Ubiquity of complex coacervation of DNA and proteins in aqueous solution.

We report complex coacervation between a primarily hydrophobic protein, elastin, and a strong polyanion DNA (2 kbp) in aqueous and salty solutions at room temperature, 25 °C. The associative interaction at fixed elastin and varying DNA concentration, thereby maintaining a mixing ratio of r = [DNA] : [elastin] = 0.0027 to 0.093, was probed. What distinguishes this study from protein-DNA coacervation reported earlier is that the protein used here was mostly a hydrophobic polyampholyte with low linear charge density, and its complementary polyelectrolyte, DNA, concentration was chosen to be extremely small (1-35 ppm). The interaction profile was found to be strongly hierarchical in the mixing ratio, defined by three distinct regions: (i) Region I (r < 0.02) was defined as the onset of primary binding leading to condensation of DNA; (ii) Region II (0.02 < r < 0.08) indicated secondary binding which led to the formation of fully charge neutralized complexes signaling the onset of coacervation; and (iii) Region III (0.08 < r < 0.12) revealed growth of insoluble complexes of large size facilitating liquid-solid phase separation. The degree of complex coacervation was suppressed in the presence of a monovalent salt implying that screened Coulomb interactions governed the binding. Small angle neutron scattering data attributed an amorphous structure to the coacervates. The elastin-DNA system belongs to a rare class of interacting biopolymers where very weak electrostatic interactions may drive coacervation, thereby implying that coacervation between DNA and proteins may be ubiquitous.

pH responsive doxorubucin loaded zein nanoparticle crosslinked pectin hydrogel as effective site-specific anticancer substrates.

Herein, we report pH-responsive hydrogels of hierarchically self-assembled protein (zein, in the form of its nanoparticles of size 80-120 nm) and polysaccharide (pectin), where gelation occurred below pH 3 in the absence of crosslinkers, which we used for encapsulation and release of anticancer drug, Doxorubicin (DOX) in the cell nucleus. These nanoparticles, spherical in shape, in addition to helping in the formation of gel network also encapsulate the drug and pectin layer adsorbed on the surface of these nanoparticle allows for the drying, redispersion and enhanced swelling. A monovalent salt-dependent study performed in the concentration range of 1-100 mM clearly showed the associative interaction between the zein nanoparticles and pectin chains were hydrophobic in nature. FTIR results confirmed the loading of the drug inside the nanoparticles. Melting profile studies of these gels revealed that encapsulation of drug did not change the thermo-physical properties. Doxorubicin drug loaded hydrogels exhibited superior cytotoxicity towards cervical cancer cell lines by inducing intracellular-antioxidative stress-based apoptosis. Confocal microscopy revealed that the hydrogels required quite less time of 4 h to completely penetrate the cells assisted by the charge specific electrostatic interaction between the negatively charged HeLa cells and positively charged crosslinks. The data, further revealed that these pH specific hydrogels were suitable for release of the drug in cell nucleus is assisted by the acidic environment of cellular organelles, and hence have a potential in cancer therapy with minimal collateral damage to healthy cells.

Fluorescent complex coacervates of agar and in situ formed zein nanoparticles: Role of electrostatic forces.

Herein, the complex coacervation between in situ formed spherical fluorescent zein nanoparticles and polyanion agar as function of mixing ratio (R=[Agar]/[Zein]) was investigated. This interaction yielded two distinguishable regions (at pH 5.4): Region I (R < 0.2), where fully charge neutralized soluble complexes with protein denaturation was noticed, and Region II (R > 0.2), where overcharged complexes were formed, with R = 0.2 defining the optimum binding. Small angle neutron scattering studies demonstrated that in the low-q region, nanoparticles formed the crosslink junctions and in the persistence regime of high-q region, the data captured the cross-sectional radius ( = 3.5 nm) for agar-zein complexes. The coacervates became more viscoelastic in salt-free samples because both the low frequency storage modulus and crosslink density were found to decrease with mixing ratio. Systematic decrease in storage modulus with ionic strength (0-0.01 M) implied screened Coulomb interaction was responsible for the observed coacervation. Further, we seek to find universality in complex coacervation of zein nanoparticle with biopolymers, and polysaccharides in particular.

Effect of organic and inorganic salt environment on the complex coacervation of in situ formed protein nanoparticles and DNA.

Complex coacervation was noticed between in situ formed protein (a primarily hydrophobic Zein protein with pI = 6.2) nanoparticles (size 80-120 nm) and ds-DNA (a high charge density polyanion), in the ionic liquid (IL) solutions of 1-ethyl-3-methyl imidazolium chloride [C2mim][Cl], and 1-octyl-3-methyl imidazolium chloride [C8mim][Cl], in the studied ionic strength range of I = 10-4 to 6 × 10-1 M, which was extended to strong monovalent 1:1 electrolyte (NaCl) to explore the commonality between the organic and inorganic salt (ionic) environment on coacervation. The salt dependent coacervation profile was monitored from the measured turbidity of the interacting solution, and zeta potential, (ζ) and apparent hydrodynamic radius (Rh) of interpolymer complexes, which depicted the following three discernible interaction regimes common to all the salts: (i) Region-I: I = 0.0001-0.01 M, primary binding, (ii) Region-II, I = 0.01-0.1 M, secondary binding, and (iii) Region-III, I = 0.1-0.6 M, saturation binding. The free-energy and the network density calculations favored preferential coacervation in [C2mim][Cl] samples. Nonetheless, commonality in the overall ionic strength dependent coacervation profiles could still be observed.

Mixing ratio dependent complex coacervation versus bicontinuous gelation of pectin with in situ formed zein nanoparticles.

We report on the competitive phenomenon of complex coacervation versus bicontinuous gelation between pectin (P, a polyanionic carbohydrate, [P] = 0.01-2% (w/v)) and zein nanoparticles (Z, a hydrophobic protein and a weak polyampholyte, [Z] = 0.1 and 0.5% (w/v), in an ethanolic solution of effective concentration 4 and 27% (v/v)), which was studied below (pH ≈ 4), and above (pH ≈ 7.4) the pI (≈ 6.2) of zein at room temperature, 25 °C. The uniqueness of this study arises from the interaction protocol used, where the pectin used was in the extended polyelectrolyte (persistence length ≈ 10 nm) conformation while zein was used as a charged globular nanoparticle (size ≈ 80-120 nm) that was formed in situ. Their mixing ratio, r = [P] : [Z] (w/w), was varied from 0.02 to 4.0 (for [Z] = 0.5% (w/v)), and from 0.1 to 7.5 (for [Z] = 0.1% (w/v)) in the ionic strength range 10-4 to 10-2 M NaCl. Zeta potential data revealed that at pH ≈ 4, the complementary binding condition, r = 1 : 1 (equivalent to 1 : 5 molecule/nanoparticle) demarcated the coacervate from the gel region. The measured rigidity (G0, low frequency storage modulus) of these materials revealed the following: for r < 1, (low pectin content samples, coacervate region) the material had lower values of Gcoac0, whereas for r > 1, an excess of pectin facilitated gelation with Ggel0 ≫ Gcoac0. Above pI, surface patch binding caused associative interactions and complex coacervation though both biopolymers had similar net charge. The network density was used as a descriptor to distinguish between the coacervate and gel samples. Their microstructures were probed by small angle neutron scattering (SANS), and viscoelastic properties by rheology. Simple modeling shows that formation of the interpolymer complex was favored in higher protein containing samples. Mixing ratio dependent selective coacervation (a kinetic process) and bicontinuous gelation (a thermodynamic process) are rarely seen to coexist in biopolymer interactions.

Does periodontal inflammation affect glycosylated haemoglobin level in otherwise systemically healthy individuals? - A hospital based study.

BACKGROUND AND OBJECTIVES: Microbial biofilm and host susceptibility play an important role in the initiation and progression of periodontitis. Periodontitis is considered the sixth complication of diabetes mellitus and a bidirectional relationship exists between diabetes and periodontitis. This cross-sectional observational study was undertaken to evaluate the glycosylated haemoglobin (HbA1c) level in chronic periodontitis. METHODS: The study involved 100 subjects. The case group consisted of 50 subjects with chronic periodontitis and the control group consisted of 50 periodontally healthy subjects. Periodontal parameters including plaque index, oral hygiene index, modified gingival index, probing pocket depth, and clinical attachment level were measured and recorded. Systemic parameters like Body Mass Index (BMI), Waist Hip Ratio (WHR), C- Reactive Protein (CRP), Glycosylated haemoglobin (HbA1c), lipid profile, fasting blood sugar, post prandial blood sugar and serum albumin were assessed in all subjects. RESULTS: The mean HbA1C for the case group was 6.27±1.5 and for the control was 5.36±0.4 and the difference was statistically significant (p = 0.001). The mean FBS, PPBS, LDL, WHR, CRP was statistically significant between groups (p ≤0.05). Periodontal parameters like PI, OHI, MGI, PD and CAL were significantly higher in the case group than the control group (p value ≤ 0.05). The multivariate linear regression model with the dependent variable HbA1c showed chronic periodontitis was significantly associated with HbA1c level. CONCLUSION: In chronic periodontitis patients (otherwise systemically healthy) the presence of periodontal inflammation affected the glycosylated haemoglobin level and they were in prediabetes stage. Therefore, it is plausible that the prediabetes stage might be reduced via appropriate periodontal therapy.

Solvent hydrophobicity induced complex coacervation of dsDNA and in situ formed zein nanoparticles.

Zein, a predominantly hydrophobic protein, was sustained as a stable dispersion in ethanol-water (80 : 20, % (v/v)) binary solvent at room temperature (25 °C). Addition of aqueous dsDNA solution (1% (w/v)) to the above dispersion prepared with the protein concentration of Czein = 0.01-0.5% (w/v) caused a concomitant change in ethanol content from 14-35% (v/v), which in turn generated zein nanoparticles in situ of size 80-120 nm increasing with water content. The subsequent associative interaction between DNA (polyanion; 2000 bps) and the positively charged zein nanoparticles, (at pH = 4) was driven by Coulombic forces, and by the solvent hydrophobicity due to the ethanol content of the binary solvent. Experimentally, two interesting regions of interaction were observed from turbidity, zeta potential, particle sizing, and viscosity data: (i) for Czein < 0.2% (w/v), zein nanoparticles of size 80 nm bind to dsDNA (primary complex) causing its condensation (apparent hydrodynamic size decreased from ≈2100 to 560 nm), and (ii) for 0.2% < Czein < 0.5% (w/v) larger nanoparticles (>80 nm) were selectively bound to primary complexes to form partially charge neutralized interpolymer soluble complexes (secondary complexes), followed by complex coacervation. During this process, there was depletion of water in the vicinity of the nucleic acid, which was replaced by hydration provided by the ethanol-water binary solvent. Equilibrium coacervate samples were probed for their microstructure by small angle neutron scattering, and for their viscoelastic properties by rheology. The interplay of solvent hydrophobicity, electrostatic interaction, and zein nanoparticle size dependent charge neutralization had a commensurate effect on this hitherto unexplored coacervation phenomenon.

Hydrophobic hydration and anomalous diffusion of elastin in an ethanolic solution.

Elastin is an important structural protein that confers elasticity to tissues. It is widely used in the biosynthesis of human elastic tissues and exhibits interesting properties. This study reports an insight into the unusual dispersion and anomalous diffusion of elastin in an ethanolic solution. Due to its complex hydrophobic structure, its dispersibility was found to be sensitive towards the hydrophobicity of the solvent. Electrophoresis measurements (zeta-potential data) revealed that its net polarity changed from an anionic to a cationic state with the decreasing solvent hydrophobicity (ethanol content in the solvent). An interesting transition temperature of ∼297 K was observed above which the hydrophobic interactions among the protein molecules became dominant. Double-layer repulsion between protein molecules competes with attractive hydrophobic interactions and causes molecular self-organization. A DLVO-based theoretical model showed that hydrophobic interactions were facilitated by a binary solvent (ethanol-water), and the repulsive double layer screening provided sufficient energy to overcome the interactions between hydrophobic domains in the protein molecule and allow the self-assembly to occur.