The synthesis and characterisation of a novel dendritic system for gene delivery

There has been increasing interest in recent years in gene delivery. We report the synthesis of non-viral delivery systems composed of variations of the cell penetrating peptide TAT, a nuclear localisation signal peptide and dendritic polylysine. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilising gel shift analysis, isothermal titration calorimetry, particle size analysis, zeta potential and transmission electron microscopy. These techniques indicated the successful formation of complexes between the peptide dendrimer and DNA.     

Cationic PLA nanoparticles for DNA delivery: comparison of three surface polycations for DNA binding, protection and transfection properties

Biodegradable cationic nanoparticles (cNP) made of poly(lactide) (PLA) have been shown to be promising carrier systems for in vivo DNA delivery and immunization. In previous work, we have described a versatile approach for the elaboration of cationic PLA cNP based on the use of pre-formed particles and subsequent adsorption of a model polycation, the poly(ethylenimine) (PEI). Here, we evaluated two more polycations, chitosan and poly(2-dimethyl-amino)ethyl methacrylate (pDMAEMA)) to determine the most suitable one for the development of PLA cNP as DNA carriers. Cationic PLA-PEI, PLA-chitosan and PLA-pDMAEMA nanoparticles were compared for interaction with plasmid DNA and, more importantly, with regards to the biological properties of bound DNA. pDMAEMA coating yielded the most positively charged nanoparticles with the highest DNA binding capacity (32 mg/g). Loaded with DNA, all three cNP were in the same size range ( approximately 500 nm) and had a negative zeta potential (-50 mV). PLA-chitosan was the only cNP that released DNA at pH 7; the two others required higher pH. Adsorption and release from cNP did not alter structural and functional integrity of plasmid DNA. Moreover, DNA coated onto cNP was partially protected from nuclease degradation, although this protection was less efficient for PLA-chitosan than others. The highest transfection efficiency in cell culture was obtained with PLA-pDMAEMA carriers. We have shown that at least three different cationic polymers (chitosan, PEI, pDMAEMA) can be used for the production of PLA-based particulate DNA carriers and most probably other cationic polymers can also be used in the same purpose. PLA-pDMAEMA cNP were the most promising system for DNA delivery in this in vitro study. Our future work will focus on the in vivo evaluation of these gene delivery systems.     

Study of Stimuli-Sensitivities of Amphiphilic Modified Star Poly[N,N-(Dimethylamino)ethyl Methacrylate] and Its Ability of DNA Complexation

Cationic star polymers as drug/gene carriers have attracted increasing attention. Herein, environmental sensitivities and star-shaped architecture were combined into a well-defined gene carrier system, and further amphiphilic modification was conducted. The thermo- and pH-sensitive behaviors of poly[N,N-(dimethyl amino)ethyl methacrylate] (PDMAEMA) star polymers before and after amphiphilic modification in aqueous solutions were investigated. The observed lower critical solution temperature (LCST) increased with increasing arm length due to the increase of hydrophilicity. A significant decrease of the LCSTs with increasing pH and ionic-strength of the solution was found. The incorporation of MMA and BA lipophilic moieties decreased the LCST of the cationic star polymer due to enhanced hydrophobic interaction. As a potential gene carrier, the performance of star polymers on binding DNA was primarily evaluated via particle size, zeta potential, and gel retardation measurements. All the complexes formed stable small particles with diameter of 100～150 nm at N/P ratio higher than 3.0. The electrostatic screening effect and facilitation for the interaction of complexes with cell membrane induced by the incorporation of lipophilic units were expected to enhance the gene transfection efficiency.     

Investigation of complexes formed by interaction of cationic gemini surfactants with deoxyribonucleic acid

Cationic gemini surfactants, N,N-bis(dimethylalkyl)-alpha,omega-alkanediammonium dibromide [C(m)H(2m+1)(CH(3))(2)N(+)(CH(2))(s)N(+)(CH(3))(2)C(m)H(2m+1) x 2 Br(-), or m-s-m], have proven to be effective synthetic vectors for gene delivery (transfection). Complexes (lipoplexes) of gemini compounds, where m = 12, s = 3, 12 and m = 18 : 1(oleyl), s = 2, 3, 6, with DNA have been investigated using isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM) and circular dichroism (CD) techniques. The results show that lipoplex properties depend on the structural properties of the gemini surfactants, the presence of the helper lipid dioleoylphosphatidylethanolamine (DOPE), and the titration sequence. ITC data show that the interaction between DNA and gemini surfactants is endothermic and the observed enthalpy vs. charge ratio profile depends upon the titration sequence. Isoelectric points (IP) of lipoplex formation were estimated from the zeta potential measurements and show good agreement with the reaction endpoints (RP) obtained from ITC. DLS data indicate that DNA is condensed in the lipoplex. AFM images suggest that the lipoplex morphology changes from isolated globular-like aggregated particles to larger-size aggregates with great diversity in morphology. This change is further accentuated by the presence of DOPE in the lipoplexes. The results are interpreted in terms of some current models of lipoplex formation.     

PEI-g-MPEG/白细胞介素-10基因传递系统转染背根神经节细胞的体外性能研究

探索非病毒基因载体聚乙二醇-聚乙烯亚胺共聚物(PEI-g-MPEG)介导白细胞介素-10(Interleukin-10,IL-10)体外转染原代培养背根神经节细胞(dorsal root ganglion cells,DRGs)的效果.采用本实验室设计合成的PEI-g-MPEG,与同时携带增强型绿色荧光蛋白报告基因及IL-10基因的真核表达质粒DNA(pDC316-EGFP/IL-10)形成复合物,以脂质体(lipofectamine)复合体系Lipo/pDNA为对照,通过溴乙啶(ethidiumbromide,EB)排斥实验、凝胶阻滞电泳实验、粒径与电位的测定及扫描电镜等实验方法观察PEI-g-MPEG/pDNA的复合效果.并且检测了复合物对DRGs的毒性、转染效果及IL-10的蛋白表达情况.结果表明,PEI-g-MPEG在N/P(PEI-g-MPEG所含的氮原子和质粒DNA中磷原子的摩尔比)为5时可完全复合pDNA;随着N/P的增大,PEI-g-MPEG/pDNA复合物的粒径逐渐减小,而表面电位逐渐增大;在N/P为15时报告基因转染效果和IL-10蛋白表达情况较好,复合物的形貌呈大小均一的球形.PEI-g-MPEG/IL-10基因传递系统对于神经病理性疼痛的基因治疗具有潜在应用价值.     

Bioreducible crosslinked low molecular weight branched PEI-PBLG as an efficient gene carrier

Low molecular weight polyethylenimine-poly(gamma-benzyl L-glutamate) (PEI-PBLG) was crosslinked by N,N′-cystamine-bisacrylamide (CBA) to get the polymer named as CBA-PEI-PBLG (CPP). CPP not only inherits PEI-PBLG’s amphiphilic advantages, but also possesses reducible properties. CPP can complex with DNA to form nanoparticles. CPP/DNA complex particles were characterized by particle size and zeta potential analysis. The result showed that the complex particles have suitable size and surface charges for gene delivery. And gel retardation assay also prove CBA-PEI-PBLG has proper condensing ability for DNA. CPP has good reducible property, and also has good biocompatibility because of introducing PBLG segment. The cytotoxicity of CPP was evaluated using MTT assay, and the results showed CPP has lower cytotoxicity compared with PEI 25 K. The transfection properties were characterized in different cells by using plasmid DNA as a reporter. CPP showed higher transfection efficiencies and lower cytotoxicity in HeLa cells. This was attributed to bioreducible and biocompatibility properties of the CPP. These results suggested that CPP is a promising low-toxic, highly effective non-viral gene carrier.     

Tailor-made poly(amidoamine)s for controlled complexation and condensation of DNA

A set of polymer carriers for DNA delivery was synthesized by combining monodisperse, sequence-defined poly(amidoamine) (PAA) segments with poly(ethylene oxide) (PEO) blocks. The precise definition of the PAA segments provides the possibility of correlating the chemical structure (monomer sequence) with the resulting biological properties. Three different PAA-PEO conjugates were synthesized by solid-phase supported synthesis, and the cationic nature of the PAA segments was systematically varied. This allows for the tailoring of interactions with double-stranded plasmid DNA (dsDNA). The potential of the PAA-PEO conjugates as non-viral vectors for gene delivery is demonstrated by investigating the dsDNA complexation and condensation properties. Depending on the applied carrier, a transition in polyplex (polymer-DNA ion complex) structures is observed. This reaches from extended ring-like structures to highly compact toroidal structures, where supercoiling of the DNA is induced. An aggregation model is proposed that is based on structural investigations of the polyplexes with atomic force microscopy (AFM) and dynamic light scattering (DLS). While the cationic PAA segment mediates primarily the contact of the carrier to the dsDNA, the PEO block stabilizes the polyplex and generates a "stealth" aggregate, as was suggested by Zeta potentials that were close to zero. The controlled aggregation leads to stable, single-plasmid complexes, and stabilizes the DNA structure itself. This is shown by ethidium bromide intercalation assays and DNase digestion assays. The presented PAA-PEO systems allow for the formation of well-defined single-plasmid polyplexes, preventing hard DNA compression and strongly polydisperse polyplexes. Moreover carrier polymers and the resulting polyplexes exhibit no cytotoxicity, as was shown by viability tests; this makes the carriers potentially suitable for in vivo delivery applications.     

Lipid carrier systems for targeted drug and gene delivery

For effective chemotherapy, it is necessary to deliver therapeutic agents selectively to their target sites, since most drugs are associated with both beneficial effects and side effects. The use of lipid dispersion carrier systems, such as lipid emulsions and liposomes, as carriers of lipophilic drugs has attracted particular interest. A drug delivery system can be defined as a methodology for manipulating drug distribution in the body. Since drug distribution depends on the carrier, administration route, particle size of the carrier, lipid composition of the carrier, electric charge of the carrier and ligand density of the targeting carrier, these factors must be optimized. Recently, the lipid carrier system has also been applied to gene delivery systems for gene therapy. However, in both drug and gene medicine cases, a lack of cell-selectivity limits the wide application of this kind of drug and/or gene therapy. Therefore, lipid carrier systems for targeted drug and gene delivery must be developed for the rational therapy. In this review, we shall focus on the progress of research into lipid carrier systems for drug and gene delivery following systemic or local injection.     

Preparation and characterization of m PEG grafted chitosan micelles as 5-fluorouracil carriers for effective anti-tumor activity

The objective of this study was to investigate the potential of methoxy polyethylene glycol（m PEG）grafted chitosan（m PEG-g-CS） to be used as a drug carrier. m PEG-g-CS was successfully synthesized by one-step method with formaldehyde. The substitution degree of m PEG on chitosan was calculated by elemental analysis and was found to be（3.23 0.25）%. m PEG-g-CS self-assembled micelles were prepared by ultrasonic method with the controlled size of 178.5–195.1 nm and spherical morphology. Stable dispersion of the micelles was formed with the zeta potential of 2.3–30.2 m V. 5-Fluorouracil（5-FU）, an anticancer chemotherapy drug, was used as a model drug to evaluate the efficiency of the new drug delivery carrier. The loading efficiency of 5-FU was（4.01 0.03）%, and the drug-loaded m PEG-g-CS self-assembled micelle showed a controlled-release effect. In summary, the m PEG-g-CS self-assembled micelle is proved to be a promising carrier with controlled particle size and controlled-release effect. Therefore, it has great potential for the application as 5-FU carriers for effective anti-tumor activity.     

阳离子基因载体的pH敏感遮蔽体系的制备及表征

合成了一种pH敏感的遮蔽体系-谷氨酸苄酯/谷氨酸共聚物(PBLG-co-PGA), 用于对DNA/阳离子基因载体复合物颗粒表面正电荷的遮蔽, 以提高其在体内的稳定性. 研究表明, PBLG-co-PGA (PGA(x), x为PGA占共聚物中摩尔百分数)具有pH敏感性. 并以pH敏感点接近生理pH值的PGA(60)为遮蔽体系进行研究. PGA(60)能够对DNA/PEI(1:1)复合物颗粒表面正电荷进行有效遮蔽. 凝胶阻滞电泳显示, 用PGA(60)对DNA/PEI复合物进行不同比例遮蔽, 没有发生与DNA的链交换作用. MTT细胞毒性测试表明, PGA(60)和三元复合物DNA/PEI/PGA(60) 在测试范围内几乎没有细胞毒性. 荧光素酶转染实验表明, 部分遮蔽后转染效率有所提高; 用PGA(60)对DNA/PEI复合物完全遮蔽为负电后, 由于同细胞表面的电荷排斥作用, 三元复合物不易被细胞内吞, 导致不发生细胞转染. 因其合适的pH响应性, PGA(60)将可能成为一种能随pH值的变化, 实现对聚阳离子基因载体进行电荷遮蔽/智能释放的遮蔽材料.     

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.     

细胞膜仿生修饰树枝状聚酰胺-胺的研究

利用2-丙烯酰氧基乙基磷酸胆碱的双键与树枝状聚酰胺-胺表面的氨基进行Michael加成反应,实现树枝状聚酰胺-胺表面的磷酸胆碱仿生修饰,修饰过程用FTIR、1H-NMR进行了表征.体外细胞活性测定和细胞形貌观察证实磷酸胆碱仿生修饰有效地改善了聚酰胺-胺树枝状聚合物的生物相容性;修饰后的聚酰胺-胺树枝状聚合物表面剩余的氨基仍然可以有效的与DNA复合,有可能作为一种潜在的基因载体得到广泛应用.     

A thermodynamic solution model for calcium carbonate: Towards an understanding of multi-equilibria precipitation pathways

Thermodynamic solubility calculations are normally only related to thermodynamic equilibria in solution. In this paper, we extend the use of such solubility calculations to help elucidate possible precipitation reaction pathways during the entire reaction. We also estimate the interfacial energy of particles using only solubility data by a modification of Mersmann’s approach. We have carried this out by considering precipitation reactions as a succession of small quasi-equilibrium states. Thus possible equilibrium precipitation pathways can be evaluated by calculating the evolution of surface charge, particle size and/or interfacial energy during the ongoing reaction. The approach includes the use of the Kelvin’s law to express the influence of particle size on the solubility constant of precipitates, the use of Nernst’s law to calculate surface potentials from solubility calculations and relate this to experimentally measured zeta potentials. Calcium carbonate precipitation and zeta potential measurements of well characterised high purity calcite have been used as a model system to validate the calculated values. The clarification of the change in zeta potential on titration illustrates the power of this approach as a tool for reaction pathway prediction and hence knowledge based tailoring of precipitation reactions.     

Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery

We recently reported the parallel synthesis of 140 degradable poly(beta-amino esters) via the conjugate addition of 20 primary or secondary amine monomers to seven different diacrylate monomers. To explore possible structure/function relationships and further characterize this class of materials, we investigated the ability of each DNA-complexing polymer to overcome important cellular barriers to gene transfer. The majority of vectors were found to be uptake-limited, but complexes formed from polymers B14 and G5 displayed high levels of internalization relative to "naked" DNA (18x and 32x, respectively). Effective diameter and zeta potential measurements indicated that, in general, small particle size and positive surface charge led to higher internalization rates. Of the 10 DNA/polymer complexes with the highest uptake levels, all had effective diameters less than 250 nm and nine had positive zeta potentials. Lysosomal trafficking was investigated by measuring the pH environment of delivered DNA. Complexes prepared with polymers G5, G10, A13, B13, A14, and B14 were found to have near neutral pH measurements, suggesting that they were able to successfully avoid trafficking to acidic lysosomes. This work highlights the value of parallel synthesis and screening approaches for the discovery of new polymers for gene delivery and the elucidation of structure/function relationships for this important class of materials.     

阳离子聚合物基因转染载体的研究进展

安全有效的基因载体是实现基因治疗的必要条件,由于阳离子聚合物易于合成和改性,无免疫原性,可以方便地与DNA形成紧密的超分子复合物,保护DNA免受核酸酶的降解,并促进其进入细胞,从而成为非病毒基因载体中的一个重要类型;但阳离子聚合物基因载体,对细胞具有电荷相关的毒性,转染效率低于病毒载体,这成为限制其进入临床使用的瓶颈.本文从提高阳离子聚合物作为基因载体时的转染效率及降低其毒性方面综述了阳离子聚合物基因载体的研究进展,归纳了改善阳离子聚合物基因载体转染特性的八种方法,预测了阳离子聚合物基因载体的发展前景.     

Biodegradable starburst carbon chain polymer-protein and lysine dendrite conjugates: adjustment of immune properties,DNA bonding and delivery

In the past decade, the development of gene therapy technology has focused on the design of new nonviral carriers for gene delivery. Proteins modified with polyethyleneimine^[1] or polylysine^[2] as well as dendrites^[3] have shown to be perspective carriers for DNA targeted delivery. The usage of protein conjugates as carriers of biologically active compounds will depend on the adjustment of their immune properties. To investigate this we have prepared starburst carbon chain polymer/protein conjugates containing low molecular weight biologically active compounds, salsolinol and bradykinin, in the polymer moieties and studied their immune properties. We have shown that chemical structure of the polymer moiety determines the conjugate biodegradation as well as their immune properties. The starburst poly(N-vinylimidazole) transferring poly(N-vinylimidazole) and polylysine 3G lysine dendrite conjugates have been prepared. The study of their ability to bind DNA and to guarantee its targeted delivery have shown that they are perspective DNA carriers.     

Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery

The development of targeted vehicles for systemic drug delivery relies on optimizing both the cell-targeting ligand and the physicochemical characteristics of the nanoparticle carrier. A versatile platform based on modification of gold nanoparticles with thiolated polymers is presented in which design parameters can be varied independently and systematically. Nanoparticle formulations of varying particle size, surface charge, surface hydrophilicity, and galactose ligand density were prepared by conjugation of PEG-thiol and galactose-PEG-thiol to gold colloids. This platform was applied to screen for nanoparticle formulations that demonstrate hepatocyte-targeted delivery in vivo. Nanoparticle size and the presence of galactose ligands were found to significantly impact the targeting efficiency. Thus, this platform can be readily applied to determine design parameters for targeted drug delivery systems.Modified gold nanoparticles are a suitable model for nanoparticle-based gene carriers.     

The adsorption of cationic and amphoteric copolymers on glass surfaces: zeta potential measurements,adsorption isotherm determination,and FT Raman characterization

The adsorption of cationic and amphoteric copolymers onto controlled pore glass (CPG) powders has been studied by measurement of the powder particle zeta (zeta) potential, by determination of the adsorption isotherm, and by FT Raman measurements of the polymer-coated powder. The cationic polymers consisted chiefly of homopolymers of dimethyldiallylammonium chloride (DMDAAC) or copolymers of DMDAAC and acrylamide. The amphoteric polymers studied included copolymers of DMDAAC and acrylic acid. The comonomer ratio was varied to explore the dependence of cationic charge density on the extent and effect of adsorption. Both types of polymers adsorb onto the anionic glass surface via an ion-exchange mechanism. Consequently, a correspondingly higher mass of a low-charge-density copolymer adsorbs than of a cationic homopolymer. The presence of the anionic portion in the amphoteric polymers does not significantly alter this picture. The zeta potential, however, reflects the overall nature of the polymer. Cationic polymers effectively neutralize the glass surface, while amphoteric polymers leave the zeta potential net negative. Adsorption isotherms, determined via the depletion technique using colloidal titration, were used to "calibrate" a FT Raman method. The latter was used to determined the amount of adsorbed polymer under solution conditions in which colloidal titration could not be performed.     

Modulation of surface charge, particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery

This work investigates the polyanion initiated gelation process in fabricating chitosan-TPP (tripolyphosphate) nanoparticles in the size range of 100-250 nm intended to be used as carriers for the delivery of gene or protein macromolecules. It demonstrates that ionic gelation of cationic chitosan molecules offers a flexible and easily controllable process for systematically and predictably manipulating particle size and surface charge which are important properties in determining gene transfection efficacy if the nanoparticles are used as non-viral vectors for gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, chitosan to TPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface charge, and tendency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The chitosan-TPP nanoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticles was found to be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of polyhedrons (e.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process. This study demonstrates that systematic design and modulation of the surface charge and particle size of chitosan-TPP nanoparticles can be readily achieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio.     

壳聚糖及其衍生物递送miRNA/siRNA的研究进展

微小RNA(microRNA,miRNA)和短链干扰RNA (small interfering RNA,siRNA)是两类具有调节基因表达功能的内源性非编码性小RNA分子.它们已成为多种疾病的潜在治疗药物,逐渐被应用于基因治疗中,而将小RNA应用于基因治疗亟需一种安全高效的递送载体.壳聚糖及其衍生物作为一种可降解、低...