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1.
Poly(2-(diethylamino)ethyl methacrylate) coated magnetic nanoparticles (PDEA-MNPs) were synthesized as a new gene nanocarrier to delivery plasmids (pEGFPN1 and pRL-TK) into human hepatoma (Hep G2) cells. The PDEA-MNPs shows the pH-sensitive property. These nanoparticles are positively charged at acidic pH and negatively charged at neutral or alkaline pH. The PDEAMNPs exhibited a low cytotoxicity in Hep G2 cells. PDEA-MNPs could bind and protect DNA from DNase I degradation. The transfection study demonstrated that the PDEA-MNPs could carry plasmid into Hep G2 cells and exhibited a high gene transfection efficiency. These results indicated that the novel magnetic nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.  相似文献   

2.
Gu W  Wang T  Zhu J  Shi Y  Liu H 《Biophysical chemistry》2003,104(1):79-94
Four 10-ns molecular dynamics (MD) simulations of the human prion protein domain (HuPrP 125-228) in explicit water solution have been performed. Each of the simulations mimicked a different environment of the protein: the neutral pH environment was simulated with all histidine residues neutral and bearing a ND proton and with other titratable side chains charged, the weakly acidic environment was simulated with all titratable side chains charged, the strongly acidic environment was simulated with all titratable side chains protonated. The protein in neutral pH environment was simulated at both ambient (298 K) and higher (350 K) temperatures. The native fold is stable in the neutral pH/ambient temperature simulation. Through out all other simulations, a quite stable core consisted of 10-20 residues around the disulfide bond retain their initial conformations. However, the secondary structures of the protein show changes of various degrees compared to the native fold, parts of the helices unfolded and the beta-sheets extended. Our simulations indicated that the heat-induced unfolding and acid-induced unfolding of HuPrP might follow different pathways: the initial stage of the acid-induced unfolding may include not only changes in secondary structures, but also changes in the tertiary structures. Under the strongly acidic condition, obvious tertiary structure changes take place after 10-ns simulation, the secondary structure elements and the loops becoming more parallel to each other, resulting in a compact state, which was stabilized by a large number of new, non-native side chain-side chain contacts. Such tertiary structure changes were not observed in the higher temperature simulation, and intuitively, they may favor the further extension of the beta-sheets and eventually the agglomeration of multiple protein molecules. The driving forces for this tertiary structure changes are discussed. Two additional 10-ns MD simulations, one with Asp202 protonated and the other with Glu196 protonated compared to the neutral pH simulation, were carried out. The results showed that the stability of the native fold is very subtle and can be strongly disturbed by eliminating a single negative charge at one of such key sites. Correlations of our results with previous experimental and theoretical studies are discussed.  相似文献   

3.
Although various gene delivery techniques are available, their application in zebrafish cell cultures has not been extensively studied. Here, we report that nucleofection of zebrafish primary embryonic fibroblasts results in higher transfection efficiency in comparison to other non-viral gene delivery methods. The transfection was performed using green fluorescent protein (GFP) gene constructs of a different size. Greatest DNA uptake was obtained with 4.9-kb plasmid, resulting in 43% GFP positive cells. Nucleofection with 7.4-kb pH2B-GFP plasmid followed by geneticin (G418) selection was successfully used to establish a cell line expressing nuclear histone 2B-GFP fusion protein. Efficient transfection of zebrafish fibroblasts by nucleofection offers a non-viral technique of plasmid delivery and can be used to overexpress genes of interest in these cells.  相似文献   

4.
α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.  相似文献   

5.
The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pKapp of membrane insertion.  相似文献   

6.
The mycobacterial enzyme pyrazinamidase (PZase) is the target of key tuberculosis drug, pyrazinamide. Mutations in PZase cause drug resistance. Herein, three point mutations, W68G, L85P, and V155G, were investigated through over 8 µs of molecular dynamics simulations coupled with essential dynamics and binding pocket analysis at neutral (pH = 7) and acidic (pH = 4) ambient conditions. The 51-71 flap region exhibited drastic displacement leading to enlargement of binding cavity, especially at the lower pH. Accessibility of solvent to the active site of the mutant enzymes was also reduced. The protonation of key surface residues at low pH results in more contribution of these residues to structural stability and integrity of the enzyme and reduced interactions with solvent molecules, which acts as a cage, keeping the enzyme together. The observed results suggest a pattern of structural alterations due to point mutations in PZase, which is consistent with other experimental and theoretical investigations and, can be harnessed for drug design purposes.  相似文献   

7.
Information on the interaction strength between small interfering RNA (siRNA) and chitosan can contribute to the understanding of the formation and stability of chitosan/siRNA nanoparticles used as siRNA delivery systems for gene silencing. In this study, we utilize atomic force microscopy to obtain force spectroscopy results of the interaction strengths between siRNA and chitosan measured in physiological phosphate buffered saline buffer at different pH. The force measurements revealed that the adhesive interactions decreased in force strength and force frequency as the pH was increased from 4.1 to 6.1, 7.4, and 9.5, exhibiting distinct multimodal distributions of the interaction forces between siRNA and chitosan molecules at acidic pH and only negligible adhesive forces were observed at neutral or high pH. The strong pH dependence of siRNA-chitosan interactions can provide a convincing rationale for siRNA/chitosan complex formation and nanoparticle stability under low acidic conditions. These findings demonstrate that the use of force spectroscopy for the adhesive force measurements allows an evaluation of the complexing ability between siRNA and chitosan that can be utilized to predict nanoparticle stability.  相似文献   

8.
Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.  相似文献   

9.
Chen J  Tian B  Yin X  Zhang Y  Hu D  Hu Z  Liu M  Pan Y  Zhao J  Li H  Hou C  Wang J  Zhang Y 《Journal of biotechnology》2007,130(2):107-113
The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.  相似文献   

10.
Neuroserpin is a member of the serpin superfamily. Point mutations in the neuroserpin gene underlie the autosomal dominant dementia, familial encephalopathy with neuroserpin inclusion bodies. This is characterized by the retention of ordered polymers of neuroserpin within the endoplasmic reticulum of neurons. pH has been shown to affect the propensity of several serpins to form polymers. In particular, low pH favors the formation of polymers of both α1‐antitrypsin and antithrombin. We report here opposite effects in neuroserpin, with a striking resistance to polymer formation at acidic pH. Mutation of specific histidine residues showed that this effect is not attributable to the shutter domain histidine as would be predicted by analogy with other serpins. Indeed, mutation of the shutter domain His338 decreased neuroserpin stability but had no effect on the pH dependence of polymerization when compared with the wild‐type protein. In contrast, mutation of His119 or His138 reduced the polymerization of neuroserpin at both acidic and neutral pH. These residues are at the lower pole of neuroserpin and provide a novel mechanism to control the opening of β‐sheet A and hence polymerization. This mechanism is likely to have evolved to protect neuroserpin from the acidic environment of the secretory granules.  相似文献   

11.
We have previously shown that modification with succinylated poly(glycidol) (SucPG) provides stable egg yolk phosphatidylcholine (EYPC) liposomes with pH-sensitive fusogenic property. Toward production of efficient pH-sensitive liposomes, in this study, we newly prepared three carboxylated poly(glycidol) derivatives with varying hydrophobicities by reacting poly(glycidol) with glutaric anhydride, 3-methylglutaric anhydride, and 1,2-cyclohexanedicarboxylic anhydride, respectively, designated as GluPG, MGluPG, and CHexPG. Correlation between side-chain structures of these polymers and their respective abilities to sensitize stable liposomes to pH was investigated. These polymers are soluble in water at neutral pH but became water-insoluble in weakly acidic conditions. The pH at which the polymer precipitated was higher in the order SucPG < GluPG < MGluPG < CHexPG, which is consistent with the number of carbon atoms of these polymers' side chains. Although CHexPG destabilized EYPC liposomes even at neutral pH, attachment of other polymers provided pH-sensitive properties to the liposomes. The liposomes bearing polymers with higher hydrophobicity exhibited more intense responses, such as content release and membrane fusion, at mildly acidic pH and achieved more efficient cytoplasmic delivery of membrane-impermeable dye molecules. As a result, modification with appropriate hydrophobicity, MGluPG, produced highly potent pH-sensitive liposomes, which might be useful for efficient cytoplasmic delivery of bioactive molecules, such as proteins and genes.  相似文献   

12.
The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.  相似文献   

13.
An interesting observation was found during our continued studies on the hydrolysis of ibuprofen esters by Candida rugosa lipase (CRL). An important role is played by pH in the stereospecific hydrolysis of these esters. The flap region of CRL plays a significant role in the access of the substrate to the active site of the enzyme. At pH 5.6, 48% of the methyl ester and 5% of the butyl ester of ibuprofen were hydrolysed in 5.5 h, whereas at pH 7.2, 9% of methyl ester and 45% of the butyl ester of ibuprofen was hydrolysed in a identical reaction time using CRL. This lead us to assume that CRL prefers the methyl ester of ibuprofen as a substrate at an acidic pH and the butyl ester of ibuprofen at a neutral pH. Therefore, in order to understand the role of pH in the substrate selection by CRL for the esters of ibuprofen we used the crystallographic coordinates of the open form of the CRL (1CRL) for molecular dynamics (MD) simulations under acidic and neutral conditions for 2 ns using GROMACS. The final structures obtained after simulation in acidic and neutral conditions were compared with the energy-minimized structure, and the root-mean-square deviations (r.m.s.ds) were calculated. The r.m.s.d. of the CRL flap at neutral pH was found to be greater than that of the CRL flap at acidic pH. The extent to which the flap opens at neutral pH allowed the bulkier substrate, the butyl ester of ibuprofen, to diffuse into the active site and provides the best enzyme-substrate fit for this specific substrate. At acidic pH there is a decreased opening of the flap thereby accommodating a more compact substrate, namely the methyl ester of ibuprofen. Thus, simulation experiments using MD provide reasonable insight for the pH-dependent substrate selectivity of CRL in aqueous environments.  相似文献   

14.
Apoptosis and cell cycle progression in HL60 cells irradiated in an acidic environment were investigated. Apoptosis was determined by TUNEL staining, PARP cleavage, DNA fragmentation, and flow cytometry. The majority of the apoptosis that occurred in HL60 cells after 4 Gy irradiation took place after G(2)/M-phase arrest. When irradiated with 12 Gy, a fraction of the cells underwent apoptosis in G(1) and S phases while the rest of the cells underwent apoptosis in G(2)/M phase. The apoptosis caused by 4 and 12 Gy irradiation was transiently suppressed in medium at pH 7.1 or lower. An acidic environment was found to perturb progression of irradiated cells through the cell cycle, including progression through G(2)/ M phase. Thus it was concluded that the suppression of apoptosis in the cells after 4-12 Gy irradiation in acidic medium was due at least in part to a delay in cell cycle progression, particularly the prolongation of G(2)/M-phase arrest. Irradiation with 20 Gy indiscriminately caused apoptosis in all cell cycle phases, i.e. G(1), S and G(2)/M phases, rapidly in neutral pH medium and relatively slowly in acidic pH medium. The delay in apoptosis in acidic medium after 20 Gy irradiation appeared to result from mechanisms other than prolonged G(2)/ M-phase arrest.  相似文献   

15.
Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.  相似文献   

16.
Mucoadhesive properties of cross-linked high amylose starch derivatives   总被引:2,自引:0,他引:2  
Acetate (Ac-), aminoethyl (AE-) and carboxymethyl (CM-)derivatives of cross-linked high amylose starch (HASCL-6) were previously shown to control, over more than 20h, the release of drugs from highly loaded (up to 60% drug) monolithic tablets. It was now of interest to evaluate their mucoadhesive characteristics in view of further utilization in buccal or vaginal transmucosal delivery. The present study shows that ionic AE-HASCL-6 and CM-HASCL-6 derivatives exhibit higher mucoadhesive properties than neutral HASCL-6 and Ac-HASCL-6, suggesting that the ionic groups introduced on cross-linked starch chains play a role in the bioadhesion process. The adhesiveness seemed related to capillary attraction forces. Surface adhesion parameters were calculated for slabs based on the mentioned polymers and corroborated with their swelling behavior at various pH changes. The positively charged AE-derivatives presented a higher adhesion at acidic pH, being thus recommended for vaginal delivery, whereas the negatively charged derivatives (CM-HASCL-6) exhibited a better adhesion at neutral pH, being thus more appropriate for buccal delivery.  相似文献   

17.
Chen J  Qiu X  Ouyang J  Kong J  Zhong W  Xing MM 《Biomacromolecules》2011,12(10):3601-3611
This study develops novel pH and reduction dual-sensitive micelles for the anticancer drug doxorubicin (DOX) delivery owing to the fact that the tumor tissues show low pH and high reduction environment. These sub-100 nm micelles present a core-shell structure under physiological conditions, but quickly release the loaded drugs responding to acidic and reductive stimuli. With disulfide bonds in each repeat unit of poly(β-amino ester)s, the novel copolymer was synthesized via Michael addition polymerization from 2,2'-dithiodiethanol diacrylate, 4,4'-trimethylene dipiperidine, and methoxy-PEG-NH(2). DOX released faster from micelles in a weakly acidic environment (pH 6.5) than at pH 7.4 or in the presence of a higher concentration (5 mM) of reducing agent (DTT). The release is even more effective in a scenario of both stimuli (pH 6.5 and 5 mM DTT). MTT assay showed that the DOX-loaded micelles had a higher cytotoxicity for HepG2 tumor cells than DOX at higher concentrations, and that blank micelles had a very low cytotoxicity to the tumor cells. Confocal microscopy observation showed that the micelles can be quickly internalized, effectively deliver the drugs into nuclei, and inhibit cell growth. These results present the copolymer as a novel and effective pH and reduction dual-responsive nanocarrier to enhance drug efficacy for cancer cells.  相似文献   

18.
Summary 5-Aminolevulinic acid(ALA) production by Rhodobacter sphaeroides was investigated at various pH with levulinic acid addition using a volatile fatty acids medium prepared from the mandarin orange peel supplemented with glycine. At neutral pH (6.8 and 7.0), extracellular ALA production was up to 16 mM, while low production of ALA(less than 3.5 mM) was observed at acidic pH (lower than 6.5) and less than 3.9 mM of ALA produced at alkaline pH (higher than 7.5). The higher ALA synthase activity observed at neutral pH might enhance the ALA production compared with that observed in acidic and alkaliphilic cultures.  相似文献   

19.
The pH low insertion peptide (pHLIP) is an important tool for drug delivery and visualization of acidic tissues produced by various maladies, including cancer, inflammation, and ischemia. Numerous studies indicate that pHLIP exists in three states: unfolded and soluble in water at neutral pH (State I), unfolded and bound to the surface of a phosphatidylcholine membrane at neutral pH (State II), and inserted across the membrane as an α-helix at low pH (State III). Here we report how changes in lipid composition modulate this insertion scheme. First, the presence of either anionic lipids, cholesterol, or phosphoethanolamine eliminates membrane binding at neutral pH (State II). Second, the apparent pKa for the insertion transition (State I → State III) is increased with increasing content of anionic lipids, suggesting that electrostatic interactions in the interfacial region modulate protonation of acidic residues of pHLIP responsible for transbilayer insertion. These findings indicate a possibility for triggering protonation-coupled conformational switching in proteins at membrane interfaces through changes in lipid composition.  相似文献   

20.
A neutral cross-linked dendritic system is described for use of delivering small interfering RNA (siRNA) for targeted gene silencing. By replacing the terminal amines with hydrazide groups and N-acetylgalactosamine (GalNAc) ligands, cationic polyamidoamine (PAMAM) dendrimers were transformed into neutral glycosylated carriers for siRNA delivery. Mainly owing to the pH sensitivity and the proton-absorption capability of the tertiary amines within the interior branches, these PAMAM derivatives showing neutrality under physiological conditions (pH 7.4) formed complexes with siRNA through electrostatic interactions at pH 5. Cross-linking procedures via reactions with glutaraldehyde were established, and cytocompatible cross-linked systems loaded with siRNA obtained, with the particulate properties variable with the cross-linking condition and the GalNAc level in the dendritic carrier. In vitro cellular uptake and RNAi experiments showed that the cross-linked dendritic systems with an appropriate level of GalNAc composition effectively interacted with HepG2 cells and inhibited the expression of a luciferase reporter gene. Neutral cross-linked dendritic systems provide a new paradigm for designing siRNA delivery systems with biocompatibility and targeting capability.  相似文献   

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