nitrosothiols (RSNO) (S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), and S,S'-dinitrosobucillamine (Buc(NO)₂)); and (iii) diethylamine NONOate (DEA/NO). SA-SNO was purified by size exclusion chromatography and the S-nitrosation site and the rate were studied by mass spectrometry and Griess–Saville assay, respectively. Then, SA-SNO was characterized by spectrofluorimetry, dynamic light scattering, and circular dichroism. Finally, SA-SNO reactivity with citrate stabilized gold nanoparticles (AuNP-citrate) was investigated via determination of NO release.

Results: S-nitrosation rates of SA were 90.1?±?3.3, 76.8?±?2.7, 80.3?±?3.2, 84.8?±?5.0, and 15.4?±?1.9% (n?=?5), when SA was reacted with acidified NaNO₂, CysNO, GSNO, Buc(NO)₂, and DEA/NO, respectively. The physicochemical characterization indicated that the resulting product corresponded to a mono-S-nitrosothiol (on cysteine-34), and the conformational construction remained unchanged. Stability studies showed that the NO content was preserved over 1 week. AuNP-citrate reacted with SA-SNO with increase of its hydrodynamic diameter but preservation of SNO bond.

Conclusions: SA-SNO prepared and stored under the reported conditions affords a well-defined reference suitable for in vitro studies.     

Intracellular protein delivery and gene transfection by electroporation using a microneedle electrode array

The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals.     

Synthesis and characterization of copper nanoparticles

Reduction of copper salt by sodium citrate/SFS and myristic acid/SFS leads to phase pure Cu nanoparticles. However, a similar reaction with hydrazine hydrate (HH) and sodium formaldehyde sulfoxylate (SFS) in polymer afforded only a mixture of Cu₂O and Cu. Copper nanoparticles so-prepared were characterized by UV-Visible spectroscopy, X-ray diffraction measurements (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Freshly prepared solutions showed an absorption band at about 600 nm due to surface plasmon resonance (SPR). XRD analysis revealed all relevant Bragg's reflection for fcc crystal structure of copper metal. The particle size by use of Scherrer's equation is calculated to be about 30 nm. TEM showed nearly uniform distribution of the particles in PVA.     

Design,characterization, and evaluation of intranasal delivery of ropinirole-loaded mucoadhesive nanoparticles for brain targeting

Context: Parkinson disease (PD) is a common, progressive neurodegenerative disorder, characterized by marked depletion of striatal dopamine and degeneration of dopaminergic neurons in the substantia nigra.

Objective: The purpose of the present study was to investigate the possibility of targeting an anti-Parkinson’s drug ropinirole (RH) to the brain using polymeric nanoparticles.

Materials and methods: Ropinirole hydrochloride (RH)-loaded chitosan nanoparticles (CSNPs) were prepared by an ionic gelation method. The RH-CSNPs were characterized for particle size, polydispersity index (PDI), zeta potential, loading capacity, entrapment efficiency in vitro release study, and in vivo distribution after intranasal administration.

Results and discussion: The RH-CSNPs showed sustained release profiles for up to 18?h. The RH concentrations (% Radioactivity/g) in the brain following intranasal administration (i.n.) of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The concentration of RH was highest in the liver (7.210?±?0.52), followed by kidneys (6.862?±?0.62), intestine (4.862?±?0.45), and lungs (4.640?±?0.92) in rats following i.n. administration of RH-CSNPs. Gamma scintigraphy imaging in rats was performed to ascertain the localization of drug in the brain following intranasal administration of formulations. The brain/blood ratios obtained (0.251?±?0.09 and 0.386?±?0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively) at 0.5?h are indicative of direct nose to brain transport, bypassing the blood–brain barrier (BBB).

Conclusion: The novel formulation showed the superiority of nose to brain delivery of RH using mucoadhesive nanoparticles compared with other delivery routes reported earlier.     

Synthesis and characterization of CdS nanoparticles

In the present work a chemical reduction method is followed to grow CdS nanoparticles at room temperature with varying the amount of reducing agent sodiumborohydride. The dispersed samples in ethanol are characterized using electron diffraction techniques. Simultaneously optical absorption, photoluminescence and longtime photorelaxation of these samples are studied at room temperature. An increase in band gap is observed in each case as compared to bulk CdS. Also particle size decreases with increased amount of reducing agent. Simultaneously long time relaxation increases with decrease of stoichiometric ratio of reducing agent. An attempt is made to correlate the structural, optical, electrical and opto-electrical properties.     

Decaarginine-PEG-artificial lipid/DNA complex for gene delivery: nanostructure and transfection efficiency

Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.