Calorimetric study on pH-responsive block copolymer grafted lipid bilayers: rational design and development of liposomes

This study is focused on chimeric advanced drug delivery nanosystems and specifically on pH-sensitive liposomes, combining lipids and pH-responsive amphiphilic block copolymers. Chimeric liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and two different forms of block copolymers, i.e. poly(n-butylacrylate)-b-poly(acrylic acid) (PnBA-b-PAA) at 70 and 85% content of PAA at six different molar ratios, each form respectively. PAA block exhibits pH-responsiveness, because of the regulative group of –COOH. –COOH is protonated under acidic pH (pK_a ca. 4.2), while remains ionized under basic or neutral pH, leading to liposomes repulse and eventually stability. Lipid bilayers were prepared composed of DPPC and PnBA-b-PAA. Experiments were carried out using differential scanning calorimetry (DSC) in order to investigate their thermotropic properties. DSC indicated disappearance of pre-transition at all chimeric lipid bilayers and slight thermotropic changes of the main transition temperature. Chimeric liposomes have been prepared and their physicochemical characteristics have been explored by measuring the size, size distribution and ζ-potential, owned to the presence of pH-responsive polymer. At percentages containing medium to high amounts of the polymer, chimeric liposomes were found to retain their size during the stability studies. These results were well correlated with those indicated in the DSC measurements of lipid bilayers incorporating polymers in order to explain their physicochemical behavior. The incorporation of the appropriate amount of these novel pH-responsive block copolymers affects thus the cooperativity, the liposomal stabilization and imparts pH-responsiveness.     

Release of indomethacin from pH-sensitive pullulan acetate microsphere

We studied the pH-sensitive indomethacin (IND) delivery system using pullulan. Hydrophobic pullulan acetate was prepared by chemical modification of hydrophilic pullulan and pullulan acetate microsphere was made by a solvent evaporation method. The size of microspheres was below 5 μm, and the drug loading efficiencies of microspheres were approximately 78 and 65% at the initial amount of drug 40 and 80 mg, respectively. The microsphere showed pH-sensitive swelling behavior in PBS buffer. After 15 hrs, the swelling of the microsphere at pH 7.4 was approximately 20 times greater than that at pH1.2. The pH of the medium significantly influenced on thein vitro release rate. The released amount of drug at pH 7.2 was approximately 90 times greater than that at pH 1.2. The shape of microspheres at pH 1.2 were maintained sphere forms, but at pH 7.4 were disintegrated. The pH-sensitive IND release pattern was due both to the pH-sensitive diffusion of IND from the microspheres and to the release of the drug from the surface which underwent disintegration after swelling, due to the chemical composition of the microspheres and the pH of the release media.     

Effect of Trace Levels of Nigericin on Intracellular pH and Acid-Base Transport in Rat Renal Mesangial Cells

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH _i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH _i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH _i decrease elicited by removing extracellular Na⁺, (ii) an increase in acid extrusion during the pH _i increase caused by elevating extracellular [K⁺], and (iii) an acid shift in the pH _i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH _i dependence of Na-H exchange, computed by adding the pH _i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999     

Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films

Alpha-amylase was covalently immobilized onto maleic anhydride copolymer films preserving activity. The initial activity of the immobilized layers strongly depended on the immobilization solution, and on the physicochemical properties of the copolymer film. Higher enzyme loading (quantified by amino acid analysis using HPLC) and activity (measured by following starch hydrolysis) were attainable onto hydrophilic, highly swelling 3-D poly(ethylene-alt-maleic anhydride) (PEMA) copolymer films, while immobilization onto hydrophobic poly(octadecene-alt-maleic anhydride) (POMA) copolymer films resulted in low content enzyme layers and lower activity. No significant activity was lost upon dehydration/re-hydration or storage of enzyme containing PEMA copolymer layers in deionised water for up to 48 h. In contrast, α-amylase decorated POMA films suffered a significant activity loss under those conditions. The distinct behaviours may be attributed to the different intrinsic physicochemical properties of the copolymer films. The compact, hydrophobic POMA films possibly favours hydrophobic interactions between the hydrophobic moieties of the protein and the surface, which may result in conformational changes, and consequent loss of activity. Surprisingly, residual activity was found after harsh treatments of active α-amylase PEMA based layers revealing that immobilization onto the hydrophilic polymer films improved the stability of the enzyme.     

MXene‐Based Mesoporous Nanosheets Toward Superior Lithium Ion Conductors

Although solid polymer electrolytes have some intrinsic advantages in synthesis and film processing compared with inorganic solid electrolytes, low ionic conductivities and mechanical moduli hamper their practical applications in lithium‐based batteries. Here, an efficient strategy is developed to produce a unique solid polymer electrolyte containing MXene‐based mesoporous silica nanosheets with a sandwich structure, which are fabricated via controllable hydrolysis of tetraethyl orthosilicate around the surface of MXene‐Ti₃C₂ under the direction of cationic surfactants. Such unique nanosheets not only exhibit individual, thin, and insulated features, but also possess abundant functional groups in mesopores and on the surface, which are favorable for the formation of Lewis acid–base interactions with anions in polymer electrolytes such as poly(propylene oxide) elastomer, enabling the fast Li⁺ transportation at the mesoporous nanosheets/polymer interfaces. As a consequence, a solid polymer electrolyte with high ionic conductivity of 4.6 × 10^?4 S cm^?1, high Young's modulus of 10.5 MPa, and long‐term electrochemical stability is achieved.     

Preparation of pH-sensitive poly(glycidol) derivatives with varying hydrophobicities: their ability to sensitize stable liposomes to pH

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.     

Antiproliferative and Apoptotic Effects of pH-Responsive Veratric Acid-Loaded Polydopamine Nanoparticles in Human Triple Negative Breast Cancer Cells

Veratric acid (VA) is plant-derived phenolic acid known for its therapeutic potential, but its anticancer effect on highly invasive triple-negative breast cancer (TNBC) is yet to be evaluated. Polydopamine nanoparticles (nPDAs) were chosen as the drug carrier to overcome VA's hydrophobic nature and ensure a sustained release of VA. We prepared pH-sensitive nano-formulations of VA-loaded nPDAs and subjected them to physicochemical characterization and in vitro drug release studies, followed by cell viability and apoptotic assays on TNBC cells (MDA-MB-231 cells). The SEM and zeta analysis revealed spherical nPDAs were uniform size distribution and good colloidal stability. In vitro drug release from VA-nPDAs was sustained, prolonged and pH-sensitive, which could benefit tumor cell targeting. MTT and cell viability assays showed that VA-nPDAs (IC50=17.6 μM) are more antiproliferative towards MDA-MB-231 cells than free VA (IC50=437.89 μM). The induction of early and late apoptosis by VA-nPDAs in the cancer cells was identified using annexin V and dead cell assay. Thus, the pH response and sustained release of VA from nPDAs showed the potential to enter the cell, inhibit cell proliferation, and induce apoptosis in human breast cancer cells, indicating the anticancer potential of VA.     

Highly Efficient Polymer Tandem Cells and Semitransparent Cells for Solar Energy

Highly efficient tandem and semitransparent (ST) polymer solar cells utilizing the same donor polymer blended with [6,6]‐phenyl‐C₆₁‐butyric acid methyl ester (PC₆₁BM) and [6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PC₇₁BM) as active layers are demonstrated. A high power conversion efficiency (PCE) of 8.5% and a record high open‐circuit voltage of 1.71 V are achieved for a tandem cell based on a medium bandgap polymer poly(indacenodithiophene‐co‐phananthrene‐quinoxaline) (PIDT‐phanQ). In addition, this approach can also be applied to a low bandgap polymer poly[2,6‐(4,4‐bis(2‐ethylhexyl)‐4H‐cyclopenta[2,1‐b;3,4‐b′]dithiophene)‐alt‐4,7‐(5‐fluoro‐2,1,3‐benzothia‐diazole)] (PCPDTFBT), and PCEs up to 7.9% are achieved. Due to the very thin total active layer thickness, a highly efficient ST tandem cell based on PIDT‐phanQ exhibits a high PCE of 7.4%, which is the highest value reported to date for a ST solar cell. The ST device also possesses a desirable average visible transmittance (≈40%) and an excellent color rendering index (≈100), permitting its use in power‐generating window applications.     

Structural and conformational studies of polyriboadenylic acid in neutral and acid solution

Raman spectra of solutions of polyriboadenylic acid have been studied in the pH range of 7.2–5.2. Bands are identified which are sensitive to the characteristics of poly(rA) in the single-and double-stranded helical forms. Thermal melting profiles were obtained as a function of pH to monitor simultaneously the changes in (1) the phosphodiester backbone, (2) the base-stacking interactions, (3) the perturbation of the PO unit, and (4) the degree of protonation at the N-1 position in the adenine base. The temperature dependence of the intensity ratio of the bands at 725 and 705 cm^?1 appears to be sensitive to the noncooperative and the cooperative thermal-melting process for the single-and double-stranded forms of poly(rA), respectively. Concurrently, bands diagnostic of the degree of protonation reveal that the cooperative melting process for the “acid” poly(rA) clearly involves deprotonation. The progressive perturbation of the 1100 cm^?1 band with an increasing degree of protonation of poly(rA) is consistent with earlier suggestions regarding a PO-(6)-NH₂ interaction in the double-helical form of poly(rA). The stability of the double-helix parallels the degree of protonation over the pH range studied as reflected in the t_m values, which increase linearly with decreasing pH.     

Polymer-supported biopolymer synthesis: 5. Ultra-high load solid (gel) phase peptide synthesis—the stepwise elaboration of quasi-homogeneous peptide gel networks?

An approach to ultra-high load solid (gel) phase peptide synthesis is described in which a bead-form phenolic core polymer, crosslinked poly[N-{2-(4-hydroxyphenyl)ethyl}-acrylamide], is used as a support matrix at near theoretical maximum loading. Consecutive repeating units of the core polymer carry peptide chains undergling stepwise elongation. Synthesis proceeds through a series of solvated networks, which consist mainly of protected peptide. The solvated networks are deemed to be quasi-homogeneous, insofar as each has a regular covalent framework and each is believed to be uniformly distributed throughout the gel beads. Illustrative synthesis of two fully-protected acylpeptide hydrazide segments, corresponding to dynorphin(6–12) and to β_h-endorphin (18–26), are described.     

Physicochemical Characterization and Study of in vitro Interactions of pH-sensitive Liposomes with the Complement System

Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG₂₀₀₀-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non–long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non–pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum.     

Block copolymerization of vinyl compounds by the terminal-group activation of poly(α-amino acids) and the characterization of block copolymers

The terminal amino groups of polysarcosine, poly(γ-benzyl l-glutamate), and poly(ε-benzyloxycarbonyl-l-lysine) were haloacetylated. The mixture of the terminally haloacetylated poly(α-amino acid) and styrene or methyl methacrylate was photoirradiated in the presence of Mn₂(CO)₁₀, or heated with Mo(CO)₆, yielding A-B-A-type block copolymers consisting of poly(α-amino acid) (the A component) and vinyl polymer (the B component). The block copolymers were characterized, and the present investigation revealed that the thermally initiated polymerization of vinyl compounds by the trichloroacetyl poly(α-amino acid)/Mo(CO)₆ system was the most suitable for the synthesis of the α-amino acid/vinyl compound block copolymers. The A-B-A type block copolymers showed higher antithrombogenicity than the corresponding homopolymers. In particular, a film of the A-B-A-type block copolymer of poly[Glu(OBzl)] and polystyrene possessed a microphase-separated structure and did not induce a conformational change of fibrinogen adsorbed, leading to a high antithrombogenicity.     

Biosynthesis of copolyesters consisting of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids from 1,3-butanediol or from 3-hydroxybutyrate by Pseudomonas sp. A33

Pseudomonas sp. A33 and other isolates of aerobic bacteria accumulated a complex copolyester containing 3-hydroxybutyric acid (3HB) and various medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from 3-hydroxybutyric acid or from 1,3-butanediol under nitrogen-limitated culture conditions. 3HB contributed to 15.1 mol/100 mol of the constituents of the polyester depending on the strain and on the cultivation conditions. The accumulated polymer was a copolyester of 3HB and 3HA_MCL rather than a blend of poly(3HB) and poly(3HA_MCL) on the basis of multiple evidence. 3-Hydroxyhexadecenoic acid and 3-hydroxyhexadecanoic acid were detected as constituents of polyhydroxyalkanoates, which have hitherto not been described, by¹³C nuclear magnetic resonance or by gas chromatography/mass spectrometric analysis. In total, ten different constituents were detected in the polymer synthesized from 1,3-butanediol by Pseudomonas sp. A33:besides seven saturated (3HB, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydrohexadecanoate) three unsaturated (3-hydroxydodecenoate, 3-hydroxytetradecenoate and 3-hydrohexadecanoate) hydroxyalkanoic acid constituents occured. The polyhydroxyalkanoate synthase of Pseudomonas sp. A33 was cloned, and its substrate specificity was evaluated by heterologous expression in various strains of P. putida, P. oleovorans and Alcaligenes eutrophus.     

On the amino-acid-sequence distribution in benzylglutamate-methionine copolymers prepared from N-carboxyanhydrides

The amino-acid-sequence distribution in poly(γ-benzyl-L -glutamate, L -methionine) prepared by polymerization of the respective N-carboxyanhydrides has been investigated. This copolymer was converted first to poly(L -glutamic acid, L -methionine), which was subsequently cleaved by treatment with cyanogen bromide. The resulting material was fractionated into oligomers of (glutamic acid)_n-homoserine whose relative molar amounts were determined quantitatively. The results have been compared with those for a random incorporation of the methionine in a γ-benzylglutamate host polymer. Fairly close agreement has been found.     

A Study of stepwise conformational changes in poly(β-benzyl-L-aspartate) with the helix–coil transition by means of proton and carbon-13 NMR

The helix–coil transition for poly(β-benzyl-L -aspartate) [poly(Asp[OBzl])] in solvent mixtures of trifluoroacetic acid/deuterated chloroform (F₃AcOH/CDCl₃) was studied by means of proton and carbon-13 nmr. Conformational fixation of the side chain occurs before the coil–helix transition of the backbone, when neighboring phenyl rings face each other. Another type of conformational fixation occurs in the side chain after the coil–helix transition of the backbone. These conformational changes of the side chain are due to the changes of the strength of the interaction between the side-chain ester group and the F₃AcOH molecule. In the absence of F₃AcOH (coil-forming solvent), the polymer has a rather rigid structure in which the side chain may wrap around the backbone. These conformational changes of the polymer are closely related to the changes of the interaction between the polymer and F₃AcOH molecules.     

Chimeric aptamers in cancer cell-targeted drug delivery

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets (“apatopes”) with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer–aptamer, aptamer–nonaptamer biomacromolecules (siRNAs, proteins) and aptamer–nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.     

In Situ Formation of MoO3 in PEDOT:PSS Matrix: A Facile Way to Produce a Smooth and Less Hygroscopic Hole Transport Layer for Highly Stable Polymer Bulk Heterojunction Solar Cells

A solution‐processed neutral hole transport layer is developed by in situ formation of MoO₃ in aqueous PEDOT:PSS dispersion (MoO₃‐PEDOT:PSS). This MoO₃‐PEDOT:PSS composite film takes advantage of both the highly conductive PEDOT:PSS and the ambient conditions stability of MoO₃; consequently it possesses a smooth surface and considerably reduced hygroscopicity. The resulting bulk heterojunction polymer solar cells (BHJ PSC) based on poly[2,3‐bis‐(3‐octyloxyphenyl)quinoxaline‐5,8‐diyl‐alt‐thiophene‐2,5‐diyl] (TQ1):[6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PC₇₀BM) blends using MoO₃‐PEDOT:PSS composite film as hole transport layer (HTL) show considerable improvement in power conversion efficiency (PCE), from 5.5% to 6.4%, compared with the reference pristine PEDOT:PSS‐based device. More importantly, the device with MoO₃‐PEDOT:PSS HTL shows considerably improved stability, with the PCE remaining at 80% of its original value when stored in ambient air in the dark for 10 days. In comparison, the reference solar cell with PEDOT:PSS layer shows complete failure within 10 days. This MoO₃‐PEDOT:PSS implies the potential for low‐cost roll‐to‐roll fabrication of high‐efficiency polymer solar cells with long‐term stability at ambient conditions.     

Effects of deuterium on the thermal stability of the poly A-poly U helix

The effect of deuterium on the thermal stability of the polynecleotide double helix formed by the homopolymers polyadenylie acid (poly A)and polyuridylic acid (poly U)has been invertigated by measuring the best capacity as a function of temperature in an automatic scanning adiabatic calorimeter. Hydrogen-bounded and deuterium-bonded helical conformations of the polynecleotides used have been melted in H₂O and D₂O₂ respectively, as solvent. Within the limits of experimental error, there is no dfference in the measured enthallpy change accompanying the helix-random coil transition. The enthalpy change ΔH is 6.6 Kcal/MBP ub any case. The half-conversion temperatures T_m differ by two degrees. T_m for poly (AU) in H₂O is 45.8°C, T_m for poly (AU) in D₂is 47.7.°C.     

Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T ₂ and T ₂*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as “probeless” magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T ₂- and T ₂*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin’s stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T ₂ relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T ₂ relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.     

Helix–coil transition for poly(L-glutamic acid) in water–ethanol solutions

Potentiometric titrations and some complementary optical rotation data are presented for solutions of poly(L - glutamic acid) (PGA) in several H₂O–ethanol mixtures. The data allow the determination of the intrinsic pK (pK₀), slope of the apparent. pK (pK_app), versus degree of ionization curves and of the enthalpy of ionization as a function of ethanol concentration. The variation of the degree of ionization at which the helix–coil transformation occurs with ethanol and temperature is also determined. Finally free energy, enthalpy, and intropy changes associated with the helix–coil transformation for the uncharged conformers are determined from the titration curves. The effect of the ethanol is to increase the stability of the helical conformation of PGA for both the charged and the uncharged forms of the polymer. The stabilization of the uncharged helix is essentially an entropic effect.