“Smart” Drug Carriers: PEGylated TATp-Modified pH-Sensitive Liposomes

To engineer drug carriers capable of spontaneous accumulation in tumors and ischemic areas via the enhanced permeability and retention (EPR) effect and further penetration and drug delivery inside tumor or ischemic cells via the action of the cell-penetrating peptide (CPP), we have prepared liposomes simultaneously bearing on their surface CPP (TAT peptide, TATp) moieties and protective PEG chains. PEG chains were incorporated into the liposome membrane via the PEG-attached phosphatidylethanolamine (PE) residue with PEG and PE being conjugated with the lowered pH-degradable hydrazone bond (PEG-HZ-PE). Under normal conditions, liposome-grafted PEG “shielded” liposome-attached TATp moieties since the PEG spacer for TATp attachment (PEG₁₀₀₀) was shorter than protective PEG₂₀₀₀. PEGylated liposomes are expected to accumulate in targets via the EPR effect, but inside the “acidified” tumor or ischemic tissues lose their PEG coating due to the lowered pH-induced hydrolysis of HZ and penetrate inside cells via the now-exposed TATp moieties. This concept is shown here to work in cell cultures in vitro as well as in ischemic cardiac tissues in the Langendorff perfused rat heart model and in tumors in experimental mice in vivo.     

Mesoscopic simulation studies on micellar phases of Pluronic P103 solution

The microphase separation dynamics of the triblock copolymer surfactant P103 [(ethylene oxide)₁₇(propylene oxide)₆₀(ethylene oxide)₁₇] was investigated by a dynamic variant of mean-field density functional theory. Different self-assembled aggregates, spherical micelles, micellar clusters and disk-like micelles, are explored in the solution. The spherical micelle above critical micelle concentration (CMC) is a dense core consisting mainly of PPO and a hydrated PEO swollen corona, and is in good agreement with the experimental results concerning their structures. At a concentration of 10–15%, micellar clusters with a larger PPO core form as a result of coalescence among spherical micelles. At concentrations above 16% by volume, a series of disk-like micelles come into being. The order parameters show that spherical micelles are easily formed, while the micellar clusters or disk-like micelles need a longer time to reach steady equilibrium. The results show that mesoscopic simulation can augment experimental results on amphiphilic polymers, and provide some mesoscopic information at the mesoscale level. Figure Coalescence of Micelles with time evolution for 15% vol system. □ represents spherical micelle that coalesce. (a) 180 μs, (b) 190 μs, (c)225 μs, and (d) 250 μs     

Facile purification of mono-PEGylated interleukin-1 receptor antagonist and its characterization with multi-angle laser light scattering

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was chemically conjugated with succinimidyl carbonate monomethoxyl polyethylene glycols of 5 kDa (SC-PEG_5k) and 10 kDa (SC-PEG_10k) molecular weight. A facile purification of the conjugates was achieved by one-step cationic exchange chromatography. The purity of mono-PEGylated protein was greater than 95%. The purified conjugate was characterized by multi-angle laser light scattering (MALLS) for determining the apparent gyration radius (r_g) and hydrodynamic radius (r_h). MALLS results showed that the conjugation of PEG markedly enhanced r_g and r_h of parent protein (r_g: from 15.7 to 48.2 nm for the PEG_5k and 81.9 nm for the PEG_10k; r_h: from 4.2 to 58.4 nm for the PEG_5k and 102.3 nm for the PEG_10k). The PEGylated rhIL-1ra retained 44.6% of binding activities to the cell receptor for PEG_5k and 40.2% for PEG_10k, compared to the original protein.     

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 2³ + 4 were performed to evaluate the following parameters: molar mass of PEG (M_PEG), concentration of PEG (C_PEG), concentration of citrate buffer (C_Cit), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of M_PEG = 10,000 g/mol, C_PEG = 22 wt%, C_Cit = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 μmol Trolox equivalent/mg protein, for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 μmol Trolox/mg protein). Nevertheless, the IC₅₀ values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.     

Positioning of the Alzheimer Aβ(1–40) peptide in SDS micelles using NMR and paramagnetic probes

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid β-peptide Aβ(1–40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn²⁺ ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two α-helices induced in Aβ(1–40), comprising residues 15–24, and 29–35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn²⁺ showing that these regions are positioned mostly outside the micelle. The central helix (residues 15–24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This α-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn²⁺, and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and ¹⁵N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Aβ(1–40) show that the size of SDS micelle is not significantly changed by interaction with Aβ(1–40). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physicochemical characterization of the staphylolytic LysK enzyme in complexes with polycationic polymers as a potent antimicrobial

Staphylococcus aureus causes many serious visceral, skin, and respiratory diseases. About 90% of its clinical strains are multi-drug resistant, but the use of bacteriophage lytic enzymes offers a viable alternative to antibiotic therapy. LysK, the phage K endolysin, can lyse S. aureus when purified and exposed externally. It has been investigated in its complexes with polycationic polymers (poly-l-lysines (PLLs) of molecular weights 2.5, 9.6, and 55.2 kDa and their block copolymers with polyethylene glycol PLL₁₀–PEG₁₁₄, PLL₃₀–PEG₁₁₄, and PLL₃₀–PEG₂₃) as a basis for creating active and stable antimicrobial. Complexing with polycationic PLLs produces a stabilizing effect on LysK due to structure ordering in its molecules and break-down of aggregates as a result of electrostatic interaction. The stability of LysK in the presence of PLL–PEG block copolymers improves by both electrostatic and hydrophobic mechanisms. Complexes of LysK with 2.5, 9.6, 55.2 kDa poly-l-lysines and PLL₃₀–PEG₁₁₄ have demonstrated sufficient stability at the temperatures of physiological activity (37 °C) and storage (4 °C and 22 °C).     

Catalytic activity of lignin peroxidase and partition of veratryl alcohol in AOT/isooctane/toluene/water reverse micelles

The activity of lignin peroxidase (LiP) and the partition of its optimum substrate veratryl alcohol (VA) in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/toluene/water reverse micelles were studied in this paper to understand the microheterogeneous effect of the medium on the catalytic properties of LiP hosted in the reverse micelle. Results showed that LiP from Phanerochaete chrysosporium could express its activity in the reverse micelles, but its activity depended, to a great extent, on the composition of the reverse micelles. Optimum activity occurred at a molar ratio of water to AOT (ω₀) of 11, a pH value of 3.6, and a volume ratio of isooctane to toluene of 7–9. Under optimum conditions, the half-life of LiP was circa 12 h. The dependence of LiP activity on the volume fraction of water in the medium (θ), at a constant ω₀ value of 11, indicated that VA was mainly solubilized in the pseudophase of the reverse micelle. Based on the pseudobiphasic model and the corresponding kinetic method, a linear line can be obtained in a plot of apparent Michaelis constant of VA vs θ, and the partition coefficient of VA between the pseudophase and the organic solvent phase was determined to be 35.8, which was higher than that (22.3) between bulk water and the corresponding mixed organic solvent. H₂O₂ inhibited LiP at concentrations higher than 80 μM; this concentration value seems to be different from that in aqueous solution (about 3 mM). The differences mentioned above should be ascribed to the microheterogeneity and the interface of the AOT reverse micelle.     

Catalytic performance and thermostability of chloroperoxidase in reverse micelle: achievement of a catalytically favorable enzyme conformation

The catalytic performance of chloroperoxidase (CPO) in peroxidation of 2, 2′-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) and oxidation of indole in a reverse micelle composed of surfactant-water-isooctane-pentanol was investigated and optimized in this work. Some positive results were obtained as follows: the peroxidation activity of CPO was enhanced 248% and 263%, while oxidation activity was enhanced 215% and 222% in cetyltrimethylammonium bromide (CTABr) reverse micelle medium and dodecyltrimethylammonium bromide (DTABr) medium, respectively. Thermostability was also greatly improved in reverse micelle: at 40°C, CPO essentially lost all its activity after 5 h incubation, while 58–76% catalytic activity was retained for both reactions in the two reverse micelle media. At 50°C, about 44–75% catalytic activity remained for both reactions in reverse micelle after 2 h compared with no observed activity in pure buffer under the same conditions. The enhancement of CPO activity was dependent mainly on the surfactant concentration and structure, organic solvent ratio (V _pentanol/V _isooctane), and water content in the reverse micelle. The obtained kinetic parameters showed that the catalytic turnover frequency (k _cat) was increased in reverse micelle. Moreover, the lower K _m and higher k _cat/K _m demonstrated that both the affinity and specificity of CPO to substrates were improved in reverse micelle media. Fluorescence, circular dichroism (CD) and UV–vis spectra assays indicated that a catalytically favorable conformation of enzyme was achieved in reverse micelle, including the strengthening of the protein α-helix structure, and greater exposure of the heme prosthetic group for easy access of the substrate in bulk solution. These results are promising in view of the industrial applications of this versatile biological catalyst.     

Layer-by-layer Assembly of Poly(lactic-co-glycolic acid)-b-poly(l-lysine) Copolymer Micelles

Biocompatible, biodegradable polyionic micelles were used as a building component for layer-by-layer (LbL) assembly that can produce drug-loaded nanolayers. To prepare the polycationic micelles, poly(lactic-co-glycolic acid)-b-poly(l-lysine) [PLGA-b-P(Lys)] copolymers were synthesized. In an aqueous phase, PLGA-b-P(Lys) copolymers were self-assembled to form spherical micelles with the inner core of poly(lactic-co-glycolic acid) (PLGA) and the cationic outer shell of P(Lys). The micelles were characterized by zeta potential, dynamic light scattering, and nuclear magnetic resonance. PLGA-b-P(Lys) micelles showed the positive zeta potential values in a broad range of pH (3–11), indicating the high stability of the polyionic micelles with the outer shell of positive charges. Cationic polymeric micelles were coated on the surface via electrostatic interactions with the oppositely charged polyelectrolyte, poly(sodium 4-styrenesulfonate). Formation of multiple micelle layers was monitored using quartz crystal microbalance in situ, and the surface topology of the layers was characterized by atomic force microscopy ex situ, as the number of micelle layer was increased. The multiple micelle layers were stable, and the thickness of micelle layer grew as the number of LbL coating increased. The approach described in this work can be used for the development of the biocompatible, biodegradable, drug-loaded bioactive nanocoatings.     

Lipid membrane expansion and micelle formation by polymer-grafted lipids: scaling with polymer length studied by spin-label electron spin resonance

Spin-label electron spin resonance (ESR) spectroscopy and auxiliary optical density measurements are used to study lipid dispersions of N-poly(ethylene glycol)-dipalmitoyl phosphatidylethanolamine (PEG:5000-DPPE) mixed with dipalmitoyl phosphatidylcholine (DPPC). PEG:5000-DPPE bears a large hydrophilic polymer headgroup (with approximately 114 oxyethylene monomers) and is commonly used for steric stabilization of liposomes used in drug delivery. Comparison is made with results from mixtures of DPPC with polymer lipids bearing shorter headgroups (approximately 45 and 8 oxyethylene monomers). ESR spectra of phosphatidylcholine spin-labeled on the 5-C atom position of the sn-2 chain are shown to reflect the area expansion of the lipid membranes by the lateral pressure exerted in the polymer brush, in a way that is consistent with theory. The lipid chain packing density at the onset of micelle formation is the same for all three PEG-lipids, although the mole fraction at which this occurs differs greatly. The mole fraction at onset scales inversely with the size of the polymer headgroup, where the experimental exponent of 0.7 is close to theoretical predictions (viz. 0.55-0.6). The mole fraction of PEG-lipid at completion of micelle formation is more weakly dependent on polymer size, which conforms with theoretical predictions. At high mole fractions of PEG:5000-DPPE the dependence of lipid packing density on mole fraction is multiphasic, which differs qualitatively from the monotonic decrease in packing density found with the shorter polymer lipids. Lipid spin-label ESR is an experimental tool that complements theoretical analysis using polymer models combined with the lipid equation of state.     

Micelles obtained by aggregation of gemini surfactants containing the CCK8 peptide and a gadolinium complex

Two gemini surfactants, [C18CysL5CCK8]₂ and [C18CysDTPAGlu]₂, containing, respectively, the CCK8 peptide and the DTPAGlu chelating agent or its gadolinium complex have been prepared by linking lipophilic chains through a disulfide bond between two cysteine residues. The two surfactants aggregate in water solution forming pure or mixed micelles, with a critical micellar concentration in the 5 × 10⁻⁶–5 × 10⁻⁵ mol kg⁻¹ range, as measured by fluorescence spectroscopy. As indicated by small-angle neutron scattering, the shape and size of the micelles are influenced by the temperature: increasing temperature leads to progressive reduction of the size of the supramolecular aggregates. Cylindrical structures found at lower temperatures (10–40 °C) evolve into ellipsoidal micelles at 50–80 °C. Furthermore, the surface-exposed CCK8 peptide changes its conformation above a transition temperature of approximately 45 °C, going from a β-sheet to a random-coil structure, as indicated by circular dichroism measurements. The mixed aggregate obtained by coaggregation of the two gemini-based amphiphilic compounds, [C18CysDTPAGlu(Gd)]₂ and [C18CysL5CCK8]₂ in 70:30 molar ratio, represents the first example of a peptide-containing gemini surfactant as a potential target-selective contrast agent in MRI. In fact, it presents a high relaxivity value of the gadolinium complex, 21.5 mM⁻¹ s⁻¹, and the CCK8 bioactive peptide exposed on the external surface is therefore capable of selective targeting of the cholecystokinin receptors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein precipitation by polyethylene glycol: a generalized model based on hydrodynamic radius

PEGs for protein precipitation are usually classified by molecular weight. The higher molecular weight precipitants are more efficient but result in higher viscosity. Following empirical evidence that the precipitation efficiency is more comprehensively characterized by PEG hydrodynamic radius (r_h,PEG) than molecular weight, this paper proposes a model to explicate the significance of r_h,PEG. A general expression was formulated to characterize the PEG effect exclusively by r_h,PEG. The coefficients of a linearized form were then fitted using empirical solubility data. The result is a simple numerical relation that models the efficiency of general-shaped PEG precipitants as a function of r_h,PEG and protein hydrodynamic radius (r_h,prot). This equation also explains the effects of environmental conditions and PEG branching. While predictions by the proposed correlation agree reasonably well with independent solubility data, its simplicity gives rise to potential quantitative deviations when involving small proteins, large proteins and protein mixtures. Nonetheless, the model offers a new insight into the precipitation mechanism by clarifying the significance of r_h,PEG. This in turn helps to refine the selection criterion for PEG precipitants.     

Surfactant-induced conformational transition of amyloid β-peptide

Accumulating evidence suggests that Aβ_1–42–membrane interactions may play an important role in the pathogenesis of Alzheimer’s disease. However, the mechanism of this structural transition remains unknown. In this work, we have shown that submicellar concentrations of sodium dodecyl sulfate (SDS) can provide a minimal platform for Aβ_1–42 self-assembly. To further investigate the relation between Aβ_1–42 structure and function, we analyzed peptide conformation and aggregation at various SDS concentrations using circular dichroism (CD), Fourier transform infrared spectroscopy, and gel electrophoresis. These aggregates, as observed via atomic force microscopy, appeared as globular particles in submicellar SDS with diameters of 35–60 nm. Upon sonication, these particles increased in disc diameter to 100 nm. Pyrene I ₃/I ₁ ratios and 1-anilinonaphthalene-8-sulfonic acid binding studies indicated that the peptide interior is more hydrophobic than the SDS micelle interior. We have also used Forster resonance energy transfer between N-terminal labeled pyrene and tyrosine (10) of Aβ_1–42 in various SDS concentrations for conformational analysis. The results demonstrate that SDS at submicellar concentrations accelerates the formation of spherical aggregates, which act as niduses to form large spherical aggregates upon sonication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interactions of inorganic mercury with phospholipid micelles and model membranes. A31P-NMR study

The binding of inorganic mercury Hg(II) to phospholipid headgroups has been investigated by phosphorus-31 nuclear magnetic resonance of phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in water micellar and multilamellar phases. HgCl₂ triggers the aggregation of phospholipid micelles, leading to a lipid-mercury precipitate that is no longer detectable by high-resolution³¹P-NMR. The remaining signal area corresponds to micelles in the soluble fraction and is a non-linear function of the initial mercury-to-lipid molar ratio. Kinetics of micelle aggregation are exponential for the first 15 min and show a plateau tendency after 120 min. Apparent Hg(H) affinities for phospholipid headgroups are in the order: PE > PS > PC. The same binding specificity is observed when HgCl₂ is added to (1:1) mixtures of different micelles (PE + PC; PS + PC). However, mercury binding to mixed micelles prepared with two lipids (PE/PC or PS/PC) induces the aggregation of both lipids. Hg(II) also leads to a³¹P-NMR chemical shift anisotropy decrease of PC, PS and mixed (1:1) PE/PC multilamellar vesicles and markedly broadens PS spectra. This indicates that HgCl₂ binding forces phospholipid headgroups to reorient and that the concomitant network formation leads to a slowing down of PS membrane collective motions. Formation of a gel-like lamellar phase characterized by a broad NMR linewidth is also observed upon HgCl₂ binding to PE samples both in fluid (L) or hexagonal (H_II) phases. The PE hexagonal phase is no longer detected in the presence of HgCl₂. Mixed PE/PC dispersions remain in the fluid phase upon mercury addition, indicating that no phase separation occurs. Addition of excess NaCl leads to the appearance of the non-reactive species HgCl _inf4 ^sup2– and induces the reversal of all the above effects.Abbreviations A(t) time-dependence of peak area - A₄₀ peak area at t=40 min - 1/ rate of peak area decrease - isotropic chemical shift - isotropic chemical shift change - chemical shift anisotropy - DPPC dipalmitoylphosphatidylcholine - Hg(II) inorganic mercury - NMR nuclear magnetic resonance - pCl –log [Cl^–] - PC phosphatidylcholine - PE phosphatidylethanolamine - PL phospholipid - PS phosphatidylserine - R_i mercury-to-lipid molar ratio - MLV multilamellar vesicles - SUV small unilamellar vesicles     

Impact of bifunctional chelators on biological properties of <Superscript>111</Superscript>In-labeled cyclic peptide RGD dimers

The present study describes the synthesis and biological evaluation of ¹¹¹In(DOTA-3P-RGD₂) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD₂ = PEG₄-E[PEG₄-c(RGDfK)]₂; PEG₄ = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), ¹¹¹In(DTPA-3P-RGD₂) (DTPA = diethylenetriaminepentaacetic acid) and ¹¹¹In(DTPA-Bn-3P-RGD₂) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α_vβ₃ expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the ¹¹¹In-labeled 3P-RGD₂. IC₅₀ values of DOTA-3P-RGD₂, DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂ were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against ¹²⁵I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting ¹¹¹InCl₃ with the RGD peptide conjugates in NH₄OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD₂, successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15–20 min. For DTPA-3P-RGD₂ and DTPA-Bn-3P-RGD₂, the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for ¹¹¹In(DOTA-3P-RGD₂) and ~200 mCi/mg (or ~400 mCi/μmol) for ¹¹¹In(DTPA-3P-RGD₂). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both ¹¹¹In(DTPA-3P-RGD₂) and ¹¹¹In(DTPA-Bn-3P-RGD₂) showed a much faster tumor washout and poorer T/B ratios than ¹¹¹In(DOTA-3P-RGD₂). The tumor uptake of ¹¹¹In(DOTA-3P-RGD₂) is integrin α_vβ₃- and RGD-specific. ¹¹¹In(DOTA-3P-RGD₂) is metabolically stable while only ~25% of ¹¹¹In(DTPA-Bn-3P-RGD₂) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that ¹¹¹In(DTPA-3P-RGD₂) can be an effective integrin α_vβ₃-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.     

Synthesis of a New Tri-Branched PEG-IFNα2 and Its Impact on Anti Viral Bioactivity

Efficacy of proteins can be enhanced by using polyethylene glycol (PEG) conjugation (PEGylation) to the protein molecules. Mobile non-toxic PEG chains conjugated to bio-therapeutics increase their hydrodynamic volume and in turn can prolong their plasma retention time and increase their solubility. An important aspect of PEGylation is the selection of PEG molecule with suitable structure and molecular weight. In this study, conceiving the idea that branched PEG-conjugates show superior efficacy over the linear PEG-conjugates, a tri-branched PEG-interferon (mPEG₃L₂-IFN) was synthesized by reacting a 30 KDa tri-branched mPEG₃L₂-NHS reagent with IFN to improve its pharmacokinetic properties and reduce the loss of in vitro bioactivity (which is generally exhibited by PEGylation) of the conjugated protein to some extent. The PEGylation procedure was optimized in terms of concentration and molar ratios of reactants, reaction time, temperature and pH conditions of the reaction mix. The conjugate was purified by cation exchange chromatography and characterized by SDS-PAGE and SE-HPLC. Trypsin digestion of mPEG₃L₂-IFN indicated a single site specificity of PEGylation. Anti viral bioactivity of mPEG₃L₂-IFN was found to be 2.38 × 10⁷ IU/mg which is approximately 9.52% of native IFNα2 (2.5 × 10⁸ IU/mg) and better than PEGasys from Roche Pharma. Therefore, it is reported that the tri-branched mPEG₃L₂-NHS reagent has the potential to be used to conjugate proteins for the promising therapeutic results.     

Photostability of quantum dot micelles under ultraviolet irradiation

Phospholipid quantum dot micelles are useful for bio‐applications because of their amphiphilicity and exceptional biocompatibilities. We investigated the uptake of phospholipid [polyethylene glycol (PEG), biotin, and folic acid terminated] modified CdSe/ZnS quantum dot micelles by cancer cells and its photostability under ultrviolet light in the C spectrum (UV‐C) (254 nm) or UV‐A (365 nm) light irradiation. The stability of micelles to the exposure of UV‐C and UV‐A light was assessed. Biotin‐modified quantum dot micelles give photoluminescence enhancement under UV‐C light irradiation. Folate modified micelle under UV‐C and UV‐A results show considerable photoluminescence enhancement. Photoluminescence lifetime measurements showed 7.04, 8.11 and 11.42 ns for PEG, folate, and biotin terminated phospholipid micelles, respectively. Folate and biotin‐modified quantum dot micelles showed excellent uptake by HeLa cells under fluorescence confocal microscopy. Phospholipid CdSe/ZnS quantum dot micelles can be potentially used for diagnosis and treatment of cancer in the future.     

A Stable Isotope Tracer Study of the Influences of Adjacent Land Use and Riparian Condition on Fates of Nitrate in Streams

The influence of land use on potential fates of nitrate (NO₃ ⁻) in stream ecosystems, ranging from denitrification to storage in organic matter, has not been documented extensively. Here, we describe the Pacific Northwest component of Lotic Intersite Nitrogen eXperiment, phase II (LINX II) to examine how land-use setting influences fates of NO₃ ⁻ in streams. We used 24 h releases of a stable isotope tracer (¹⁵NO₃-N) in nine streams flowing through forest, agricultural, and urban land uses to quantify NO₃ ⁻ uptake processes. NO₃ ⁻ uptake lengths varied two orders of magnitude (24–4247 m), with uptake rates (6.5–158.1 mg NO₃-N m⁻² day⁻¹) and uptake velocities (0.1–2.3 mm min⁻¹) falling within the ranges measured in other LINX II regions. Denitrification removed 0–7% of added tracer from our streams. In forest streams, 60.4 to 77.0% of the isotope tracer was exported downstream as NO₃ ⁻, with 8.0 to 14.8% stored in wood biofilms, epilithon, fine benthic organic matter, and bryophytes. Agricultural and urban streams with streamside forest buffers displayed hydrologic export and organic matter storage of tracer similar to those measured in forest streams. In agricultural and urban streams with a partial or no riparian buffer, less than 1 to 75% of the tracer was exported downstream; much of the remainder was taken up and stored in autotrophic organic matter components with short N turnover times. Our findings suggest restoration and maintenance of riparian forests can help re-establish the natural range of NO₃ ⁻ uptake processes in human-altered streams.     

Effects of carbon source,polyethylene glycol and abscisic acid on secondary embryo induction and maturation in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) microspore-derived embryos

In this study, the effects of carbon sources, abscisic acid (ABA) either alone or in combination with polyethylene glycol (PEG) were evaluated on secondary embryo (SE) induction and maturation in rapeseed microspore-derived embryos (MDE) of cultivars Global, PF₇₀₄ and Option. Among various carbon sources tested (sucrose, glucose, fructose and sorbitol), the use of 0.3 M (300 mOsml⁻¹) glucose and 0.2 M (200 mOsml⁻¹) sorbitol in SE induction medium (for cultivars Global and PF₇₀₄) and sorbitol at 0.2 and 0.3 M (200 and 300 mOsml⁻¹, for cultivar Option), induced the highest secondary embryogenesis percentage (%SE). The highest number of SEs per each MDE (SE/MDE) was observed with 0.2 M (200 mOsml⁻¹) sorbitol in cultivar Global and with 0.3 M (300 mOsml⁻¹) glucose in cultivars PF₇₀₄ and Option. In another part of this study, the effect of different concentrations of ABA (0, 20, 40, 60, 80 and 100 μM) and of a combined use of ABA (0 and 40 μM) and PEG 4000 or PEG 6000 at 15 g l⁻¹ (3.75 and 2.5 mOsml⁻¹, respectively) was examined on induction and maturation of SEs. In the first experiment, the use of ABA in SE induction medium reduced the mean number of SE/MDE in the three studied cultivars, whereas use of 40–80 μM ABA in SE induction medium increased the percentage of mature SEs in each cultivar. The combined use of PEG with or without ABA also reduced the mean number of SE/MDE compared with control, but resulted in significant enhancement of the percentages of mature SEs for the three cultivars.     

Synthesis of protoporphyrin–lipids and biological evaluation of micelles and liposomes

Protoporphyrin IX (PPIX) lipids were synthesized by introducing a long alkyl chain, such as C13, C15, and C17, at each vinyl group on PPIX via hydrobromination. The PPIX lipids exhibited a water-soluble property by forming their micelles in water and the PPIX–lipid micelles showed relatively low cytotoxicity toward HeLa cells (IC₅₀ = 151.7–379.9 μM) without light irradiation. PL-C17 liposomes (post-inserted liposomes) were readily prepared by adding PL-C17 micelle solution to the liposome solution. The IC₅₀ values of PPIX, PL-C17 micelles, and PL-C17 liposomes toward HeLa cells were 0.53, 5.65, and 12.9 μM, respectively, after irradiation with a xenon lamp in the 400–800 nm range for 2 min. PL-C17 liposomes were selectively accumulated in the Golgi apparatus in cells.