Poly(ethylene oxide) grafted with short polyethylenimine gives DNA polyplexes with superior colloidal stability, low cytotoxicity, and potent in vitro gene transfection under serum conditions

Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ～ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.     

Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions

The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.     

Folding of the alphaII-spectrin SH3 domain under physiological salt conditions

The SH3 domain has often been used as a model for protein folding due to its typical two-state behaviour. However, recent experimental data at low pH as well as molecular dynamic simulations have indicated that the folding process of SH3 probably is more complicated, and may involve intermediate states. Using both kinetic and equilibrium measurements we have obtained evidence that under native-like conditions the folding of the spectrin SH3 domain does not follow a classic two-state behaviour. The curvature we observed in the Chevron plots is a strong indication of a non-linear activation energy relationship due to the presence of high-energy intermediates. In addition, circular dichroism measurements indicated that refolding after thermal denaturation did not follow the same pattern as thermal unfolding but rather implied less cooperativity and that the refolding transition increased with increasing protein concentration. Further, NMR experiments indicated that upon refolding the SH3 domain gave rise to more than one conformation. Therefore, our results suggest that the folding of the SH3 domain of αII-spectrin does not follow a classical two-state process under high-salt conditions and neutral pH. Heterogeneous folding pathways, which can include folding intermediates as well as misfolded intermediates, might give a more reasonable insight into the folding behaviour of the αII-spectrin SH3 domain.     

Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions

Folding mechanisms of functional RNAs under idealized in vitro conditions of dilute solution and high ionic strength have been well studied. Comparatively little is known, however, about mechanisms for folding of RNA in vivo where Mg²⁺ ion concentrations are low, K⁺ concentrations are modest, and concentrations of macromolecular crowders and low-molecular-weight cosolutes are high. Herein, we apply a combination of biophysical and structure mapping techniques to tRNA to elucidate thermodynamic and functional principles that govern RNA folding under in vivo–like conditions. We show by thermal denaturation and SHAPE studies that tRNA folding cooperativity increases in physiologically low concentrations of Mg²⁺ (0.5–2 mM) and K⁺ (140 mM) if the solution is supplemented with physiological amounts (∼20%) of a water-soluble neutral macromolecular crowding agent such as PEG or dextran. Low-molecular-weight cosolutes show varying effects on tRNA folding cooperativity, increasing or decreasing it based on the identity of the cosolute. For those additives that increase folding cooperativity, the gain is manifested in sharpened two-state-like folding transitions for full-length tRNA over its secondary structural elements. Temperature-dependent SHAPE experiments in the absence and presence of crowders and cosolutes reveal extent of cooperative folding of tRNA on a nucleotide basis and are consistent with the melting studies. Mechanistically, crowding agents appear to promote cooperativity by stabilizing tertiary structure, while those low molecular cosolutes that promote cooperativity stabilize tertiary structure and/or destabilize secondary structure. Cooperative folding of functional RNA under physiological-like conditions parallels the behavior of many proteins and has implications for cellular RNA folding kinetics and evolution.     

Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides

The potential use of antisense and siRNA oligonucleotides as therapeutic agents has elicited a great deal of interest. However, a major issue for oligonucleotide-based therapeutics involves effective intracellular delivery of the active molecules. In this Survey and Summary, we review recent reports on delivery strategies, including conjugates of oligonucleotides with various ligands, as well as use of nanocarrier approaches. These are discussed in the context of intracellular trafficking pathways and issues regarding in vivo biodistribution of molecules and nanoparticles. Molecular-sized chemical conjugates and supramolecular nanocarriers each display advantages and disadvantages in terms of effective and nontoxic delivery. Thus, choice of an optimal delivery modality will likely depend on the therapeutic context.     

Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions.

Various oligonucleotides containing 8-methylguanine (m8G) have been synthesized and their structures and thermodynamic properties investigated. Introduction Of M8G into DNA sequences markedly stabilizes the Z conformation under low salt conditions. The hexamer d(CGC[M8G]CG)2 exhibits a CD spectrum characteristic of the Z conformation under physiological salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m8G]CG)2 adopts a Z structure with all guanines in the syn conformation. The refined NMR structure is very similar to the Z form crystal structure of d(CGCGCG)2, with a root mean square deviation of 0.6 between the two structures. The contribution of m8G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m8G-containing oligomers. The presence of m8G in d(CGC[m8G]CG)2 stabilizes the Z conformation by at least deltaG = -0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was further stabilized by increasing the number of m8Gs incorporated and destabilized by incorporating syn-A or syn-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the chemically less reactive m8G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate syn-G conformation.     

Poly(glycoamidoamine)s for gene delivery: stability of polyplexes and efficacy with cardiomyoblast cells

Polymeric vectors have potential as nucleic acid delivery vehicles for novel gene therapy and oligonucleotide treatments for cardiovascular disease. In this report, poly(glycoamidoamine)s that contain four secondary amines and either two or four hydroxyl units in the repeat unit with D-glucarate (D4), meso-galactarate (G4), D-mannarate (M4), and l-tartarate (T4) stereochemistry have been investigated for their pDNA-binding affinity, DNase protection effect, and polyplex stability in the presence of salt and serum. Also, the luciferase gene delivery and cellular internalization of polyplexes formed with these polymers have been investigated with rat cardiomyoblast [H9c2(2-1)] cells. The results demonstrate that the number of hydroxyl groups and the stereochemistry affect the biological properties. Polymers T4 and G4 have higher pDNA binding affinity, protect pDNA from nuclease degradation, and do not release pDNA in the presence of serum. Polymers D4 and M4 bind pDNA with lower affinity, which allows for some pDNA degradation and release in the presence of serum. Although T4 forms the most stable polyplexes, vector G4 reveals the highest luciferase gene expression in serum-free media and the greatest cellular internalization of fluorescein-labeled pDNA both in serum-free and serum-supplemented media. The results of these studies indicate that the polymer-DNA binding affinity, nuclease protection capability, and polyplex stability are important parameters to facilitate effective pDNA delivery with poly(glycoamidoamine)s in cultured cardiomyoblast cells. The carbohydrate type also plays an important role to increase cellular uptake and gene expression where the polymer with the galactarate stereochemistry (in G4) is found to be the most effective vector for pDNA delivery to cardiomyoblast cells in vitro.     

Effect of pendant group structure on the hydrolytic stability of polyaspartamide polymers under physiological conditions

We describe the synthesis of metal chelating polymers based on polyaspartamide and polyglutamide backbones as carriers for (111)In in radioimmunoconjugates. These polymers [PAsp(DTPA), PGlu(DTPA)] have a biotin end group and diethylenetriaminepentaacetic acid (DTPA) chelators attached to the primary amines of the diethylenetriamine (DET) pendant groups of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] and of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]glutamide} [PGlu(DET)]. Like Asn-containing proteins and polypeptides, polyaspartamides undergo uncatalyzed degradation under model physiological conditions (10 mM phosphate buffer, pH 7.4, 150 mM NaCl). We studied the uncatalyzed degradation of the polyaspartamide polymers by size exclusion chromatography and found that the degradation rate was sensitive to the nature of the pendant groups. The metal-free polymer underwent somewhat slower degradation than the corresponding polymers in which the DTPA groups were saturated with Eu(3+) or In(3+), but even after 14 days, substantial fractions of the polymers survived. We conclude that these polymers undergo negligible degradation on the time scale (24-48 h) of radioimmunotherapy treatment of tumors with (111)In. From a mechanistic perspective, we note that these degradation rates are on the order of the deamidation rates reported [J. Peptide Res. 2004, 63, 426] for Asn-containing pentapeptides, with half-times on the order of 10 days, but much slower than the rapid decay (hours) reported recently [Biomaterials 2010, 31, 3707] for poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} itself. This variation in degradation rate can be explained in terms of the influence of positive charges on the pendant group enhancing the acidity of the side-chain amide nitrogen of the aspartamide repeat unit. The DET pendant group is positively charged at pH 7, but in indium-loaded PAsp(DTPA) this charge is offset by the net negative charge of the DTPA-In complex.     

Non-enzymatic deamidation and autofragmentation of lysozyme and albumin under physiological conditions

Nonenzymic deamidation of amides in proteins (lysozyme and albumin) under conditions which imitate physiological ones has been experimentally established with subsequent asparagine-dependent autofragmentation of the polypeptide chain.     

Effect of aspartic acid on physiological characteristics and gene expression of salt exclusion in Tartary buckwheat under salt stress

Salt-tolerant variety Chuanqiao No. 1 and salt-sensitive variety Chuanqiao No. 2 of Tartary buckwheat were used as experimental materials. The effect of aspartic acid on seed germination, physiological characteristics of seedlings and gene expression of salt exclusion in Tartary buckwheat were studied under NaCl stress of 150 mM. The results showed that the aspartic acid treatment could restore the seed germination rate and root vigor of seedlings to the control with non-damage level in salt-tolerant Tartary buckwheat variety under salt stress, and the salt-sensitive variety was increased greatly. Spraying aspartic acid had some protective effects on cell membrane of leaves in Tartary buckwheat under salt stress, and the protective effects were more obviously on salt-sensitive variety, and that could restore the activity of SOD and CAT of leaves to the control level in salt-tolerant Tartary buckwheat variety under salt stress, and the activity of antioxidant enzymes in salt-sensitive variety was increased significantly. The relative expression of FtNHX1 and FtSOS1 genes was increased significantly under salt stress, and that of FtNHX1 gene in salt-tolerant and salt-sensitive varieties was reached the maximum expression level at 12 h and 24 h respectively, while that of FtSOS1 gene in salt-tolerant and salt-sensitive varieties was reached the maximum expression level at 12 h, and the salt-tolerant variety was increased greatly. After spraying aspartic acid, the relative expression of FtNHX1 and FtSOS1 genes was increased more obviously. The relative expression of FtNHX1 gene in salt-tolerant and salt-sensitive varieties was reached the maximum expression level at 12 h, while that of FtSOS1 gene was reached the maximum expression level at 12 h and 24 h respectively, and that in salt-tolerant variety was increased especially more, indicating that spraying aspartic acid on gene expression of salt exclusion in salt-tolerant variety of Tartary buckwheat has a better effect under salt stress.     

Principles of membrane stability and phase behavior under extreme conditions

Biological membranes consist of a complex assortment of lipids and proteins. The arrangement of the components, particularly in regard to their lateral disposition in the plane of the membrane under physiological conditions, is dependent on the phase behavior of the different membrane lipids and the way that this behavior is modified by interaction with other membrane components and electrolytes in the aqueous medium. Irreversible phase separation of components within the membrane may result from exposure to extreme environmental conditions including temperature, pressure, or electrolyte concentration. The principles underlying the phase-mixing behavior of model membrane systems can be used to provide useful information about the factors that determine the stability of biomembranes under physiological and non-physiological conditions. These data are reviewed and used to predict events that take place when membranes are exposed to environmental stress.     

Seasonal variations of physiological characteristics and thermal sensation under identical thermal conditions

Seasonal variations of human thermal characteristics were inspected in thermal comfort and when constantly indoors. Metabolic rate, tympanic temperature, skin temperature, body fat, body weight and thermal sensation were measured under identical thermal conditions in a chamber over the course of one year. Experiments were carried out for each subject in both summer and winter. Six subjects were measured 35 times in summer and 45 times in winter. one subject was measured weekly for 14 months. Measurements for analyses were taken 40-60 min after entrance into the chamber. Results revealed the following. 1) For all subjects, the metabolic rate, tympanic temperature and body fat were lower in summer than in winter; thigh skin temperatures were higher in summer than in winter. The averaged individual ratio of seasonal difference was 11.9% for metabolic rate, 14.9% for body fat, 1.8% for thigh temperature and 0.53% for tympanic temperature. Seasonal differences of about 10% in metabolic rate were maintained in this study. 2) Seasonal variations of the variables were examined for phase relationships against the outdoor temperature. 2-1) Metabolic rate, thermal sensation, body weight and body fat changed in reverse phase, whereas skin temperature was in-phase. 2-2) Skin temperature lagged by about one month in both summer and winter. Body fat also lagged by about one month in summer, but corresponded to the phase in winter. Metabolic rates were also in-phase in winter but led about three months in summer. Thermal sensations lagged by about three months in winter but were in-phase in summer. Body weight was in-phase in summer and winter. 2-3) Summer disorders were observed particularly in seasonal variations of metabolic rates, tympanic temperature, skin temperatures, and thermal sensation, thereby suggesting that the effect of temperature exposure was altered by air-conditioner use.     

Awareness of physiological responding under stress and nonstress conditions in temporomandibular disorders

To test the hypothesis that individuals with temporomandibular disorders (TMD) have deficits in proprioceptive awareness, 20 TMD patients were compared with 20 nonpain individuals matched to the TMD patients on age and gender. Left and right frontalis, masseter, and temporalis were monitored, as were forearm extensor, heart rate, and skin conductance while the participants viewed a nonstressful film segment. Following the film segment, participants provided self-reports of these physiological responses. This sequence was repeated for a second, stressful film segment and for a third, non-stressful film segment. Correlations between physiological activity and self-report were used as measures of proprioceptive awareness. The results indicated that TMD subjects were most accurate in their awareness of facial muscle activity during the stress condition and least accurate in the two nonstress periods. Control subjects increased their accuracy of awareness at each time period, showing significantly greater accuracy than the TMD group in the last, nonstress period. TMD subjects more accurately perceived activity of the nonfacial muscle variables in the two nonstress periods than during the stress period. These findings may provide a mechanism for understanding clinical observations showing that TMD patients with myofascial pain engage in high levels of parafunctional oral activity without awareness.Portions of this study were supported by the Weldon Spring Foundation and by a grant from the National Institute of Dental Research (DE 11017). I thank Drs. Ernest G. Glass and Edward Mosby for their assistance in identifying patients. I also thank Natalie Beltgens, Stella Brown, Linda Coates, Elbert Darden, Ben Javid, and Susan Sherrick for their assistance in recruiting nonpain participants and in helping carry out this study.     

Elucidation of physiological and biochemical mechanisms of an endemic halophyte Centaurea tuzgoluensis under salt stress

In this study, physiological and biochemical responses of Centaurea tuzgoluensis, a Turkish endemic halophyte, to salinity were studied. Therefore, the changes in shoot growth, leaf relative water content (RWC), ion concentrations, lipid peroxidation, hydroxyl (OH^.) radical scavenging activity, proline (Pro) content, and antioxidant system [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR)] were investigated. The 60 days (d) old C. tuzgoluensis seedlings were subjected to 0, 150 and 300 mM NaCl for 7 d and 14 d. The relative shoot growth was generally did not change in the 150 mM NaCl, but reduced with 300 mM NaCl stress at 7 d and 14 d. RWC was higher in 150 mM NaCl-treated leaves than that of 300 mM NaCl. Salinity decreased K⁺/Na⁺ ratio, but increased Na⁺, Cl^?, Ca⁺² and Na⁺/Cl^? ratio in the leaves. On the other hand, it did not change or increase the K⁺ content at 150 and 300 mM NaCl, respectively. MDA content in the 150 and 300 mM NaCl-treated leaves remained close to control at 7 d. This was related to enhanced activities of SOD, CAT, APX and GR enzymes, and their isoenzymes especially Fe-SOD in the leaves. On the other hand, the higher sensitivity to 300 mM NaCl at 14 d was associated with inadequate increase in antioxidant enzymes and the decreased OH radical scavenging activity. All these results suggest that C. tuzgoluensis has different antioxidant metabolisms between short- (7 d) and long-term (14 d) salt treatments and salinity tolerance of C. tuzgoluensis might be closely related to increased capacity of antioxidative system to scavenge reactive oxygen species (ROS) and accumulation of osmoprotectant proline under salinity conditions.     

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.     

Testing oligothiophene fluorophores under physiological conditions. Preparation and optical characterization of the conjugates of bovine serum albumin with oligothiophene N-hydroxysuccinimidyl esters

Bovine serum albumin (BSA) was reacted with linear and newly synthesized branched oligothiophene N-hydroxysuccinimidyl ester fluorophores (TSEs) in moderately basic carbonate buffer solution. Optically stable BSA-TSE conjugates were obtained with a degree of labeling depending on experimental conditions. Conjugates with high fluorophore to BSA ratios (F/BSA = 8) displayed fluorescence quantum yields in the range of 10-30% in water at pH = 7.2, comparable to the quantum yield (25%) of the BSA-FITC conjugate prepared under the same conditions and with the same degree of labeling.     

盐胁迫下水杨酸及其衍生物对小黑麦幼苗生理特性的影响

盐胁迫下,适当浓度的两种酚酸均能降低幼苗叶片电导率和丙二醛(MDA)含量,增加根系活力。在相同处理浓度下．5-磺基水杨酸提高幼苗生理特性的效果比水杨酸好。