Pluronic block copolymer micelles: structure and dynamics

Transcription

1 11/9/2013 A review on Pluronic block copolymer micelles: structure and dynamics 1. Introduction By Mohammad Atif Faiz Afzal Poly-ethylene oxide and poly-propylene oxide tri-block copolymers, commercially known as Pluronics or Poloxamers have been studied extensively due to its wide applications in drug delivery, synthesis of mesoporous materials, emulsification, lubrication, foaming, detergency, cleaning and dispersion stabilization. Few of the Pluronic structures are also approved by US Food and Drug Administration (FDA) as injectable materials for human body and therefore Pluronics have been extensively studied as drug delivery systems in the last decade. Pluronics play a vital role in cancer therapy by carrying and delivering anti-cancer drugs to specified targets including lung, breast, ovarian, gastric and oesophagus tumors. Pluronics are amphiphilic copolymers containing hydrophilic and hydrophobic blocks arranged in a triblock structure. The hydrophilic and hydrophobic blocks consist of poly-ethylene oxide (PEO) and polypropylene oxide (PPO) respectively and the number of these hydrophilic and hydrophobic units in the polymeric chain characterizes the properties of Pluronics. Due to the amphiphillic properties, Pluronics are known to self-assemble into micelles in aqueous solution above a critical polymer concentration called critical micelle concentration (cmc). The study on the micellization of Pluronic block copolymers started in the early 1990 s. The primary problem was to understand the structure and the dynamics of micelle formation. A productive insight on the micellization process was obtained when Alexandridis et al explained the process on the basis of thermodynamics [1]. They discovered that the process of micellization is entropy driven and this is considered as a major breakthrough in the field of Pluronics. During the same time Linse et al explained the process of micellization on the basis of mean field lattice theory [2]. The above mentioned authors also studied cmc, critical micellization temperature (cmt), the aggregation number and the hydrodynamic radius of various Pluronic block copolymers in aqueous solutions. These findings lay the ground work for further research on Pluronic block copolymer micelles. Later, extensive research has been performed to study the phase behavior of these micelles. In early 2000 s, vast efforts were invested in trying to manipulate the structure, cmc and cmt of Pluronic block copolymer micelles by changing the solvent composition or by adding some additives. For example, the effect of urea [3], effect of using mixed aqueous solutions [4-6], non-aqueous polar solvents[4], organic solvents[7] and the effect of addition of different salts[8], ionic and non-ionic surfactants[9, 10] on the structure of the micelles was explored. Subsequently, molecular simulations were conducted by other scientists to further understand the structural properties and interactions in micellar solutions[11-13] [14]. In this report, a comprehensive review of the structure and dynamics of Pluronic micelles is presented. The fundamentals of Pluronics synthesis, thermodynamics of micelle formation, molecular 1

2 structure and solvent quality effects on the micelle structure and dynamics have been reviewed. Various applications of Pluronic micelles are also reported, with drug delivery being the main focus. 2. Pluronic Micelles 2.1 Chemistry of Pluronics Pluronics (also known poloxamers) are commercially available and can be obtained in a range of molecular weights and PPO/PEO ratios. Pluronics are synthesized by anionic polymerization of alkyl oxide in the presence of an alkaline catalyst, generally sodium or potassium hydroxide. Anionic polymerization was first discovered in 1956 by Szwarc and since then it has been used to synthesize a variety of block copolymers [15]. In case of Pluronics, the polymerization is proceeded by first adding propylene oxide and then subsequently adding ethylene oxide to low molecular weight propylene glycol (molecular weight less than 750). After the reaction, the catalyst is neutralized and removed. Figure 1 shows the sequence of reactions producing PEO-PPO-PEO triblock copolymer [16]. Pluronics with varying molecular weight can be produced using this method. Figure 1:- sequence of reactions showing the formation PEO-PPO-PEO triblock copolymer Pluronic block copolymers exist as unimers in the aqueous medium below CMC and above CMC these unimers aggregate to form micelles in a very narrow concentration range and are in equilibrium with the unassociated unimers. Pluronic copolymers form micelles at much lower concentration when compared to low molecular weight surfactants. Micelles formed from Pluronics consist of a swollen core of lyophobic PPO blocks and flexible corona of lyophilic PEO blocks. Micellization of Pluronics is characterized by CMC, critical micelle temperature (CMT), molecular weight of the micelle and the aggregation/association number. Micelles are also characterized based on their size and shape which includes the radius of gyration (R g), radius of core (R c), thickness of corona (L), hydrodynamic radius (R h) and the ratio R g/r h [17]. The structure of micelles can be determined by using various characterization techniques depending on the characteristics we are looking for. Table 1 shows the listings of different techniques used for different micelle characteristics [18]. 2

3 Table 1:- Experimental techniques for micelle characterization [18] Techniques TEM (Transmission Electron Microscope) SANS (Small Angle Neutron Scattering) and SAXS (Small Angle X-Ray Scattering) SLS (Static Light Scattering) DLS (Dynamic Light Scattering) SEC (Size Exclusion Chromatography) Ultracentrifgation Fluorescence techniques NMR (Nuclear Magnetic Resonance) Viscometry Stop flow techniques Micelle Characteristics Shape, size Molecular weight (weight average), R g, Rcore, macrolattice structures, Molecular weight (weight average), R g R h R h, dynamics of micellar equilibrium Micelle density, molecular weight (Z average), micelle/unimer weight ratio Chain dynamics, CMC, hybridization of micelles Chain dynamics R h, intrinsic viscosity Kinetics of micelle formation and dissociation As Pluronics can exist in a large range of molecular weight and hydrophilic lipophilic balance (HLB), a nomenclature is given to clearly differentiate different Pluronics. The notation of Pluronics starts with a letter and followed by two or three digit number. The letter indicates the phase of Pluronic which includes either P (for paste), L (for liquid) or F (for flakes), whereas the last digit signifies the percentage of ethylene oxide and the first one or two digits of the number when multiplied by 300 gives the approximate molecular weight of PPO block (Vaughn, T.H.,1951). For example, Pluronic P105 represents a Pluronic in the paste form with 40% ethylene oxide and molecular weight of PPO block being on the order of Table 2 shows the commercially available Pluronics provided by BASF along with their properties [19]. It can be seen that a very wide distribution of molecular weight from 1630 to is available. Table 2:- Properties of Pluronic PEO-PPO-PEO copolymers [19]. A: copolymer. B: average molecular weight. C: PEO wt.%. D: melting pour point ( O C). E: viscosity (Brookfield) (cps; liquids at 25 O C, pastes at 60 O C, solids at 77 O C). F: surface tension at 0.1%, 25 O C (dyn cm -1 ). G: foam height (mm)(rossmiles, 0.1% at 50 O C). H: cloud point in aqueous 1% solution (:C). l: HLB (hydrophilic-lipophilic balance). A B C D E F G H I L F >100 >24 L L L L L L P F >100 >24 L P

4 F >100 >24 P P F >100 >24 F >100 >24 F >100 >24 P P PI F >100 >24 L P F > Thermodynamics of Micelle formation Thermodynamics of micelle formation has been well studied by Alexandridis et al [1, 4, 19, 20]. Micellization of Pluronics in aqueous solutions is essentially entropy driven and obeys closed association model. The closed association model assumes that there is equilibrium between aggregated structure (micelle) and the dispersed monomers. The thermodynamic analysis of micelle formation can be explained by two different models: the phase separation model and the mass-action model. According to the phase separation model, micelles are formed at CMC which are considered as a new phase and assumes that the concentration of monomers to be equal to CMC. Mass-action model considers that the monomers and micelles are in association-dissociation equilibrium. Moroi [21] has evaluated the relationships between thermodynamic entities and the CMC based on mass action model. He obtained the following equations for Gibbs free energy of micellization, ΔG m and enthalpy of micellization, ΔH m as ΔG m = (1 + m N ) RT ln(x cmc) + RT ln(2n(n + m)) (1) N ΔH m = RT 2 [(1 + m N ) ( ln(x cmc) ) T P + ln(x cmc ) ( (m N ) T ) + { [(1 N P ) ln{2n(n + m)}] } ] (2) T where T is the absolute temperature of the experiment, R is the gas constant, m is the no. of counter ions bound to the micelle, N is the aggregation number and XCMC is the CMC expressed in mole fraction units. The second term in equation 6 and the third term in the equation 7 have very less weightage and can be neglected [21, 22] and written as follows ΔG m = (1 + m N ) RT ln(x cmc) (3) P 4

5 ΔH m = RT 2 [(1 + m N ) ( ln(x cmc) ) T P + ln(x cmc ) ( (m N ) T ) ] (4) P In case of non-ionic surfactants like Pluronics, the equations for ΔG m and ΔH m are given as ΔG m = RT ln(x cmc ) (5) ΔH m = RT 2 ( ln(x cmc) ) T P The relationship between the CMC and T can be obtained by fitting to the equation used by Kresheck[23] which is given as (6) ln(x cmc ) = a + b + c ln T (7) T m b = a + + c ln T (8) N T Where a,b, c, a,b and c are constants that will be determined by fitting the experimental data. Using the equation 12 and 13, ΔH m can be evaluated as ΔH m = RT 2 [(1 + m N ) ( b T 2 + c b ) + ( T T 2 + c T ) ln X cmc] (9) The change in entropy of micellization S m will be obtained based on the following equation S m = ΔH m ΔG m T (10) In the case of block copolymers, it has been shown that the enthalpy of micellization is depended on critical micellization temperature given by H=R[ ln(x)/ (1/T CMT)] (11) where T CMT is the critical micelle temperature, and X is the concentration expressed in mole fraction [24]. When the unimers of Pluronic system form aggregates, the hydrogen bonding structure in water is restored and the entropy of water increases outweighing the entropy loss due to localization of monomers. The contribution of entropy plays a major role in micellization process in aqueous solution with enthalpy playing the minor role [1]. The formation of micelles depends on the aqueous solubility of the PPO and PEO segments, which is in turn dependent on the temperature. As the hydrophobic effect increases with temperature, there is a high tendency to form micelles at higher temperatures. As the temperature increases above CMT the solubility of PEO group decreases and becomes more hydrophobic because of the conformational changes in EO segments and at a certain temperature PEO groups aggregate giving rise to phase separation, which is referred to as cloud point [25, 26]. 5

6 The experimental results using the isothermal titration calorimeter shows that the heat of micellization decreases monotonically with an increase in temperature [26]. In contrast, some studies have shown that the heat of micellization monotonically increases with increase in temperature. Very recently, it was shown that the heat of micellization increases with temperature to reach a maximum and then decrease along with an increase in temperature. Figure 2 shows the variation of enthalpy of micellization of F68 and P65 with temperature [26]. Some studies also show the thermodynamics of Pluronic micelle in presence of salts [3, 27], surfactants [28, 29], mixed micelle solution [9] and in different solvents [4, 30, 31]. Figure 2:- Variation of the heat of micellization of the Pluronics F108 (filled circle), F98 (circle), F88 (filled inverted triangle), and F68 (triangle) as a function of Tm [26] 3. Pluronic Micelle structure 3.1 Shape and Size The structure of micelles plays an important role in many applications, for example in the synthesis of mesoporous materials the structure of micelles determines the pore structure in the material. The size of micelles in Pluronic aqueous solution is of the order nm and therefore the scattering techniques that are appropriate to use is SANS and SAXS [32]. As the electron densities of PEO and PPO blocks is similar to that of water, it is difficult to characterize using SAXS and thus SANS is best technique to characterize micellar structure of Pluronics in aqueous solutions. The primary parameters that are used to study the structure of Pluronic micelles are the aggregation number of micelle, hard sphere volume fraction, the core radius and the stickiness factor between micelles [33]. As an example, at 50 O C Pluronic P85 micelles have a core radius of about 40 A O and contains 40% water while the corona region extends from A O with less than 10% polymer fraction [34]. Theoretical predictions about the structure of the micelles were presented by Linse and Hatton based on the mean field lattice theory [2, 19]. According to this theory, the core was assumed to be filled 6

7 with lyophobic block and the outer layer (corona) filled exclusively with lyophilic block. The theory also assumed that the segment densities is constant in different regions and no solvent penetration to the core was allowed. Using this theory and minimizing the free energy various micelle properties like micellar size, aggregation number, CMC etc., were determined. These models give the scaling relations for aggregation number and micellar size. Two other models were developed later to study the structure of micelles: the hard sphere model and the cap-and-gown model [35, 36]. The hard sphere model was given by Mortenson et al. for the P85 micellar system and is based on the core-shell structure and a hard sphere inter-micellar interaction [35]. The cap-and-gown model is given by Liu et al. and is based on a compact and diffused corona structure [36]. A considerable amount of polydispersity is considered in the hard-sphere model to fit the scattering intensity distribution, whereas no polydispersity is required in a cap-and-gown model. 3.2 Cylindrical or Ellipsoidal structure Some studies have shown that the micelles are not spherical in shape but are prolated ellipsoidal shape. Theoretical studies on L64 studies also suggest that L64 micelles prefer a prolate ellipsoidal structure [12]. It has also been shown using SANS study that L64 micelles can be best described by ellipsoidal structures and the aspect ratios are dependent on the temperature[37]. As the temperature increases, the aspect ratio increase progressively leading to an anisotropic growth of micelles and results in a worm-like or cylindrical structures [38-40]. At higher temperatures, the association number increases leading to an increase in the micelle core radius and when the core radius exceeds the stretched length of PEO block, the micelles tend to change the shape to prolate ellipsoid. The anisotropic growth of micelles at higher temperatures (near cloud point) is responsible for increased viscosity of the copolymer solution. Due to this ellipsoidal structure of micelles, the interaction between micelles is predominantly repulsive. The change from spherical to ellipsoidal structure leads to a minimization of the interaction energy at shorter distances by alignment of the structures along the majority axis. A study on P85 shows that the structure of micelles change from spherical to prolate ellipsoid at O C temperature range[41]. At higher temperature and concentration, the combination of the two effects: structure change from spherical to prolate ellipsoid and increased hydrophobicity of PEO blocks, leads to the formation of rod like structures with hexagonal symmetry. Further increase in temperature leads to the formation of ordered lamellar structure. The change in structure of micelles from spherical to cylindrical structures can also be induced by addition of other inorganic materials [42, 43]. 3.3 Modeling and simulations An insight to the structure of micelles and micelle-micelle interactions have also been given from molecular simulations. Considering the large size of Pluronic micelles and slow kinetics of formation it is difficult to perform molecular dynamic or Monte Carlo simulations to study the structure of micelles. Therefore, modeling of Pluronic solutions is done based on self-consistent field lattice models or mean field density functional theory approaches [2]. These models assume an ideal Gaussian chain representation of Pluronic chains. Though these models give an insight on the morphology and phase, it 7

8 is difficult to incorporate some important phenomenon like hydrogen bonding structure, solvent clustering, local conformations etc. Bedrov et al have developed a coarse grained, implicit solvent (CGIS) model for Pluronic chains, which gives a more detailed structural and conformational correlations of PEO and PPO chains in aqueous solutions [13]. They have reported the results of this model on L64 Pluronics showing the inter-micellar interactions, intra-micellar structure and the structure of the micellar solutions with varying concentrations. Figure 3 shows the representative model of Pluronic L64 and F127 in CGIS model [13]. Several studies have also been performed using Monte Carlo simulations to analyze the interactions between Pluronic micelles [34, 41]. In these models, the micelles are treated as either solid spheres or hard spheres with tethered Gaussian chains. These analysis shows that the micelles are fairly independent of concentration and temperature except with a slight polydispersity in aggregation number. Yuan et al. have studied P103 structure using a dynamic variant of mean-field density functional theory [44]. They have reported that the structure of self-assembled aggregates highly depends on the polymer concentration, which are in agreement with experimental results. Polymer concentration of 10-15% result in spherical aggregates with large PPO core, while concentrations above 16% shown formation of disc like micelles. Figure 3:- Representative configurations of L64 and F127 micelles in the CGIS model [13]. 3.4 Molecular structure effects on micelle structure The structure of the micelle depends on many factors like the molecular weight of polymer chains, PEO/PPO ratio, temperature, etc. Results have shown a strong dependence of temperature on CMC of Pluronic micelles. However, at constant temperature, the CMC can be varied by varying polymer length, changing EO %, by changing from ABA to BAB type tri-block copolymers or by changing to AB di-block copolymers. Increasing the polymer length or decreasing EO % decreases the CMC at constant temperature and accordingly reduces CMT at constant concentration [2, 45]. Increasing the PPO chain length leads to an increase in micelle core radius and more dehydrated inner layers of micelle [46]. Onset of micellization for diblock copolymers is initiated at lower concentration values when compared to the diblock copolymers. Comparison of PPO-PEO-PPO bblock copolymers with PEO-PPO-PEO block copolymers showed that there is no micellization in PPO-PEO-PPO block copolymers observed up to the cloud point [1]. The size of the micelle structure also depends on the segment length of copolymers. For 8

9 example, the large segment copolymers form star-like blob structure which is not seen in small segment copolymers. 3.5 Solvent quality effects Temperature effects on micelle structure Results have shown a strong dependence of temperature on CMC of Pluronic micelles. It has been shown that the aggregation number and surface stickiness increases with temperature and the core become more dehydrated at higher temperatures [47, 48]. Also, as the temperature increases the hydrodynamic radius of micelle remains constant even though the aggregation number increases. This effect is attributed to the dehydration of PEO blocks at higher temperatures [49]. Figure 4 shows a phase phase diagram of Pluronic P85 and P123 clearly showing dependence of micelle structure on the polymer concentration and temperature [50]. As one approaches the cloud point the aggregation number increases, the core becomes more dehydrated and the stickiness factor increases. Extensive studies have been performed on the effect of pressure, ph, addition of ionic and non-ionic agents, addition of organic agents on the size and shape of the Pluronic micelles [add references]. More information on these effects is presented in the following sections. Recently, a model has been developed by Manet et.al which determines how the preparation parameters influence on the structure of micelles [42] Pressure effect Figure 4:- Phase diagrams for P85 (left) and P123 (right) [50]. The effect of pressure has been studied on various amphiphilic surfactant micelles. Few of the studies reveal that the radius of gyration decreases with increase in pressure whereas the core radius increase with increase in pressure. The effect of pressure on Pluronic F88 has been studied by Mortensen et al. which show that the increasing pressure results in melting of micellar crystals and decomposition of micelles [51]. A detailed study on the pressure-temperature phase behavior of Pluronic F108 at high concentration is given by Kostko et al [52]. Figure 5 shows the schematic of phase diagram for F108 Pluronic at high concentration of polymer [52]. It can be seen that the range of temperature spanning the gel phase decreases with increase in pressure. Also the pressure at CMC line increases with increase in temperature and a similar increase is seen at low temperature gelation boundary whereas the high temperature gel-melting phase boundary steeply decreases with temperature. This variation in phase of 9

10 micellar solution is attributed to the increased solubility of PPO blocks at higher pressure. Also, is can be seen that the cloud point is not affected with an increase in pressure. Studies on Pluronic P85 also show the similar behavior i.e. the phase transition temperature increase with increase in pressure [53]. An exception in study was that a new phase corresponding to de-mixed lamellae is observed in Pluronic P85 at high temperature and the onset temperature of this new phase decrease with increasing pressure. Figure 5:- Schematic of pressure-temperature phase behavior of Pluronic F108[52] Effect of non-aqueous and mixed solvents Extensive research has been done in the past on the effect of mixed aqueous solvents and nonaqueous solvents on the structure of micelles in Pluronic solutions. Alexandridis et al. studied the effect of non-aqueous polar solvent (formadide) on the Pluronic micelle systems for the first time [4]. In this system, the micelle radii and aggregation number of Pluronic P105 increase with increase in temperature and also the solvent quantity in the micelle core decreased with temperature increase till 15 O C. Pluronic systems form reverse micelles in most of the organic solvents [7, 54]. Guo et al. have reported the study on water-induced reverse micelle formation of Pluronic system in p-xylene [7]. Pluronic micelle structure in other non-aqueous solvents like ethylammonium nitrate, o-xylene and mixed water and other solvents like hexanol, hexylamine, formadide, urea, ethanol and glycerol have also been studied [5, 7, 43, 45, 54-56] Effect of salt It is well known from the past studies that addition of salts have a considerable change in the structure of micelles. Bahudur et al have extensively studied the Pluronic micelle structures in presence of added salts [8, 56-58]. Most of the studies have shown that the water-structure- making salts like NaCl decrease the transition temperature from spherical to rod-like structure in Pluronic micelles [59]. Addition of salts in the Pluronic solutions results in the onset of micellization process at a lower polymer concentration (at constant temperature) or at lower CMT (at constant concentration) [8, 27, 57]. The presence of salts leads to dehydration of PEO near the micelle core increasing the radius of the core as the dehydrated PEO now becomes the part of micelle core. Addition of KCl to Pluronic P84 and P104 has shown to increase in aggregation number, however the micelle radius remains constant [8]. The effect of salt addition to the structure of micelles in Pluronic solution in most of the studies is found to be analogous to the temperature effect [57]. Micellar volume fraction increases with Pluronic concentration in the presence of low salt concentration, but is independent at higher salt concentrations. The presence of salts 10

11 also reduce the gel-formation and cloud points to lower values with an exception of KCNS which increased the cloud point in several Pluronic solutions [37, 60]. This effect is observed to be more pronounced by addition of PO 4 salt followed by HaH 2PO 4, NaCl and NaBr in the decreasing order [60]. It was reported that Pluronic P85 forms two types of micelles: monomolecular micelles and polymolecular micelles. Addition of salts (NaCl and NaBr) to P85 solution resulted in the increase of polymolecular micelles while decreasing the size of these polymolecular micelles. Therefore, salts play a vital role in tuning the structure of micelles in Pluronic solutions. 4. Micelle dynamics 4.1 Relaxation processes Dynamics of micelles of conventional surfactants has been investigated long back by Aniansson et al. using chemical relaxation methods [61]. It was shown that the free surfactants constantly exchange with the surfactants in the micelles i.e., there exists a dynamic equilibrium between micelles and free surfactants and the average lifetime of the surfactant in micelle is given by T R. Apart from this surfactant, micelle equilibrium the micelles constantly form and break with a lifetime of T M. When micelles are subject to a rapid change in temperature the micellar systems respond with two relaxation times corresponding to the exchange of surfactants and micelle formation/breakup respectively. The latter process is a slow process whereas the exchange process is very fast. Based on this theory of surfactant micelles, Alexander et.al showed that block copolymer micelles dynamics is also associated with two relaxation times [62]. However, the free copolymers exchange with micelles can occur either by micelle collision where the copolymer jump from one micelle to another or by copolymer exiting one micelle and combing with another micelle [63]. Also, the exchange process in block copolymer micelles is extremely slow when compared to the surfactant micelles [63]. The disentanglement of the copolymer chains from a tangled and complex micelle structure result in slowing the relaxation process. Various characterization techniques like ultrasonic relaxation, gel chromatography, temperature jump, rheological studies and pulsed field gradient NMR are used to study the dynamics of micelles [64-67]. Zana et al. have studied the dynamics of Pluronic micelles( PF80 and L64) in aqueous solution [68]. The studies revealed two relaxation processes as similar to that of the surfactant micelles. The fast relaxation (in microsecond range) was associated with exchange process while the slow relaxation ( in millisecond range) was associated with micelle formation/break up process. The studies also shows that the association of micelles is diffusion controlled and the micelle formation/break up process is proceeded via fission/fusion reactions. The exchange process of free copolymers with micelles and the micelle formation/breakup plays a vital role many applications like formulation of lubricants, solubilization and dispersion polymerization. The lifetime T M of the micelles indeed determines the rate at which the micellar system solubilize and also determines the effectiveness in detergency. Apart from the two relaxation times, a third relaxation time was also observed at temperatures close to cloud point [64]. This third relaxation time is related to the clustering of micelles which is the initial step in phase separation. Kositza et al. first observed this relaxation time in L64 Pluronics near cloud 11

12 point [69, 70]. At higher temperatures, the micelles are fairly large and the concentration of free copolymers is low which is attested by the disappearance of first relaxation process at higher temperatures [1]. Therefore, the third relaxation process near cloud point is due to the clustering of micelles and these clusters phase separate at cloud point. The third relaxation time stays constant till the first relaxation process completely disappears. Figure 6 shows the variation of three relaxation times with temperature for various polymer concentration for L64 Pluronic system [69]. It can be seen that the first relaxation almost vanishes at higher temperatures while the third relaxation time is fairly constant for a long range of temperature and then increase significantly at higher temperatures. The delay in the relaxation time for the clustering process is attributed to the decrease in the number density of the particles, as all the free copolymers are exhausted and also due to the increased particle size [64]. Figure 6:-Relaxation lifetimes τ 1 (A), τ 2 (B), and τ 3 (C) obtained from ILTJ experiments with light scattering detection at 0.625% (O), 1.25% ( ), 2.5% (Δ), and 5% ( ) L64 (w/v) in aqueous solution as a function of temperature [64]. 4.2 Dynamics discerned from rheology measurements Rheological properties of Pluronics near the percolation threshold change abruptly exhibiting a change from simple liquid phase to a gel like elastic phase. The phase below percolation is characterized by a low viscosity values while the phase above the threshold has very high viscosity values [71]. Calculations based on sticky sphere model of Baxter have showed that the Pluronic micellar systems have well defined percolation threshold [72]. This model accurately describes the coexistence, spinodal and percolations lines for the micellar systems. Inset of figure 7 shows the Baxter s phase diagram as a function of the reduced temperature and reduced volume fraction [71]. Mallamace et al. have performed SANS study at various concentrations and temperature for L64 Pluronic system and also investigated the viscoelasticity near the percolations threshold and relate the results to the theoretical calculations [71]. They observed an abrupt increase in stickiness factor, in agreement with the Baxter s model, showing that the Pluronic systems have well defined percolation threshold. Figure 7 shows the phase diagram for L64 Pluronic, in which a well-defined percolation line can be seen in accordance with Baxter s model (inset of figure 7). With these experimental and theoretical results on L64 Pluronic system, L64 is proposed as a model system to study percolation and related dynamics of other Pluronic systems. 12

13 Figure 7:- The phase diagram, including the cmc cmt curve, the cloud point curve, the critical concentration (C c 0.05(wt%)) and the critical temperature of the system (T c 57.3 C), of the system L64 D 2O determined, by means of light scattering measurements. In the inset is reproduced the Baxter's phase diagram [71]. The percolation transition in complex fluids is generally difficult to characterize due the underlying viscoelastic nature of these complex systems. The onset of sol-gel transition occurs at the point where the elastic modulus and the viscous modulus scale identically with frequency [73]. Prior to gelation the viscous modulus dominates the entire frequency domain, whereas post gelation the elastic modulus dominates at low frequencies [74]. At higher frequencies, the static structure of gel breaks down due to the high frequency glassy dynamics. Zanten et al. showed that the diffusive wave spectroscopy (DWS)- based tracer particle micro rheology is a useful way to study the dynamics of Pluronic L64 systems, which is considered as model adhesive hard sphere (AHS) system [74]. The results showed that at higher temperatures the dynamics is dominated by the attractive intermicellar potential. The tracer microparticle rheology is more influenced by the local micelle dynamics in the near sphere region rather than the bulk mechanical properties in contrast to traditional rheometry measurements. Very recently, Zanten et al. performed DWS studies on Pluronic F108, which are considered as spherical soft spheres. The results showed dehydration of micelle corona with increase in temperature [75]. They showed that at higher temperatures the dynamics is dominated by soft repulsive intermicellar interactions. 4.3 Modelling and simulation Pluronic micellar systems are highly dynamical entities with the copolymers constantly exchanging with micelles and therefore the micellar crystal are considerably intricate structures with stable long range orders. Theoretical models like density functional model and mean field theory have been developed long back which directly study the ordered micelles [76, 77]. These theories provide a 13

14 detailed study about the structure of micelles, however they do not provide insight into the micellar dynamics. Vlimmeren et al. developed 3D mesoscale model to study the dynamic behavior of complex polymer solutions [77]. They applied these models to study the dynamic behavior of L64 and 25R4 Pluronic systems in aqueous solutions. This model is based on the mean field density functional theory for Gaussian chains. The dynamic mean-field functional model is a combination of Gaussian mean-field statistics with a coarse grained Ginzburg-Landau model for time evolution of conserved order parameters. The model study on L64 Pluronic systems showed formation of four different phases depending on the polymer concentration, which are in good agreement with the experimental results. The four phases L64 can exist are micellar, hexagonal, bicontinous and lamellar phase. The simulations showed the time scale of phase separation in the order of milli seconds. However, the final structure after phase separation contain high degree of structure defects, which is attributed to the short equilibration times. The simulations confirm the experimental studies that the formation of lamellar phase is independent of the block sequence. The studies also show that the kinetics of phase change is initiated with fast local aggregation and followed by slow rearrangement by overcoming defects in the structure. Xu et al. developed another model (MesoDyn) that is also based on dynamic density functional method to simulate phase separation kinetics of Pluronic systems [78, 79]. Their simulation results showed that as the temperature increases there is a decrease in interfacial energy, the Pluronic systems become more ordered and the overall phase separation process slows down. All the models developed based on density functional or mean field theory study the ordered micelles but do not provide good insight into dynamical degrees of freedom of the polymeric micellar systems [80]. A more realistic description of the dynamics of such systems can be given using molecular dynamics (MD). Molecular dynamics allows to define the role of a single polymer towards the dynamics of polymeric solutions. Another advantage of molecular dynamics over other theories is there is no need to define or assume thermodynamic state of the system in MD simulation. However, the longer time scales in the order of minutes to hours makes it difficult to perform MD simulations on Pluronic micellar systems. Travesset et al. studied the dynamics and equilibrium properties of Pluronic systems using MD simulations [80]. The results show that the formation of crystal and the equilibration occur polymer exchange between micelles. Pluronic system showed a bcc lattice near the disordered transition. For Pluronics with short hydrophilic blocks and at lower kinetic temperatures, fcc lattice was observed. MD simulations can be extended to understand the dynamics of many polymer nano-composites with development of more accurate models. 4.4 Solvent quality effects on dynamics Considerable amount of efforts have been given to understand the dynamics of Pluronic systems in aqueous solutions. However, very less literature is available on the study of dynamics of Pluronic micelles in non-aqueous solutions. The effect of solvent quality on the dynamics of other block copolymers has been studied by Honda et al [81]. They studied the dynamics of poly(a-methyl styrene)-blockpoly(vinylphenethya1l cohol) (PaMSb-PVPA) in benzyl alcohol. They observed that micellization is a step wise process including a fast and slow process, similar to what is observed in aqueous Pluronic systems. The fast process is related to the association of free polymers to form quasi-equilibrium micelles and the 14

15 slow process relating to the equilibration of micelles and gradual decrease in micelle number. Schlick et al. studied the dynamics of Pluronic systems in water/o-xylene mixtures using electron spin resonance spectroscopy and nitroxide spin probes [6]. They determined the local polarities in L64 reverse micelles with an emphasis in polar core with varying water concentrations. Kositza et al. studied the effect of addition of sodium dodecyl sulphate (SDS) on the dynamics of the Pluronic L64 aqueous solutions [70]. They showed the change in all the three relaxation times (τ 1, τ 2, and τ 3) with the SDS addition. It is known that the SDS addition lowers the CMT and smaller micelles with loosen structure are formed. Therefore, SDS addition caused an increase in 1/τ 1 because of the reduced energy barrier for exchange of free polymer. In contrast, SDS addition caused a decrease in 1/τ 2 and 1/τ 3. The decrease in 1/τ 2 showed an evidence for a shift from fission/fusion to a stepwise mechanism, which can be reversed by addition of NaCl. It is well known that the addition of salt to the Pluronic solution have effect on the shape of the micelles. Ganguly et al showed that the addition of salt leads to formation of rod like structures [82]. They observed that this transformation of micelles from sphere to rod is time dependent and have a strong dependence on the type of anion used as well as the copolymer composition. When the anion type was changed to more water structure making anions i.e along the Hofmeister series in the order Cl - <F - <(PO4) 3-, the rate of growth increased significantly. This dependence is due the ability of the anions to dehydrate the outer covering of the micelles, which is a major factor for the transformation from sphere to rod like structure. The molecular weight of the polymer also plays a vital role in the growth rate of the rod like structures. It was observed that the increase in copolymer molecular weight leads to a decrease in the growth rate, which is attributed to the faster restructuring ability of the low molecular weight copolymers. The growth rate to rod like structures also depends on the type of solvent used. In the presence of solvents like ethanol which have affinity towards both PPO and PEO blocks, the copolymers can restructure very quickly leading to an increased growth rate towards rod like structures. 5. Bio Applications of Pluronics 5.1 Un-modified Pluronics Nanotechnology based on polymers has become one of the fast growing and attractive fields due to its wide applications in nanomedicine. Different nanoscale systems like liposomes, micelles, nanogels, polyplexes and other nano-materials are being used in nanomedicine. Pluronics are one such example to be used in nanomedine which is a promising material for drug delivery, gene delivery and for imaging. Immense research is being done on Pluronics to ensure safe and efficient delivery of drugs and genes. Pluronic micelles have a hydrophobic core and hence act as carriers for in-soluble compounds and this transfer of compounds to the micelle core is called solubilization. Due to this solubilization property of Pluronic micelles, they have been extensively studied as potential hydrophobic drug carriers. Immense research has been undertaken in the past to understand the interaction between the Pluronic micelles and various drugs [83-85]. Controlling the release of drugs from these micelle cores is one of the major challenges recently, and therefore vast research is being done to obtain controlled drug release [58, 86-88]. Drug release is followed by the disruption of the micelle structure; therefore the focus is on 15

16 understanding the dynamics of disruption and breakage of micelle structure for controlled drug release [42, 86, 89, 90]. The size of micellar core formed from Pluronic polymers is in the range of 4-20 nm. Therefore, Pluronic micelle core can act as carrier for hydrophobic nano-particles, low molecular mass drugs, proteins and genes in aqueous solutions. Encapsulation of hydrophobic drugs is of vital importance in cancer therapy and Pluronics are promising materials for encapsulating and solubilizing hydrophobic anti-cancer drugs [91]. Various hydrophobic anticancer drugs like doxorubicin have been successfully encapsulated in the core of Pluronic micelles. Drug encapsulated micelle not only increase the solubility of drugs but also increase passive targeting of the tumor [92, 93]. 5.2 Interaction of drugs with Pluronic micelles Puronics micelles have good stability in blood and have high drug loading capacity which makes them ideal for drug delivery applications. The hydrophilic outer corona stabilizes the interface between hydrophobic drug and external medium [94]. This interaction increases metabolic stability and the blood circulation time and also prevents the burst release of drugs [95]. Drugs can be encapsulated into the core of Pluronic micelles either by physical entrapment or by chemical conjugation of drugs to the core of micelles [96]. In chemical conjugation the drug is covalently coupled to PPO blocks in the core of Pluronic micelles[97]. Some studies showed that, chemically conjugated drugs stay in the blood for a longer time and the uptake of the drugs by non-targeted organs is lowered making the delivery system more effective [98]. Many drugs can also be easily entrapped physically into the micelle core by simply mixing the drugs in the Pluronic solution. For example, drugs including Doxorubicin, Paclitaxel and Ruboxyl can be physically entrapped into the micellar core and can be released at specific target by ultrasound [97]. Drug encapsulated micellar solution can be prepared usin thin-film hydration method. The drugs are initially dissolved in ethanol using ultrasound and then Pluronic is dissolved in the solution. The solution is then evaporated to remove ethanol and the obtained drug/polymer film is then hydrated with PBS or de-ionized water and subsequently stirred to obtain a micelle solution. The unincorporated drug aggregate can be removed by centrifuging the solution [99, 100]. Another method to load the drugs is the novel nano-precipitation method [101]. In this method, the drug and polymer is dissolved in acetonitrile and the solution is subsequently added drop wise to an aqueous solution. The colloidal solution is centrifuged to remove unencapsulated drug and residual Pluronic. The solution mixture is evaporated and then hydrated with water. The solution mixture is subsequently frozen and then lyophilized for storage. In modern medicine, it is of vital importance to design drug delivery systems that can specifically target selected tissues. For example, several anti-cancer drugs like doxorubicin are fairly toxic to normal tissue and it is important to target only the tumor. Targeted delivery is also important in gene delivery where the genes are delivered to a specified part of a targeted organ. Targeted drug delivery not only reduces the side effects but also reduces the drug wastage making it more cost efficient. Pluronic micelles sequester hydrophobic drugs and thus reduce the exposure of drugs in the systemic circulation and preventing any harm to healthy cells [97]. Batrakova et al. showed that low concentrated Pluronics reverse the drug resistant behavior of cancerous cells and also result in less accumulation of doxorubicin in cells 16

17 [102]. At higher concentrations of Pluronics, monomers are still present which are in equilibrium with micelles and are still able to overcome the multidrug resistant behavior of cancerous cells. Pluronic systems have been found very effective in transport of anti-cancer drugs like doxorubicin to the targeted tissues and triggering the drug release on application of low frequency ultrasound [97]. 5.3 Pluronics blended with salts and the effect of ph Addition of salts in the Pluronic solutions results in the onset of micellization process at a lower polymer concentration (at constant temperature) or at lower CMT (at constant concentration) [8, 27, 57]. The presence of salts leads to dehydration of PEO near the micelle core increasing the radius of the core making more space for encapsulation of drugs. Therefore, the solubility of drug increases with the addition of salt. It was observed that, as the ionic strength of the solution increases the amount of drug encapsulated increases. For example, Pandit et al. studied the effect of inorganic salts and observed increased solubility of hydrophobic drug propyl paraben in presence of salts [103]. The addition of salts generates more hydrophobic microenvironment and also increases the number of micelles and therefore increasing the total volume available for solubilization of hydrophobic drugs. Studies on the another drug prednisolone also showed an increase in solubilization with increase in salt concentration in Pluronic P85 solution [60]. Figure 8 shows the increase in solubility of drug with increasing the salt concentration. The solubility of many drugs also increases considerably with a change in ph. Kadam et al. have studied the effect of ph on the solubility of hydrochlorothiazide drug [96]. They have shown that the solubility increases with decreasing ph from 6.7 to 3.7. Below the ph value of 6, the HCT drug remains as an uncharged molecule and therefore prefers to stay in the micelle core, increasing the solubility. Figure 8 shows the variation of solubility with Pluronic concentration at various ph values. It can be seen that at higher Pluronic concentrations the solubility is higher for low ph values. In another studies by Yang et al. it was shown that the hydrodynamic radius of chitosan oligosaccharide (CSO) conjugated Pluronic micelle increases from 24 to 38 nm with decrease in ph value from 7.6 to 4.75 [104]. The swelling of the CSO- Pluronic micelle is due to electro-static repulsion between the positive charged CSO chains at lower ph values. A B Figure 8:- A-Solubility of HCT with increasing salt concentration. B- Solubility of HCT vs. P103 concentration at different ph at 28 C [96]. 17

18 5.4 Pluronics blended with other polymers Many efforts are being made to modify the Pluronic structure to improve the drug-loading capacity and to obtain efficient drug delivery. One of the methods is to blend in the Pluronics with other miscible polymers to form a single phase of mixed micelles [105, 106]. Mixed micelles with Pluronics have been widely studied to provide multi-functionality to the micellar systems for obtaining controlled and targeted drug delivery. There are two types of mixed micelles that has been studied: Pluronic/Pluronic mixed micelle and Pluronic/non-Pluronic mixed micelles. Recently Fang et al. have studied the Pluronic P123/F127 mixed micelles for the encapsulation of paclitaxel (PTX), an anti-cancer drug [107, 108]. Figure 9 shows the formation of mixed micelle along with the encapsulation of PTX drug in the core of mixed micelle [107]. The mixed micelles showed increased drug loading and longer circulation time, therefore significantly enhanced the anti-cancer activity. Saxena et al. reported a new delivery system consisting of Pluronic 407/TPGS mixed micelles for delivery of gambogic acid (GA) to treat breast cancer [109]. These mixed micelle systems showed high cellular uptake of GA and 2.9 times higher toxicity towards multidrug resistant cancerous cells. Very recently, Sosnik et al. reported mixed micelles of Pluronics and poloxamines and studied the encapsulation of anti-hiv drug efavirenz [110]. The mixed micelles showed 8430 fold increase in the drug loading capacity and also displayed higher physical stability in comparison with pure poloxamines. These mixed micellar systems are highly versatile in bioavailability as shown in figure 10, and therefore are potential drug delivery systems for anti-hiv pharmacotherapy [110]. Figure 9:- Representation of the strategy of developing Pluronic P123/F127 mixed polymeric micelles [107]. 18

19 Figure 10: Schematic representation mixed polymeric micelles and encapsulation of EFV. F127:T904 displayed greater encapsulation efficiency while F127:T304 micelles hamper the incorporation of EFV when compared to only F127 micelles [110]. Many studies have also been reported on the Pluronic/PLA mixed micelles for drug delivery, which also have enhanced bioavailability and increased intracellular uptake of drugs [111, 112]. In these mixed micelles of Pluronic/PLA, Pluronics provide encapsulation stabilizing effect while the ph sensitive PLA facilitates controlled drug release in acidic cancerous environment. Pluronics/PLGA blends have been successfully used for gene delivery [113, 114]. Initially pure PLGA were used for gene delivery, however these systems were poor in release of DNA vaccines. Adding Pluronics to this delivery system facilitates controlled release and also help preserving the biological activity of genes. Pluronics are also used to hydrophilize the PLGA scaffolds to develop a polymeric matrix for pdna delivery[113]. Such PLGA scaffolds with Pluronics were observed to have higher transfection efficiency of the pdna when compared to pure PLGA scaffolds. Table 3 shows the list of modifications of Pluronics by blending with other polymers and specific applications. 19

20 Table 3:- Modification of PEO-PPO-PEO triblock copolymers by blending with other polymers Modification Unique feature Application Ref Pluronic and polyacrylic acid Oral in situ temperature-and phsensitive For the delivery of an [115, hydrogels anticancer drug, 116] Polyethylene glycolpolycaprolactone/pluronic P105 or PEG-PCL/Pluronic P105 Pluronic407/TPGS mixed micelles Folate conjugated Pluronic F127/Chitosan Core-Shell Nanoparticles poly(ethylene oxide)- poly[118]-poly(ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 Folate and β-cyclodextrin decorated Pluronic F127-bpoly(ε-caprolactone) copolymer F127-metal-drug Composite doxorubicin-loaded micelles are radio-sensitive. High cellular uptake, improved drug retention, and enhanced antitumor effect relative to free doxorubicin Increased cellular uptake and sustained release of drug More stable nano-particles and showed greater cytotoxicity towards MCF-7 cells than free DOX Increased drug loading efficiency and drug stability Biodegradable, and target specific cellular uptake 20 epirubicin. For the delivery of an anticancer drug, doxorubicin [117] Delivery of gambogic acid [109] to breast and multidrugresistant cancer High anti-cancer activity [99] In tumor accumulation [118] As nanocarriers for targeted drug delivery. [119] ph-responsive In antitumor therapies [120] coordination-bonding P123 and F127 mixed micelles High encapsulation efficiency (>90%), good stability in lyophilized form and ph-dependent in vitro release P123 and L121 Binary mixture Physical mixture of alginate and PF127 Mixed MPEG PLA/Pluronic Polyethyleneglycol- poly(dllactic-co-glycolic acid) (PEG- PLGA) and Pluronic 105 (P105) Silk sericin/ Pluronic nanoparticles Mixed micelles of hydrophobic Pluronic L81 and relatively hydrophilic Pluronic P123 High stability due to Pluronic P123 and high solubilization capacity due to Pluronic L121. Delivery of anticancer drug to melanoma As anticancer drug delivery systems [107, 108, 121] [122] Greater porosity and no degradation Selegiline skin permeation [123] Mixed micelles significantly reduced the tumor size than the control (Taxotere) The combination of PEG-PLGA and Pluronic further improved both the tumor-suppressive activity and the intracellular accumulation of DOX, Self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. Mixed micelles are very small in size, showed fairly high entrapment efficiency, loading capacity and sustained release profile for aceclofenac, a model hydrophobe. Enhanced bioavailability and to overcome multidrug resistance of docetaxel in cancer therapy. To reverse the multidrug resistance in tumor cells. Delivery of both hydrophobic and hydrophilic drugs to target sites. Controlled and targeted drug delivery [112] [124] [125] [126]

21 5.5 Chemically modified Pluronics Multifunctionality in the Pluronic systems can be induced by chemically modifying the Pluronic structure by addition of other polymers or other materials. For example, Pluronics have been chemically modified by poly-caprolactone (PCL), PLGA, polyethyleneimine and folic acid [ ]. PXT drugs are known to have good compatibility with PCL, and therefore Pluronics modified with PCL can entrap more PXT drug molecules. PCL modified Pluronics also reverse the multi-drug resistance of cancer cells, increase intracellular concentration and increase circulation time, thus making them highly effective delivery systems for sensitizing resistant tumors [128]. Figure 11 shows the scheme of the synthesis of PCL modified Pluronic P105 [128]. Polyethyleneimine (PEI) has been demonstrated to have high gene transfection efficiency, but PEI aggregate have very short circulation time which limits its applications invivo. Many methods have been developed to modify the PEI to increase its biocompatibility while retaining its gene transfection efficiency. Recently, Pluronics have also been used to enhance PEI properties [127]. PEI-Pluronic conjugates have excellent gene transfection efficiency as well as good biocompatibility. Figure 12 shows the scheme of the synthesis of PEI-Pluronic conjugates [127]. Gao et al. have also reported Pluronic modified low molecular weight PEI for degradable gene delivery systems [130, 131]. They showed that the Pluronics with high HLB homogenously distribute in the cytoplasm while Pluronics with lower HLB result in localization inside the nucleus. Figure 11:- scheme of the synthesis of poly(caprolactone)-modified Pluronic P105 polymers (C) [128]. 21

22 Figure 12:- Synthetic scheme of PEI Pluronic copolymers (PCMs) [127]. Most of the drug delivery systems for cancer therapy have very less selectivity for tumor cells, which would have high toxicity towards healthy cells. Developing drug delivery systems with selectivity for cell types not only increase drug efficiency but also reduces side effects on normal cells. Various targeting molecules like folic acid, RGD peptide, mannose and galactose can be conjugated to the drug delivery systems to increase targeted delivery. In case of Pluronic systems, folic acid is the most popular molecule used for increasing selectivity toward tumor cells. Fang et al. tested folic acid functionalized Pluronics in-vivo and reported increased bioavailability of these conjugates [108]. Figure 13A shows the schematic for the synthesis of Folic acid conjugated Pluronics. Folic acid conjugated Pluronics can be mixed with other Pluronics to form targeted mixed micelles as shown in figure 13B [108]. Multifunctional superparamagnetic particles can be produced by solubilizing iron oxide particles by folic acid conjugated Pluronics which have combined targeting, diagnosis and therapy applications [132]. Very recently, Zhou et al. reported Pluronics conjugated with folic acid and β-cyclodectrin, in which the folic acid acts as targeting molecule whereas β-cyclodectrin provide increased bioavailability [119]. Table 4 shows the list of chemical modifications done to the Pluronics and their applications. 22

23 A B Figure 13:- (A) Schematic illustration of the synthetic steps for Pluronic F127 functionalization and conjugation of folic acid. (B) Representation of the strategy of developing targeted polymeric mixed micelles [108]. Table 4:- Chemical modification of PEO-PPO-PEO triblock copolymers Modification Unique feature Application Ref Poly (ethylenimine) conjugated Pluronic copolymers A marked increase of cellular accumulation compared with Pluronic P123 micelles. Poly(caprolactone)- modified Pluronic P105 m Gold-nanoparticlecrosslinked Pluronic micelles Folate and β-cyclodextrin decorated F127-b-poly(εcaprolactone) copolymer Carboxylated PluronicF127 (F127COOH) Alginate Pluronic F127 composite copolymer Sensitize the resistant SKOV-3/PTX tumor cells. More micelle stability and cellular uptake Biodegradable, and target specific cellular uptake Alleviating potential toxicity, enhancing the stability and improving targeting efficiency of CdTe/ZnS quantum dots (QDs) in tumor Exhibited linear permeation properties for the transdermal delivery of selegiline. Enhanced anticancer activity and also used as biocompatible gene delivery carriers. Delivery of paclitaxel and treatment of the resistant ovarian tumors. High anti-cancer activity against glutathione pretreated U87 cells As nanocarriers for targeted drug delivery. In human pancreatic cancer detection and targeted drug delivery As a topical therapeutic formulation for selegiline. [127, 130, 131, 133, 134] [128] [135] [119] [136] [123] 23

24 Pluronic F127-Chitosan Nanocapsules Anti-HIF-1a antibodyconjugated Carboxylterminated Pluronic P123 Folic acid conjugated F127 decorated with polyacrylic acid-bound iron oxides Lactobionic acid (LA)- conjugated Pluronic P105 (P105) Hyaluronic acid (HA) grafted Pluronic F127 Core-Functionalized PEO- PPO-PEO Copolymer Heparin-conjugated Pluronic F-127 Loosely cross-linked poly(acrylic acid) grafted with Pluronic F127 and L92 Chitosan oligosaccharide (CSO)-g-Pluronic P103 copolymers Multiple alphacyclodextrin (alpha-cd) threaded on Pluronic coplymer Pluronic P123 modified polyamidoamine (PAMAM) Poly(diethylamino ethyl methacrylate) blocks conjugated Pluronic F127 Hydrogel of Di-acrylated Pluronic F127 Gelatin grafted Pluronic F127 PLGA-Pluronic F68 copolymer The nanocapsules are ~37 nm at 37 C and expand to ~240 nm when cooled to 4 C in aqueous solutions, exhibiting>200 times change in volume. Specifically bounds with stomach cancer MGC-803 cells MRI agent as well as diagnosis and therapeutic agent that specifically targets cancer cells that overexpress folate receptors in their cell membranes. Remarkable increase in the dissolubility for silybin in LA-P105 micelle solution Thermosensitive hydrogel with sustained release characteristics Greatly enhanced cytotoxicity for MCF-7 human breast cancer cells and high extent of cellular uptake Has good injectability and the release properties can be tuned. Pluronic L92 exhibited highly porous structure, while the microgels containing Pluronic F127 were generally larger and possessed smooth surfaces, a homogeneous structure, and lower ionexchange capacity. ph- and temperature-responsive polymeric drug carriers Low cytotoxicity and high transfection efficiency Low cytotoxicity and higher transfection efficiency These copolymers electrostatically condense plasmid DNA into nanostructures (nanoplexes) and further self-assemble above critical concentration to form thermoreversible hydrogels at physiological temperatures. No chemical degradation and maintained the gene expression Thermosensitive and excellent cell viability Can load bovine serum albumin effectively and keep it stable after it is released from the nano-particles For encapsulating small [137] therapeutic agents to treat diseases particularly when it is combined with cryotherapy Tumor targeting therapy [138] For combined targeting, diagnosis, and therapy applications [132] Target-specific delivery [139] vehicles for diverse waterinsoluble therapeutic and diagnostic agents. Anti-cancer treatment and [140] for tissue regeneration Used for overcoming the [92] multidrug resistance (MDR) effect For protein delivery [141, Delivery of anti-cancer and hydrophobic drugs 142] [143] Controlled release of [104] Doxorubicin drug for anticancer therapy Sustained gene delivery [144] Carrier for gene delivery [145] Act as both nanoscale gene delivery vectors and macroscale sustained delivery agents [146] Controlled release of [147] plasmid DNA Tissue regeneration [148] (cartilage), gene and drug delivery For protein delivery [129] 24

25 6. Non-Bio Applications of Pluronics 6.1 Unmodified Pluronics An important application of Pluronic micelles is the storage of nano-materials in the core of micelle. Micelles can act as host to many nano-particles/drugs and make stabilized dispersions of these particles in aqueous solutions. These nano-containers can help transport of particles/drugs in aqueous that are insoluble or unstable in water. For example, Pluronic micelles can act as nano-containers for magnetic nano-particles, gold nano particles, CNTS, drugs like doxorubicin, paclitexal, etc [ ]. One of the problems related to carbon nano-tubes (CNTS) is their insolubility in water due to large vander walls forces. Though there are various techniques to disperse CNTS in aqueous solutions, recently it was shown that Pluronic micelles can also be used to disperse CNTS in water [150]. Pluronic micelles can also encapsulate gold nano-particles to reduce its cytotoxity and improve stability in aqueous solution [149]. Pluronics can encapsulate superparamagnetic nano-particles for magnetic resonance imaging and nanothermotherapy [152]. Recent studies have shown that Pluronic micelles can also encapsulate quantum dots preserving the optical and colloidal stability of quantum dots in biological systems [153]. Nano-particles are highly unstable in aqueous solutions and tend to flocculate. To prevent flocculation, these nano-particles need to be kinetically stabilized by steric means to obtain stable dispersion of these nano-particles. Pluronic systems are extensively used to stabilize various nanoparticles. One of the major examples is the stabilization of magnetic nano-particles to use in magnetic resonance imaging (MRI). Pluronics decorate the outer surface of magnetic nano-particles in an aqueous solution and stabilize the particles. The polymeric chains fold and self-assemble on the particles providing dispersion stability as shown in the figure 14. Apart from stabilizing, the external corono consisting of PEO blocks provide antifouling property to the particles and helps prevent aggregation and protein adsorption [95]. The stabilization of particles is temperature dependent as the PPO and PEO blocks in Pluronics become highly hydrophobic with increase in temperature. Pluronics also influence the size of the particles, the protein binding to the particles and dispersion stability. Along with stabilization, the Pluronic/nanoparticle system can also be loaded with drugs and hence providing drug delivery and imaging properties simultaneously [154]. Figure 14:- Schematic showing stabilization of magnetic nano-particles by Pluronics [154]. 25

26 Pluronic micelles have also been widely used to synthesize mesoporous materials. Pluronics are mixed with particles in aqueous medium and the Pluronics form micellar structures which get entrapped inside the material. The particles are then subjected to calcination which decomposes the micellar structures resulting in mesoporous structure. Pluronics have been widely used to synthesize various materials including TiO 2, steel, Mn 3O 4, bioactive glass, carbon materials and silica based materials like SBA- 15, organosilicates and ferrosilicates [ ]. Thin film mesoporous structures of TiO 2, ZnO, ZrO 2 have also been successfully synthesized using Pluronics [163, 164]. The pore structure highly depends on the type of Pluronics used during the synthesis. Ardizzone et.al have reported the effect of Pluronics molecular weight and HLB on the porosity of TiO2 materials [162]. They showed that Pluronics with low molecular weight and intermediate HLB values generate larger surface areas and pore volumes. Pluronics have also been extensively used in the synthesis of nano-particles and in tuning the size/shape of the nano-particles. For example, Pluronics have used in synthesis of gold nano-particles, Uranium oxide nano-particles, hydroxyapatite nano-particles and magnetic microbeads [ ]. 6.2 Pluronics blended with other polymers It is well known that the properties of Pluronics can be significantly enhanced by addition of surfactants and other polymers. In synthesis of mesoporous silica, Pluronics can be mixed with mixed with n-butanol (BuOH) and tetraethoxysilane (TEOS) for tailoring the pore structure [170, 171]. Figure 15 shows the phase diagram of mesoporous silica with the variation of BuOH and TEOS [170]. Zhao et al. have reported a method for the synthesis of highly ordered mesoporous structures by using formaldehyde/phenol resols and Pluronics [155]. Hydrocarbon molecules like hexadecane or decane are used in the Pluronic systems for the swelling of the structure to produce larger pore size. Also, by varying the hydrocarbon molecules the pore size can be tailored from 4nm to 6 nm. The authors proposed a scheme (Figure 16) of possible interaction between Pluronics with resols which includes one-layer hydrogen bond interaction between resols and PEO segments. This organic-organic cooperative assembly as shown in figure 16 favors the formation of mesoporous carbon structure which is a very promising material for various applications. In another study, Ozdural et al. mixed SDS along with Pluronics to synthesize magnetic microbeads [165, 166]. They were successful in producing magnetic polyvinylbutyral microbeads and magnetic nitrocellulose microbeads using this Pluronic/SDS mixture. 26

27 Figure 15: Phase diagram of mesophase structures established according to the XRD measurements. Each sample is prepared with a molar ratio of P123/x TEOS/y BuOH/1.83 HCl/195 H2O [170]. Figure 16:- (A) A Possible Formation Mechanism of Mesoporous Polymers with a Variety of Structures in an Aqueous Solution under Basic Conditions, (B) Resol Anions Formed at a Relatively Strong Basic Condition, and (C) a Pore Size Swelling Process of Hydrocarbon Molecules in the P123 System [155]. 6.3 Chemically modified Pluronics Pluronics have been chemically modified to induce multi-functionality in various applications like dispersion of particles, synthesis of mesoporous structures and ph/temperature sensitive materials. Petrov et al. modified Pluronics with pyrene to produce micelles which can be used to stabilize multiwalled carbon nanotubes in aqueous media [150]. Figure 17 shows schematic of synthesis of pyrene 27

28 functionalized Pluronics and interaction with multi-walled carbon nanotube. In another study, Pluronics have been modified by amines to disperse quantum dots in aqueous media [172]. The amine terminated hydrophilic blocks were used for Gd3+ chelation and the quantum dots remain well protected in the core with excellent optical and colloidal stability. Park and co-workers have modified Pluronics in various ways as shown in figure 18 to produce ph and temperature sensitive materials [173]. In another study, Park et al. have reported spiropyran conjugated Pluronics to develop calorimeter detector [174]. Figure 17:- Synthesis of pyrene-functionalized stabilized polymeric micelles and their non-covalent interactions with multi-walled carbon nanotubes [150]. Figure 18:-. Phase diagram of mesophase structures. Each sample is prepared with a molar ratio of P123/x TEOS/y BuOH/1.83 HCl/195 H2O [173]. 28