Pluronic Concentration Research Articles

The effects of Pluronic block copolymers on the aggregation state of nystatin

The purpose of this work was to characterize the effect of block copolymer composition on the aggregation state of nystatin in the presence of Pluronic micelles. The critical aggregation concentrations (CACs) of nystatin were determined by dynamic light scattering (DLS). The CAC of nystatin in phosphate-buffered saline was 20 μM at 37 °C. Addition of Pluronics significantly increased the CAC of nystatin up to >350 μM at 37 °C at the concentrations studied. The CAC values corresponded directly to the size of the Pluronic hydrophobic blocks, and inversely with Pluronic critical micellization concentration (CMC). Predictably, increasing Pluronic concentration and temperature revealed increases in CACs. The micelle–water partition coefficient ( P) of nystatin was determined by nystatin fluorescence. The P for nystatin at 37 °C was calculated in F68, F98, P105, and F127 to be 15, 21, 73, and 79, respectively. Pluronic micelle core polarity experiments, determined by pyrene fluorescence, revealed decreased polarity with increasing hydrophobic block length and temperature. Thus, nystatin CACs in the presence of Pluronics correlated directly with the partition coefficients, and inversely with core polarity. These results point to the number of micelles in solution as the primary factor responsible for nystatin solubilization by Pluronics.

Pluronic F‐68 Enhanced Shoot Regeneration in Micropropagated Citrus Rootstock and Passiflora Species

AbstractThe promotory effects have been studied of the non‐ionic surfactant, Pluronic F‐68, on bud induction/shoot regeneration in epicotyl and cotyledon explants of Citrus depressa and on shoot regeneration from leaf segments of 4–6 week‐old axenic nodal segment‐derived in vitro plants of Passiflora mollissima, P. giberti and P. edulis var. flavicarpa. For epicotyls of C. depressa, supplementation of agar‐solidified MS‐based bud induction/shoot regeneration medium with 0.5% [w/v] Pluronic F‐68 significantly (P < 0.05) increased mean fresh weight gain of cultures, percentage of explants giving shoots and number of shoots per explant. The same Pluronic concentration also enhanced the mean percentage of cotyledons exhibiting bud induction and the number of buds regenerated per cotyledon explant. Fresh weight gain was unaffected across the range of concentrations (0.001–0.5% w/v) of Pluronic F‐68 evaluated for this latter explant source. For leaf explants from axenic shoot cultures of P. mollissima, supplementation of NN‐based medium, containing 3 mg/l 6‐benzyladenine and 2.0 mg/l kinetin with 0.001–0.5% [w/v] Pluronic F‐68, significantly (P < 0.05) increased mean (± s.e.m.) biomass gain by a maximum of 2.7 ± 0.1 g fresh weight (g f.wt.) over the control. Similarly, for leaf explants of P. giberti, 0.001–0.5% [w/v] Pluronic F‐68 in MS‐based medium, containing 1.0 mg/l 6‐BAP and 0.5 mg/l kinetin significantly (P < 0.05) increased mean percentage of explants undergoing shoot regeneration. For P. edulis leaf explants, mean f.wt. gain was also significantly (P < 0.05) higher with Pluronic F‐68 at 0.001–0.5% [w/v].

Determination of critical micelle concentration (CMC) of different pluronics with two different methods

Pluronics are series of closely related block copolymers of ethylene oxide and propylene oxide and are used primarily in pharmaceutical formulation as emulsifying or solubilizing agents. In this study, the crit. micelle concn. (CMC) of different pluronics such as L61, L62, L63, L64, L92, F38, and F68 was measured by two different methods. The first method is the surface tension method (ring method)​; the second one is fluorimetrical method by using DPH (1,​6 di-​Ph hexatriene)​. It was found that, by increasing the lipophilicity of the block copolymer (increase the propylene oxide moiety) the CMC value increases. Also, the fluorimetric method is very sensitive, accurate and more reproducible than surface tension method.

The effect of Pluronic on the protein release kinetics of an injectable drug delivery system

A new class of injectable controlled release depots has been prepared by incorporating materials that preferentially segregate during phase inversion. These consist of blends of poly(ethylene oxide) (PEO)/poly(propylene oxide) (PPO)/poly(ethylene oxide) (PEO) triblock copolymers (Pluronics) with poly(d,l-lactide) (PDLA)/1-methyl-2-pyrrolidinone (NMP) solutions. The effects of preferential segregation on the phase inversion dynamics and in vitro protein release kinetics were examined using dark ground imaging, high performance liquid chromatography (HPLC), scanning electron microscopy (SEM), and confocal microscopy. Variations in Pluronic concentration and molecular weight had an insignificant effect on the internal depot morphologies, however, increasing the concentration and molecular weight did result in increased phase separation rates and, surprisingly, a decrease in the magnitude of the protein burst, though the release profiles still retained a typical burst-type shape. Additionally, increasing the Pluronic concentration beyond a critical point resulted in a transition from a burst-type profile to an extended-release profile. An interpretation of these results in terms of a qualitative model for the protein release mechanism is also given.

Temperature-controlled content release from liposomes encapsulating Pluronic F127

Temperature-dependent internal content release from liposomes was examined using di-oleoylphosphatidylcholine (DOPC)/cholesterol liposomes with encapsulated Pluronic F127 molecules. The interaction of Pluronic F127 with the lipid bilayer at elevated temperature causes the release of encapsulated contents. Content release was measured using fluorescent markers of two different sizes: small, carboxyfluorescein (CF), and large, bovine serum albumin-conjugated fluorescein iso-thiocyanate (BSA-FITC). Release of CF was studied using fluorescence de-quenching, while that of BSA-FITC was studied using fluorescence emission quenching due to fluorescence resonance energy transfer (FRET). Temperature-controlled complete internal content release was achieved at a precise temperature by controlling the concentration of the encapsulated Pluronic. Increasing cholesterol % in the liposome composition resulted in a sharper transition with temperature in content release. The onset temperature of content release increased with decrease in Pluronic concentration. For the same Pluronic concentration, the onset temperature also depended on the size of the encapsulated marker and was higher for larger markers. We have established that onset of content release is determined by the critical micellar temperature (CMT) of the Pluronic. Temperature-sensitive liposomes, made stealth using di-stearoyl(polyethylene glycol 5000) phosphatidylethanolamine (DSPEG5000PE) in conjunction with Pluronic F127, had similar temperature sensitivity and efficiency in content release compared to the non-stealth liposomes.

Synthesis and Self-Assembly of Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide)-g-poly(acrylic acid) Gels

A one-step scalable synthetic procedure results in gels of poly(acrylic acid) (PAA) cross-linked by poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic). The rate of polymerization of acrylic acid in the presence of Pluronic is of 3/2 order in acrylic acid and of 1/2 order in the initiator concentration and is independent of the Pluronic concentration. The formation of cross-linked networks in the synthesis can be attributed to the hydrogen abstraction by Pluronic radicals from the propagating Pluronic−PAA copolymer. The Pluronic−PAA gels exhibit temperature- and pH-dependent swelling in aqueous solutions. Aqueous gel suspensions change the rheological behavior in a certain range of temperatures, which depend on the type of Pluronic used. Intraparticle assembly of poly(propylene oxide) segments can be invoked to explain the temperature-dependent swelling, while interparticle attractive interaction of the dangling chains of Pluronic is responsible for viscosification of the gel suspensions.

Effect of a Polymeric Surfactant on Electron Transport in HL-60 Cells

To assess the effect of a polymeric surfactant, Pluronic P-105 on the activity of electron transport chains in the mitochondria of HL-60 cells, the bioreduction rates of two membrane-localized lipophilic spin probes, 16-doxylstearic acid methyl ester (16-DSME) and 5-doxylstearic acid (5-DS), were studied. In addition, the effect of Pluronic on the bioreduction rate of the DNA-intercalating spin-labeled anthracyclin drug, Ruboxyl (Rb) was evaluated. For 16-DSME, the bioreduction kinetics was zero order with regard to the nitroxide concentration, indicating that the rate was controlled by the concentration of the reducing enzyme(s), which depends on the activity of the electron transport chains. The introduction of Pluronic at concentrations higher than 0.01% resulted in the decrease of the 16-DSME bioreduction rate. The data suggested that short-term cell incubation with Pluronic resulted in reduced activity of the electron transport chains in the mitochondria of HL-60 cells. This was corroborated by the results of an MTT assay. For 5-DS, the bioreduction kinetics was first order in the absence of Pluronic, but did not follow any simple kinetic law after a short-term cell incubation with Pluronic. For Rb, the degree of nitroxide bioreduction dropped progressively with increasing Pluronic concentration. Thus, incubating cells with polymeric surfactants modulates the intracellular energy metabolism, which can affect the rates of energy-dependent intracellular processes.

Factors affecting acoustically triggered release of drugs from polymeric micelles

A custom ultrasonic exposure chamber with real-time fluorescence detection was used to measure acoustically-triggered drug release from Pluronic P-105 micelles under continuous wave (CW) or pulsed ultrasound in the frequency range of 20 to 90 kHz. The measurements were based on the decrease in fluorescence intensity when drug was transferred from the micelle core to the aqueous environment. Two fluorescent drugs were used: doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Pluronic P-105 at various concentrations in aqueous solutions was used as a micelle-forming polymer. Drug release was most efficient at 20-kHz ultrasound and dropped with increasing ultrasonic frequency despite much higher power densities. These data suggest an important role of transient cavitation in drug release. The release of DOX was higher than that of Rb due to stronger interaction and deeper insertion of Rb into the core of the micelles. Drug release was higher at lower Pluronic concentrations, which presumably resulted from higher local drug concentrations in the core of Pluronic micelles when the number of micelles was low. At constant frequency, drug release increased with increasing power density. At constant power density and for pulse duration longer than 0.1 s, peak release under pulsed ultrasound was the same as stationary release under CW ultrasound. Released drug was quickly re-encapsulated between the pulses of ultrasound, which suggests that upon leaving the sonicated volume, the non-extravasated and non-internalized drug would circulate in the encapsulated form, thus preventing unwanted drug interactions with normal tissues.

Salt induced micellization and micelle structures of' PEO/PPO/PEO block copolymers in aqueous solution

Aqueous micellar solutions of two moderately hydrophilic polyethylene oxide/polypropylene oxide/polyethylene oxide (PEO/PPO/PEO) triblock copolymers, pluronics P84 and P104 are examined by small angle neutron scattering (SANS), viscosity and cloud point measurements. Micellar structures of pluronic P84 in aqueous solution are determined as a function of its concentrations (5 and 10 wt%) and added KCl concentrations (0–2 M). 5 wt% solutions of both the block copolymers contain spherical micelles with hydrophobic core of PPO and corona of highly hydrated ethylene oxide subchains. The presence of added neutral salt favors micellization at markedly lower concentration/temperature unlike conventional surfactants. The addition of the salt causes dehydration of ethylene oxide units from hydrated PEO shell from the side of PPO core, leading to an increase in core radius ( R C) of spherical micelles. Micellar volume fraction ( φ) increases with pluronic concentrations. It however increases at lower salt concentrations (up to 0.5 M) but is found to be independent at higher salt concentrations. Aggregation number ( N ag) of P84 in aqueous salt solutions increases from 72 in water to 141 in 2 M KCl. No growth of spherical micelles is observed in unimer-to-micelle transition region and at temperatures below 20–22°C to its cloud point (CP) as hard sphere remains almost constant ( R hs∼70–74 Å) with increasing salt concentration. Micelles remain spherical throughout the KCl concentration range studied.

Oligonucleotide and Water Self-Diffusion in Systems of Pluronic Triblock Copolymers and in Buffer Solutions by Pulsed Field Gradient Nuclear Magnetic Resonance

Pulsed field gradient nuclear magnetic resonance (PFGNMR) measurements of oligonucleotide (poly-thymidine, T1 to T30) self-diffusion coefficients in gels of Pluronic [poly(ethylene oxide) (PEO)−poly(propylene oxide) (PPO)−poly(ethylene oxide) (PEO)] micelles follow the Zimm scaling theory that predicts the diffusion coefficient to vary with solute molecular weight to the −0.5 power. The ratio of the diffusion coefficients in a 20% Pluronic gel-like liquid crystal phase to those in buffer solutions (without Pluronics) was approximately equal to 0.5 for all molecule sizes tested. PFGNMR water self-diffusion coefficient measurements in the gel phase and the solution phase of Pluronic micelles were interpreted within the context of volume averaging theory and known structural features of Pluronic micelles for various Pluronic concentrations. The result of combining the volume averaging theory and measurements of water and oligonucleotide self-diffusion indicates that the geometrical constraints of the Pluroni...

Stabilization and activation of Pluronic micelles for tumor-targeted drug delivery

In order to be used as drug carriers, Pluronic micelles require stabilization to prevent degradation caused by significant dilution accompanying IV injection. This article studies three routes of Pluronic micelle stabilization. The first route was direct radical crosslinking of micelles cores which resulted in micelle stabilization. However, this compromised the drug loading capacity of Pluronic micelles. In the second route, a small concentration of vegetable oil was introduced into diluted Pluronic solutions. This decreased micelle degradation upon dilution while not compromising the drug loading capacity of oil-stabilized micelles. The third route was a novel technique based on polymerization of the temperature-responsive LCST hydrogel in the core of Pluronic micelles. The hydrogel phase was in a swollen state at room temperature, which provided a high drug loading capacity of the system. The hydrogel collapsed at physiological temperatures which locked the core of micelles thus preventing them from fast degradation upon dilution. This new drug delivery system was called Plurogel®. Phase transitions in Plurogel® caused by variations in temperature or concentration were studied by the EPR. The effect of Pluronic concentration in the incubation medium on the intracellular uptake of two anti-cancer drugs was studied. At low Pluronic concentrations, when the drugs were located in the hydrophilic environment, drug uptake was increased, presumably due to the effect of a polymeric surfactant on the permeability of cell membranes. In contrast, when the drugs were encapsulated in the hydrophobic cores of Pluronic micelles, drug uptake by the cells was substantially decreased. This may be advantageous in the prevention of undesired drug interactions with normal cells. Ultrasonication enhanced intracellular drug uptake from dense Pluronic micelles. These findings permitted the formulation of a new concept of a localized drug delivery.

Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion.

Recently, patterned polystyrene surfaces containing hydrophobic (PS) and more hydrophilic (PSox) areas have been shown to be capable of directing cellular growth, which is mainly due to the competitive adsorption of adhesive and antiadhesive molecules. In this article, the competitive adsorption between a pluronic surfactant and fibronectin was studied on homogeneous PS or PSox substrates conditioned with mixtures containing increasing concentrations of one of the two molecules. Radiolabeling and X-ray photoelectron spectroscopy techniques showed that fibronectin adsorption increased on both surfaces if the fibronectin concentrations increased in the conditioning mixture. In contrast, fibronectin adsorption decreased on PSox and did not occur on PS surfaces when pluronic concentrations increased in the coating mixture. A comparison of these data with pheochromocytoma and Schwann cells cultured on patterned surfaces showed that the direction of cell growth on PSox areas depended first on the relative concentrations of the two components in the mixtures, and second, on their ratio; the best concentration ratio probably depends on the cell's ability to recondition its support.

Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: Relationship to cell adhesion

Recently, patterned polystyrene surfaces containing hydrophobic (PS) and more hydrophilic (PSox) areas have been shown to be capable of directing cellular growth, which is mainly due to the competitive adsorption of adhesive and antiadhesive molecules. In this article, the competitive adsorption between a pluronic surfactant and fibronectin was studied on homogeneous PS or PSox substrates conditioned with mixtures containing increasing concentrations of one of the two molecules. Radiolabeling and X-ray photoelectron spectroscopy techniques showed that fibronectin adsorption increased on both surfaces if the fibronectin concentrations increased in the conditioning mixture. In contrast, fibronectin adsorption decreased on PSox and did not occur on PS surfaces when pluronic concentrations increased in the coating mixture. A comparison of these data with pheochromocytoma and Schwann cells cultured on patterned surfaces showed that the direction of cell growth on PSox areas depended first on the relative concentrations of the two components in the mixtures, and second, on their ratio; the best concentration ratio probably depends on the cell's ability to recondition its support. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 45, 404–413, 1999.

Micellar delivery of doxorubicin and its paramagnetic analog, ruboxyl, to HL-60 cells: effect of micelle structure and ultrasound on the intracellular drug uptake

The effect of Pluronic P-105 micelle structure and ultrasound on the uptake of two anthracycline drugs, doxorubicin and its paramagnetic analogue, ruboxyl, by HL-60 cells was investigated. Pluronic micellization was studied over the temperature range of 25–42°C using the EPR and fluorescence spectroscopy. In the presence of Pluronic P-105 at concentrations corresponding to unimers (or loose aggregates), drug uptake by HL-60 cells was enhanced, apparently due to the effect of the polymeric surfactant on cell membrane permeability. At Pluronic concentrations corresponding to the formation of dense micelles with hydrophobic cores, drug uptake was substantially decreased. However, insonation with 70 kHz ultrasound enhanced the intracellular uptake of drugs encapsulated in dense Pluronic micelles. These findings may provide for developing a new technique of drug targeting by encapsulating the drug in micelles to prevent unwanted interactions with healthy cells and focusing ultrasound on a tumor to enhance drug uptake at the tumor site.

Optimizing capillary gel electrophoretic separations of oligonucleotides in liquid crystalline Pluronic F127

Micellar, liquid crystalline solutions of poly(ethylene oxide) (EO)–poly(propylene oxide) (PO)–poly(ethylene oxide) copolymers are alternatives to solutions of random coil polymers as replaceable media for capillary `gel' electrophoresis. Excellent capillary gel electrophoresis separations of oligonucleotides were obtained in the liquid crystalline phase of 18–30% solutions of Pluronic F127 (approximate molecular formula [EO] 106[PO] 70[EO] 106). These solutions are isotropic and flow freely into capillaries at reduced temperatures (0–5°C), but rapidly transform into a gel-like, cubic liquid crystalline phase of spherical micelles at room temperature and above. Separations on Pluronic gels can be tuned because polymer chain packing in the liquid crystalline phase is sensitive to polymer concentration and temperature. Optimum resolution of short oligonucleotides of sizes typically used for DNA sequencing primers or oligonucleotide therapies (≤40 nucleotides) was obtained by capillary gel electrophoresis in 25% Pluronic F127 at 30°C with an applied voltage of 500 V/cm. Decreasing the Pluronic concentration to 20% and increasing the temperature to 50°C substantially reduced analysis time with a modest decrease in resolution. Individual nucleotides in dT 12–18 and dT 19–24 standards were separated in 8 min with baseline resolution. The complex dependencies of resolution and peak shape on oligonucleotide size, Pluronic concentration, temperature, and applied voltage are described.

Antifoaming action of polyoxyethylene-polyoxypropylene-polyoxyethylene-type triblock copolymers on BSA foams

Antifoaming action and mechanism of polyoxyethylene-polyoxypropylene-polyoxyethylene-type triblock copolymers (Pluronics) on aqueous bovine serum albumin (BSA) foams were studied. Addition of Pluronic L-62 or L-61 to 4 gl −1 BSA solution in 4–100 μM concentrations had a strong antifoaming effect which increased with the Pluronic concentration. It was found that, in spite of the general view that antifoams work effectively only in the heterogeneous phase, the Pluronics acted in the homogeneous phase, i.e. when the copolymer was dissolved in the BSA solution. Surface tension measurements showed that Pluronics adsorb at the solution surface and interact with BSA in the surface layer. Foam experiments revealed that the copolymer antifoams acted primarily at the top of foams where the foam lamellae are the thinnest and the probability of film rupture increased with the film drainage time. Single film drainage observations showed that the BSA solution forms stable foams because it forms rigid foam films. Addition of Pluronic L-62 or F-68 made the protein film mobile and the film thinning rate was much faster than without Pluronic. The film drainage rate was independent of the Pluronic type and concentration, but the films were less stable with L-62 than with F-68. A possible explanation for this is that the polyoxyethylene chains of the F-68 are much longer than in the L-62 (or L-61) and thus the steric repulsion between the polyoxyethylene chains of the adsorbed polymer can produce a positive film disjoining pressure only with the F-68.

Stability of Cefazolin in Pluronic F-127 Gels

As a part of studies on the possible application of pluronic F-127 gels for drug delivery systems, an investigation was carried out to characterize cefazolin stability in aqueous pluronic F-127 gels. Pseudo-first-order rate constants for the cefazolin degradation were obtained from different concentrations of pluronic F-127 gels at temperatures of 35°, 45°, and 55°C. Rate measurements were also carried out in the gels composed of different pluronic F-127 concentrations (20%, 25%, and 30% w/v).

Structural transitions in micellar solutions of pluronic P-105 and their effect on the conformation of dissolved cytochrome C: An electron paramagnetic resonance investigation

Multimolecular micelles formed in aqueous solutions by some members of the Pluronic copolymer family have the potential to be used as drug vehicles. Structural transitions in Pluronic P-105 solutions at increasing Pluronic concentrations were monitored by a spin-probe technique. Electron paramagnetic resonance data revealed reversible transitions from Pluronic unimers to small and loose molecular aggregates to dense multimolecular micelles. The transition to multimolecular micelles started at a Pluronic concentration of about 1 wt.% and was completed at the concentration of 10 wt.%, with two populations of micelles coexisting at intermediate concentrations. Conformational changes of the membrane protein Cytochrome C in interaction with Pluronic solutions were investigated by the spinlabeling technique. The rotational correlation time of a spin label located near the surface of Cytochrome C, and the distance between the nitroxyl moiety and the heme iron of Cytochrome C depended on Pluronic concentration indicating protein conformational changes in Pluronic solutions.

Interactions between animal cells and gas bubbles: The influence of serum and pluronic F68 on the physical properties of the bubble surface

We describe a method by which the degree of bubble saturation can be determined by measuring the velocity of single bubbles at different heights from the bubble source in pure water containing increasing concentrations of surfactants. The highest rising velocities were measured in pure water. Addition of surfactants caused a concentration-dependent and height-dependent decrease in bubble velocity; thus, bubbles are covered with surfactants as they rise, and the distance traveled until saturation is reached decreases with increased concentration of surfactant. Pluronic F68 is a potent effector of bubble saturation, 500 times more active than serum. At Pluronic F68 concentrations of 0.1% (w/v), bubbles are saturated essentially at their source. The effect of bubble saturation on the interactions between animal cells and gas bubbles was investigated by using light microscopy and a micromanipulator. In the absence of surfactants, bubbles had a killing effect on cells; hybridoma cells and Chinese hamster ovary (CHO) cells were ruptured when coming into contact with a bubble. Bubbles only partially covered by surfactants adsorbed the cells. The adsorbed cells were not damaged and they also could survive subsequent detachment. Saturated bubbles, on the other hand, did not show any interactions with cells. It is concluded that the protective effect of serum and Pluronic F68 in sparged cultivation systems is based on covering the medium-bubble interface with surfaceactive components and that cell death occurs either after contact of cells with an uncovered bubble or by adsorption of cells through partially saturated bubbles and subsequent transport of cells into the foam region. (c) 1994 John Wiley & Sons, Inc.

Biomaterials for digestive surgery

Dispersions of insulin poly(isobutylcyanoacrylate) nanoparticles were obtained by anionic in situ polymerization using aqueous pluronic acid solution. Results showed a decrease in particle size diameter by increasing the pluronic acid concentration. Nanoparticles prepared in the presence of 2.5% pluronic acid resulted in particles of 85 nm average diameter and 59% intra-particular insulin load without the use of the oily core [Damge, C., Michel, M., Aprahamian, M., Couveur, P., 1988. New approach for oral administration with polycyanoacrylate nanocapsules as drug carrier. Diabetes 37, 246–251]. In vivo testing was performed on streptozocin induced diabetic rats. The subcutaneous injection of insulin nanoparticles was able to prolong its duration of hypoglycemic effect from 6 to 72 h. Effective oral absorption of the entrapped insulin was significantly better (p < 0.01) when compared with non-encapsulated insulin or the control experiments.