Materials Science and Engineering C 34 (2014) 280–286

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Ultrasonic monitoring of drug loaded Pluronic F127 micellular hydrogel phase behaviour Mario Farrugia a, Stephen P. Morgan a, Cameron Alexander b, Melissa L. Mather a,⁎ a b

Applied Optics Group, Electrical Systems and Optics Research Division, University of Nottingham, Nottingham NG7 2RD, UK School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK

a r t i c l e

i n f o

Article history: Received 24 April 2013 Received in revised form 4 August 2013 Accepted 18 September 2013 Available online 27 September 2013 Keywords: Ultrasound Hydrogel Pluronic Drug delivery

a b s t r a c t Pluronic hydrogels composed of PEO–PPO–PEO tri-block copolymers have received a lot of attention for their applicability to drug delivery. These systems can be injected into the body in a liquid form and then, in response to temperature changes, self-assemble into nano-sized micelles which ultimately aggregate to form a gel. The phase behaviour and effectiveness of Pluronic hydrogels as drug carriers is affected by the local thermal and ionic environment which is likely to be different from patient to patient. There is a current need for in vivo techniques to study the phase behaviour of Pluronic hydrogels and this work demonstrates an ultrasound approach for the study of drug loaded Pluronic F127 hydrogels. Ultrasound velocity and attenuation were both found to change with temperature and through validation with fluorescence spectroscopy it was determined that the temperature dependent micellation transition in the Pluronic solutions could be identified through relative changes in ultrasound velocity and attenuation as a function of temperature. This phase transition was more clearly detected through examination of the first and second derivatives of both ultrasound parameters with respect to temperature. Further this work demonstrates for the first time to our knowledge ultrasound characterisation studies on drug loaded Pluronics. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The use of hydrogels in biomedical applications is growing, particularly due to advances in polymer synthesis that produce stimuli sensitive materials which respond to environmental changes such as temperature and pH. Hydrogels are water-swollen polymeric networks that contain approximately 60% to 99% water, yet maintain the structural integrity of a solid due to the presence of cross-links [1]. This high water content coupled with their soft, rubbery nature and typically high permeability makes hydrogels attractive biomaterials [2]. Recently there has been great interest in using hydrogels as vehicles for delivery of drugs [3] and cells [4–6] into the body for therapeutic purposes. In particular, stimuli sensitive hydrogels that can be injected into the body in a liquid state and subsequently form a gel once in the body are favourable due to the minimally invasive route of administration [7]. Hydrogels can be produced in a number of ways and can be classified by the type of cross-links forming the polymeric network. For example, cross-links can be chemical e.g. covalent bonds, physical e.g. secondary bonds including hydrogen bonding, van der Waals forces, crystal junctions, micellar packing, etc. or hybrid e.g. a combination of ⁎ Corresponding author at: Institute of Biophysics, Imaging and Optical Science Applied Optics Group, Faculty of Engineering University of Nottingham, Nottingham NG7 2RD, UK. Tel.: +44 (0) 115 95 15337. E-mail address: [email protected] (M.L. Mather). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.018

chemical and physical cross-links. The type of cross-link affects hydrogel degradation, mechanical properties, permeability and binding affinity [1] and thus its suitability for use in different applications. Hydrogels composed of amphiphilic block co-polymers, which gel by micellar packing in response to temperature changes, have received a lot of attention for their applicability to drug and cell delivery [8–11]. These systems can flow freely in their liquid form and then, in response to temperature changes, self-assemble into nano-sized micelles and fibres which ultimately aggregate to form a gel [7]. Thus, prior to administration, and while the system is in the liquid phase, drugs and/or cells can be mixed into the solution. As temperature rises the therapeutic agents can be encapsulated in the forming micelles and eventually in the gel structure itself. This capability enables water-insoluble or poorly soluble drugs as well as labile molecules such as proteins and peptide drugs to be effectively solubilised [12]. Micellar hydrogels composed of PEO–PPO–PEO tri-block copolymers, also known as Pluronics [7], have been widely studied for drug delivery applications with some formulations being approved for therapeutic use by the United States Food and Drug Authority (FDA) [13]. Medical applications of these hydrogels include use in the treatment of wounds [14], transdermal delivery of protein and peptides (insulin, urease, bone morphogenic protein and growth factors) [15], delivery of drugs in ophthalmic applications [16], prevention of postoperative adhesions [17] and the delivery of chondrocytes to promote cartilage regeneration [18]. Continued work using Pluronic hydrogels have

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

focussed on modifying their drug release profile and reducing the polymer concentration required for gelation, for example, by altering the hydrophilicity of polymer side chains. Due to the thermoresponsive nature of Pluronic hydrogels it is important to characterise their phase behaviour prior to application. This phase behaviour involves a transition from a liquid state in which the polymer chains are dispersed as unimers towards a more complex liquid-like state with polymer micelles and finally on to a gel state due to packing of the micelles [19]. The most widely used Pluronic hydrogel has a formulation with a nominal molecular weight of 12,500 and a PEO/PPO ratio of 2:1 by weight, and is commercially known as Pluronic F127. Extensive studies of the phase behaviour of Pluronic F127 hydrogels have been reported in the literature using techniques such as light scattering, small angle neutron scattering, 1H nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, differential scanning calorimetry (DSC) and rheometry [20]. The combined results from all of these techniques have enabled a good understanding of the phase behaviour of Pluronic F127 hydrogels to be obtained. However, as the end application of these hydrogels is in the body it is pertinent that non-invasive methodologies capable of studying the phase behaviour in vivo are developed, particularly as the thermal and ionic environment is likely to be different from patient to patient and in healthy as opposed to diseased states. In this work the potential of ultrasound techniques to probe the phase behaviour of Pluronic F127 hydrogels is assessed. Ultrasound techniques have been widely used for non-invasive monitoring of physical changes in polymer solutions and gels [21–23], and in a clinical setting for medical imaging [24]. The main benefits of ultrasound characterisation studies are its non-invasiveness, speed of measurements which enable inprocess monitoring, ease of use and good sensitivity to physical changes without the use of any contrast agents. A few prior ultrasound studies of Pluronic F127 hydrogels have been carried out, for example the dependence of ultrasound velocity on Pluronic F127 concentration in aqueous solutions was studied using a sing-around technique, in which the average time delay of ultrasound pulses in a closed loop is used to determine velocity [21]. Ultrasound velocity was evaluated as a function of temperature for solutions of Pluronic P85, with a nominal molecular weight of 4600, by Glatter et al. [25]. Measurements were made using a bench top density and sound velocity meter and the observed micellation behaviour was validated with DSC. In these studies plots of the first derivative of velocity as a function of temperature displayed a peak which was reported to be an indication of micelle formation. More recently Cespi et al. have used a high resolution ultrasound spectrometer to assess its potential as a tool to characterise polymer aggregation in Pluronic F127 hydrogels [20]. The dependence of ultrasound relative velocity and attenuation on temperature was presented for a range of concentrations and compared to results of DSC. Further the first derivative of the ultrasound parameters with respect to temperature was also displayed to aid in localisation of the micellation phase transition in the hydrogel. These results agreed with those earlier reported by Glatter demonstrating that ultrasound parameters can be used to indicate micelle formation. These initial ultrasound studies suggested that ultrasound monitoring of responsive polymers should be considered in more detail. Of particular interest is investigation of measurement techniques that could ultimately be applied in a non-invasive way in a clinical setting. The aim of the work presented here is to implement a robust ultrasound method to study the phase behaviour of Pluronic F127 hydrogels on a larger scale than has previously been performed. Another goal of this work is to incorporate a model hydrophobic drug, pyrene, into the system as both a fluorescent probe to validate micelle formation and to study the uptake of the drug for controlled release applications. This fluorescent method is very sensitive to subtle changes in the microenvironment around the probe [26] and microstructural changes in the micelles at a molecular level [27]. Hence, this approach will provide greater sensitivity than calorimetry studies, which examine bulk properties. Further, this work will for the first time to our knowledge, perform ultrasound studies on drug loaded Pluronics.

281

2. Materials and methods 2.1. Preparation of samples Aqueous stock solutions of Pluronic F127 (Sigma Aldrich) were prepared using the cold method, as described in the literature [28]. In practice samples with concentrations of 14%, 16%, 18% and 20% w/w were prepared by adding appropriate amounts of polymer (in flake form) to distilled water. The aqueous Pluronic solutions were then mixed and held at 3 °C for 12 h. The fluorescent probe pyrene (Sigma Aldrich) was then incorporated into the Pluronic solutions. This involved first dissolving the hydrophobic pyrene in methanol, according to the procedure described previously [26] using a concentration of 8.09 mg/100 mL. The pyrene-methanol solution was then sonicated using a Clifton MU 1.5D sonicator until, on visual observation, the pyrene had dissolved. The solution was then left for 24 h in a fridge at 3 °C to promote homogeneous dissolution of pyrene in the methanol. Aliquots of the pyrene-methanol solution were then added to each Pluronic stock solution to produce solutions in a ratio of 5% pyrene-methanol to 95% stock solution. Thus the pyrene loaded co-polymer solutions had final Pluronic F127 concentrations of 13.3%, 15.2%, 17.1% and 19.0% w/w. This phase behaviour of Pluronic F127 solutions as a function of polymer concentration and solution temperature has been well documented [11,19,29]. With reference to the literature, the range of polymer concentrations studied in this work was chosen as it allowed the study of samples representative of solutions that only undergo micellation up to those reaching micellation concentrations sufficient for gelation over the temperatures considered.

2.2. Fluorescence spectroscopy The phase behaviour of the Pluronic solutions as a function of temperature was studied by a fluorescence technique using the molecular probe pyrene [30]. Pyrene is a hydrophobic probe which is sensitive to the polarity and viscosity of its microenvironment [31]. Of interest to this work is the ability of the pyrene fluorescent probe to form excimers in a concentration dependent way enabling the aggregation in micellar systems to be studied [32]. In this work the fluorescence spectra of pyrene loaded Pluronic solutions were obtained using a Varian Cary Eclipse fluorescence spectrophotometer at an excitation wavelength of 336 nm. For each sample concentration, 1 mL of the pyrene loaded Pluronic solution was placed in a spectrophotometer cuvette and inserted into the thermostated multi-cell holder of the spectrophotometer. The temperature of the sample compartment was then adjusted over a temperature range of 10 °C to 27 °C at steps of 1 °C. Spectra were obtained after a delay of 2 min following each temperature rise to aid equilibration of the sample temperature with that of the compartment. At each temperature the intensities of the monomer band at 385 nm and excimer band at 460 nm were measured and the ratio of the fluorescence intensities of the excimer to monomer determined to provide information about the available hydrophobic volume in the micelle cores, the concurrent changes in the local concentration of pyrene and infer the critical micelle temperature [26,30].

2.3. Tube inversion tests The critical gelation temperature of the pyrene loaded Pluronic F127 solutions was determined using a tube inversion method. Cylindrical glass bottles of diameter 20 mm and height 50 mm containing 2 mL samples of each concentration were placed in a temperature controlled water bath (Grant Sub Aqua 5) and heated over a temperature range from 17 °C to 32 °C at steps of 0.5 °C. The tubes were allowed at least 15 min at each set temperature point to equilibrate before being removed and tilted. The gelation temperature was defined as the temperature at which the sample did not flow when the tube was inverted [7].

282

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

2.4. Ultrasound measurements The experimental set-up for ultrasonic measurements is shown in Fig. 1. Pluronic F127 solutions under investigation were injected into a 7.3 mm long cylindrical, 25 mm diameter sample chamber consisting of a hollow polymer tube enclosed by 0.175 mm thick Mylar sheets at each end to act as acoustic windows. The sample chamber was placed in a temperature controlled water tank and clamped in position. Two 13 mm diameter, 2.25 MHz ultrasound unfocussed immersion transducers (Olympus) were placed in the water tank on opposite sides of the sample chamber. One transducer was used as a transmitter and the other as a receiver. Over the temperature range studied the transducer near field distance was between 63 mm and 66 mm. The spacing between the transducers was set to 250 mm, with the sample sitting at a 125 mm distance from each transducer, placing it well within the acoustic far field. The transducers were then aligned to produce a maximum transmitted signal through the sample chamber. Initially the water tank was cooled to 5 °C and subsequently heated to 30 °C at a heating rate of 6.5 °C/h. A thermocouple was inserted into the sample chamber to measure the hydrogel temperature throughout the experiment which was logged by a PicoLog TC-08 data logger. Ultrasound signals were produced by using a pulse generator (UPR, NDT Solutions) to apply 10 V, 700 ns pulses with a repetition rate of 0.1 kHz to the transmitting transducer. Signals detected by the receiving transducer were amplified (20 dB) and displayed on a digitising oscilloscope (MSO4034, Tektronix). At intervals of 0.02 °C 512 received samples were recorded and the resulting averaged signal transferred to a personal computer for storage. This procedure was performed for each sample concentration three times to assess repeatability of results. The received signals were analysed using an in-house routine written in Matlab® (Mathworks) to extract the first arriving ultrasound pulses that had propagated once and three times respectively through the sample. In all the cases a single cycle in each pulse was selected and Fourier analysed to determine the signal amplitude at the peak frequency. The propagation delay between the two signals analysed was determined through analysis of the zero crossing of each pulse. Through knowledge of the propagation delay and signal amplitudes the ultrasound velocity (v) and attenuation (α) were calculated using Eqs. (1) and (2) [33]. v¼

2d Δt

α¼−

ð1Þ   1 A1 ln ; 2d A0 R2

ð2Þ

where Δt is the propagation delay, d is the sample chamber length, R is the ultrasound reflection coefficient at the interface between the Mylar and sample and A0 and A1 are the Fourier analysed signal amplitudes at the peak frequency for the pulses that propagated once and three times respectively through the sample. The above equations were applied to the experimental data based on the assumption that the sample properties were homogeneous over the propagation path and without correction for beam spreading. In this work relative ultrasound parameters were reported by subtracting the value of each parameter at the lowest temperature to aid the comparison of results for the different polymer concentrations considered. 3. Results and discussion 3.1. Fluorescence spectroscopy For each polymer concentration, over the temperature range of 10 °C to 27 °C, the emission spectra of pyrene in the aqueous solutions of Pluronic F127 were analysed. Specifically, the intensity of the monomer fluorescence (I3), centred at 385 nm, and the excimer fluorescence (Ie), centred at 460 nm, were determined. Fig. 2 is a plot of the ratio of Ie to I3 as a function of temperature for each polymer concentration studied. This ratio can be used to obtain information about the local concentration of pyrene and hence the aggregation behaviour of the polymer [26]. In particular, a rise in temperature leads to an increase in the solubility of pyrene in the hydrophobic cores thus reducing the fluorescent losses due to quenching that occurs when the pyrene is in a hydrated region. At the starting temperature of 10 °C there is negligible excimer emission for all samples. This indicates that the polymer aggregation is not sufficient for micelles to form and that the pyrene is homogeneously distributed throughout the samples. With increasing temperature Ie/I3 gradually increases to a peak which drops off as temperature rises further. The broad nature of the observed peaks is thought to be attributed to the inherent polydispersity of Pluronic copolymers as has been previously reported [34]. The onset of the peak in the Ie/I3 results is associated with the start of excimer emission and a corresponding decrease in monomer emission, indicative of partitioning of the pyrene into localised areas [26]. As temperature increases further, the Ie/I3 ratio reaches a maximum due to reorganisation of pyrene molecules in micelles leading to a heterogeneous distribution of pyrene. As temperature rises and further micelles form, the microviscosity of the solution increases due to the micelles filling the available volume and the pyrene distribution becomes more homogeneous, thus reducing Ie/I3.

0.1

13.3 % 15.2% 17.1% 19.0 %

0.09 0.08

IE I3

0.07 0.06 0.05 0.04 0.03 10

15

20

25

Temperature (oC) Fig. 1. Experimental set up for ultrasound measurements. The sample, ultrasound transmitter and receiver are placed in a temperature controlled water bath. The transmitter is excited by a function generator. Received signals are digitised by the oscilloscope and sent to the computer. A temperature probe is placed in the sample and recorded to the computer.

Fig. 2. The ratio Ie/I3 of pyrene monomer fluorescence (I3), centred at 385 nm, and the excimer fluorescence (Ie), centred at 460 nm, as a function of temperature for each polymer concentration studied: 13.3% w/w , 15.2% w/w ; 17.1% w/w ; 19% w/w/ Peaks correspond to the CMT.

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

283

be the dominant effect based on findings in the literature that the density of micellar systems tends to decrease with increasing temperature as micelles are formed. This is due to the lower density of the hydrophobic PPO chains in the core as compared to the hydrophilic PEO chains forming the micellar shell [38]. A further increase in temperature leads to an abrupt decrease in velocity which occurs at progressively higher temperatures as Pluronic concentration decreases. From comparison of the fluorescence results it can be seen that the temperature at which the deviation in ultrasound velocity occurs, corresponds to the region where the onset of excimer fluorescence is observed. Physically this is associated with desolvation of the PPO part of the polymer which leads to unimer aggregation and ultimately progressive formation of micelles [12]. Further, it has been reported in the literature that ultrasound velocity has a dependence on the number of particles in solution at a fixed concentration [25] suggesting a dependence on the surface area of the polymer phase. Thus, as the polymer phase aggregates the total surface area will go down, so will the amount of structured water and hence compressibility will increase. This effect could explain the observed decrease in velocity following the onset of aggregation. Changes in aggregation behaviour of the polymer can be further examined by taking the first derivative of velocity with respect to temperature as shown in Fig. 3(b). The form of the displayed curves is in agreement with those reported in the literature using resonant ultrasonic methods to measure velocity [20]. From observation of the plot it is noted that the temperature at which each curve has a stationary point corresponds to the temperature region in the fluorescence study where the peak in Ie/I3 ends. To probe the behaviour of the micellation phase transition in Pluronic F127 samples further the second derivative of velocity as a function of temperature was calculated, Fig. 3(c). From comparison of these results with the fluorescence results it is observed that the first stationary point in the second derivative occurs in the same temperature region as the peak in the fluorescence spectra. Thus, this indicates that, unlike findings reported previously in the literature [20] which suggest the stationary point in the first derivative corresponds to CMT, the CMT corresponds to the first stationary point in the second derivative. Earlier work in the literature studying the

The observed shape of the Ie/I3 curve, showing an initial monotonic increase, a peak and monotonic decrease, has been reported previously and is a result of the competition between increasing aggregation, which tends to increase Ie/I3, and increasing microviscosity, which tends to decrease Ie/I3 [35]. The experimental data plotted in Fig. 2 has been fitted with a piecewise cubic Hermite interpolating polynomial to preserve the monotonicity of the data [36]. The temperature at which Ie/I3 peaks has been reported to correspond to the critical micellisation temperature (CMT) [26] and in this work will be used to assess the sensitivity of ultrasound results to identify the CMT for each sample. This approach has been used widely to study micellation in Pluronic solutions enabling subtle changes in the microenvironment around the pyrene probe and microstructural changes in the micelles at the molecular level to be studied which will provide different information to techniques relying on multi-chain, bulk responses such as DSC [27]. 3.2. Ultrasound results The mean relative ultrasound velocity (Δv) of the three repeat experiments performed for all the Pluronic concentrations studied over a range of temperatures from 5 °C to 30 °C is reported in Fig. 3(a). The repeatability of these measurements can be assessed from the standard error in the mean which was found to be 3.5%. The relative error in each velocity calculation was calculated to be 4.5% through consideration of the uncertainty in measurement of the sample chamber length and the time of flight, which depends on the acquired data length and sampling frequency of the oscilloscope. Initial inspection of Fig. 3(a) indicates that the relative ultrasound velocity of the Pluronic solutions increases with concentration. This could be attributed to decreased compressibility due to both an increased volume fraction of polymer and greater amounts of structured water. For all the concentrations studied, the relative velocity initially increases linearly with temperature. As ultrasound velocity is inversely proportional to the square root of sample density and compressibility it is thought that this linear rise in velocity is due to a decrease in density, a decrease in compressibility or a combination of the two [37]. Here density is considered to

a

b

30 20 10 0 5

10

15

20

25

0 −0.025 −0.05 −0.075 5

30

d2(ΔV)/dT2 (m s−1 oC−2)

d(ΔV)/dT (m s−1 oC−1)

ΔV (m s−1)

40

Temperature (°C)

10

15

20

25

15 10 5

20

25

Temperature (°C)

−2

5

10

30

0.04 0.02 0 −0.02 5

10

15

20

25

Temperature (°C)

15

20

25

30

Temperature (°C)

30

f

−3

d2(Δα)/dT2 (Np m−1 oC−2)

d(Δα)/dT (Np m−1 oC−1)

Δα (Np m−1)

20

15

−1

−3

30

0.06

10

0

e

d

0 5

1

Temperature (°C)

25

c

−3

x 10

0.025

2

x 10

1 0 −1 −2 5

10

15

20

25

30

Temperature (°C)

Fig. 3. The effect of temperature on relative ultrasonic velocity (a) and its first (b) and second (c) derivatives; relative ultrasonic attenuation (d) and its first (e) and second (f) derivatives. 13.3% w/w , 15.2% w/w ; 17.1% w/w ; 19% w/w/ .

284

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

to the end of the fluorescence peak. In Fig. 3(f) the first stationary point in the second derivative occurs in the same temperature region as the peak in fluorescence results corresponding to the CMT. The temperature dependent transition in the Pluronic co-polymer solutions can more clearly be seen in Fig. 4 which presents the results for the solution with a concentration of 19% w/w. In the first column dashed vertical lines are used to indicate the temperature at which the abrupt change in velocity, Fig. 4(a), and attenuation, Fig. 4(d), is observed. Similarly dashed lines are used in Fig. 4(b) and (e) to highlight the temperature at which the stationary point in the curve occurs. Finally, Fig. 4(c) and (f) shows the temperature that corresponds to the first stationary point in the second derivative. Over the temperature range studied here it is known from the literature that solutions with concentration of 19% w/w will undergo a phase transition to form a gel [19]. This is verified by results of tube tilting tests carried out which found the gelation temperatures to be 22.5 °C for the 19% (w/w) sample, 26.5 °C for the 17.1% (w/w) sample and 31 °C for the 15.2% (w/w) sample. The 13.3% (w/w) sample did not undergo gelation. The results shown in Fig. 4 reveal that at the gelation temperature a plateau is found in the second derivative of both the velocity and attenuation results for the 19% (w/w) sample as indicated by the vertical dotted lines and it is postulated that this feature could be used to identify gelation. The correspondence between the ultrasound parameters discussed above with fluorescence results are summarised in Fig. 5. The temperature at the onset of the fluorescence peak is compared to the temperature at which the deviation in the ultrasound parameter–temperature curve occurs. The temperature at the fluorescence peak is shown with the temperature related to the first stationary point in the second derivative while the temperature at which the first derivative of the ultrasound parameters has a stationary point is shown alongside the end temperature of the peak in the fluorescence study. These findings show, based on the temperature resolution used in the fluorescence studies, that good agreement between the features extracted from the ultrasound parameters and the fluorescence peak properties is obtained. It is thus concluded that the parameters extracted from ultrasonic

energetics of micelle formation identified the critical micellation concentration as occurring at the concentration corresponding to the maximum change in the gradient in the physical property–concentration curve, where such physical properties included conductivity [39]. Our results for CMT are analogous to these and we conclude that the CMT can be defined as the temperature at which the maximum change in the gradient of the ultrasound velocity–temperature curve occurs. The relationship between relative ultrasound attenuation and temperature was also investigated for all of the Pluronic F127 concentrations studied and is presented in Fig. 3(d). As in the ultrasound velocity studies the repeatability of the three experiments was assessed through calculation of the standard error in the mean which for the attenuation measurements was 5%. The uncertainty in each attenuation measurement depends on the determination of the sample chamber length and the acquisition error in the vertical and horizontal scales of the oscilloscope, which was calculated to be 5%. From inspection of Fig. 3(d) it is observed that attenuation increases with increase in Pluronic concentration which could result from a rise in the number of scattering centres as polymer concentration increases as well as an increase in solution viscosity. For all the curves in Fig. 3(d) attenuation initially increases monotonically. With further temperature rise an abrupt change in attenuation is observed at a temperature which corresponds to the region where the onset of excimer fluorescence and the non-linear increase in ultrasound velocity are observed. As temperature rises further attenuation continues to climb. It is thought that this increase is associated with the drastic increase in solution viscosity, of several orders of magnitude, associated with micelle growth [38]. In addition, scattering losses could also contribute to attenuation due to the heterogeneity of the sample with concurrent existence of unimers, micelles and water molecules [20]. As the micellation process concludes the system becomes more homogeneous reducing scattering losses and overall attenuation. As in the case of the ultrasound velocity results, the first and second derivatives of attenuation as a function of temperature were calculated. Fig. 3(e) displays the results for the first derivative with curves being characterised by a stationary point at the temperature corresponding

a

30 20 10

10

15

20

25

30

−0.025 −0.05 −0.075

5

Temperature (°C)

15 10 5

10

15

20

25

Temperature (°C)

20

25

0 −1 −2 −3

30

5

10

30

0.04 0.02 0 −0.02

5

10

15

20

25

Temperature (°C)

15

20

25

30

Temperature (°C)

e

0.06

d(Δα)/dT (Np m−1 oC−1)

Δα (Np m−1)

20

0 5

15

1

Temperature (°C)

d

25

10

30

f

−3

d2(Δα)/dT2 (Np m−1 oC−2)

0 5

0

c

−3

x 10

d2(ΔV)/dT2 (m s−1 oC−2)

d(ΔV)/dT (m s−1 oC−1)

ΔV (m s−1)

40

b

0.025

2

x 10

1 0 −1 −2

5

10

15

20

25

30

Temperature (°C)

Fig. 4. The temperature dependence for the solution with concentration of 19% w/w of relative ultrasonic velocity (a) and its first (b) and second (c) derivatives; and relative ultrasonic attenuation (d) and its first (e) and second (f) derivatives. Temperatures associated with micellation are represented by a dashed line; and the gelation temperature highlighted by a dotted line .

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

20

Fluorescence Data Velocity Data Attenuation Data

19

Temperature (oC)

18 Peak End

17 Peak

16 Peak Onset

15 14 13 12 13

14

15

16

17

18

19

Pluronic Concentration (%(w/w)) Fig. 5. The correspondence between the ultrasound parameters and fluorescence results. The fluorescence peak onset is compared to the temperature at which the deviation in the ultrasound parameter–temperature curve occurs. The fluorescence peak is shown with the temperature related to the first stationary point in the second derivative while the temperature at which the first derivative of the ultrasound parameters has a stationary point is shown alongside the temperature of the peak end in the fluorescence study.

monitoring of Pluronic F127 hydrogels can be used to identify the micellation phase transition in the hydrogel which is necessary for the design of hydrogel drug delivery systems. In addition, the ultrasonic results obtained from the 19% solution suggest that the gel phase transition can also be detected.

285

ultrasound results than DSC results used previously as these rely on large scale changes in the sample to occur and are thus less sensitive to changes in micelle aggregation. A major application area of Pluronic hydrogels is in the delivery of therapeutic agents into the body. As such, it is pertinent that methodologies capable of studying their phase behaviour in vivo are developed. The ultrasound techniques used in this work could feasibly be adapted for application in a clinical setting. The through transmission set up using transmitting and receiving transducers could be used to reconstruct ultrasound tomography images of local velocity and attenuation. In this instance only the first arriving pulse would be required rather than signals from multiple reflections within the sample. The time evolution of changes in these images could then be used to track the polymer aggregation. Alternatively, currently used diagnostic ultrasound systems could be used in a pulse-echo format where the ultrasound transducer is used both to transmit and receive signals with the resulting images being analysed as a function of time to probe the hydrogel phase behaviour. Based on the results of this study it is concluded that ultrasound techniques may have a key role to play in the in vivo study and design of hydrogel systems used for therapeutic purposes. Acknowledgements The authors wish to acknowledge the financial contributions from EPSRC for the funding of Dr M Farrugia through a ‘remedi’ Doctoral Training Award (Grant Ref: GR/T07549) and for Dr M L Mather who is funded through a Career Acceleration Fellowship (Grant Ref: EP/ J001953/1). References

4. Conclusion The findings of this work provide evidence that widely accessible ultrasound equipment can be used in a simple arrangement to assess the temperature induced phase changes in Pluronic F127 hydrogels. The ultrasound studies carried out here are of direct relevance to those using Pluronic hydrogels in drug delivery applications as, unlike previous studies, a model hydrophobic drug, pyrene, was incorporated into all the samples. Through validation with a fluorescence spectroscopy technique it was determined that the temperature dependent micellation phase transition in Pluronic solutions could be identified through analysis of the relative changes in ultrasound velocity and attenuation as a function of temperature. The phase transition was more clearly detected through examination of the first and second derivatives of both ultrasound parameters with respect to temperature. In particular from comparison with the fluorescence results it was observed that an abrupt change in the ultrasound parameters occurs in the temperature region corresponding to the onset of the fluorescence peak. The first stationary point in the second derivative occurs at the temperature where the peak in the fluorescence spectra was observed and the stationary point in the first derivate occurs in the temperature region where the fluorescence peak ends, see Fig. 5. This demonstrates the previously reported broad transition process of polymer unimers to micelles. Further, unlike findings reported earlier in the literature [20] which suggest that the stationary point in the first derivative corresponds to CMT, this work indicates that the CMT corresponds to the temperature at the first stationary point in the second derivative. This may in part be due to the different approaches used for validation of ultrasound measurements. The previous studies performed by Glatter and Cespi used DSC for validation while our study used fluorescence spectroscopy results. The use of fluorescence spectroscopy in conjunction with pyrene is a well-established method to study micellation in Pluronics, particularly as it enables changes on a microscopic scale to be monitored. It is thus considered that the fluorescence studies of micellation used in this work are more suited to validation of the

[1] J. Kopecek, Hydrogel biomaterials: a smart future? Biomaterials 28 (34) (2007) 5185–5192. [2] W.E. Hennink, C.F. van Nostrum, Novel crosslinking methods to design hydrogels, Adv. Drug Deliv. Rev. 54 (2002) 13–36. [3] Q. Hou, et al., In situ gelling hydrogels incorporating microparticles as drug delivery carriers for regenerative medicine, J. Pharm. Sci. 97 (9) (2008) 3972–3980. [4] Y. Hong, et al., Collagen-coated polylactide microcarriers/chitosan hydrogel composite: injectable scaffold for cartilage regeneration, J. Biomed. Mater. Res. 85 A (2007) 628–637. [5] Y. Hori, et al., Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy, Biomaterials 29 (2008) 3671–3682. [6] J.S. Temenoff, A.G. Mikos, Injectable biodegradable materials for orthopedic tissue engineering, Biomaterials 21 (2000) 2405–2412. [7] S.H. Lee, et al., Enzyme-mediated cross-linking of Pluronic copolymer micelles for injectable and in situ forming hydrogels, Acta Biomater. 7 (4) (2011) 1468–1476. [8] J.C. Gilbert, J. Hadgraft, A. Bye, L.G. Brookes, Drug release from Pluronic F-127 gels, Int. J. Pharm. 32 (2–3) (1986) 223–228. [9] J.C. Gilbert, J.L. Richardson, M.C. Davies, K.J. Palin, The effect of solutes and polymers on the gelation properties of Pluronic F-127 solutions for controlled drug delivery, J. Control. Release 5 (2) (1987) 113–118. [10] S. Miyazaki, S. Takeuchi, C. Yokouchi, M. Takada, Pluronic F-127 gels as a vehicle for topical administration of anticancer agents, Chem. Pharm. Bull. 32 (10) (1984) 4205–4210. [11] J.J. Escobar-Chavez, M. López-Cervantes, A. Naik, Y.N. Kalia, D. Quintanar-Guerrero, A. Ganem-Quintanar, Applications of thermo-reversible pluronic F-127 gels in pharmaceutical formulations, J. Pharm. Pharm. Sci. 9 (3) (2006) 339–358. [12] G. Bonacucina, M. Cespi, G. Mencarelli, G. Giorgioni, G.F. Palmieri, Thermosensitive self-assembling block copolymers as drug delivery systems, Polymers 3 (2) (2011) 779–811. [13] W. Zhang, et al., Synthesis and characterization of thermally responsive Pluronic f127–chitosan nanocapsules for controlled release and intracellular delivery of small molecules, ACS Nano 4 (11) (2010) 6747–6759. [14] R.M. Nalbandian, R.L. Henry, H.S. Wilks, Artifical skin. II. Pluronic F-127 silver nitrate or silver lactate gel in the treatment of thermal burns, J. Biomed. Mater. Res. 6 (1972) 583–590. [15] B. Jeong, S.W. Kim, Y.H. Bae, Thermosensitive sol–gel reversible hydrogels, Adv. Drug Deliv. Rev. 54 (1) (2002) 37–51. [16] H.J. Jung, A. Chauhan, Temperature sensitive contact lenses for triggered ophthalmic drug delivery, Biomaterials 33 (7) (2012) 2289–2300. [17] A. Steinleitner, H. Lambert, C. Kazensky, B. Cantor, Poloxamer 407 as an intraperitoneal barrier material for the prevention of postsurgical adhesion formation and reformation in rodent models for reproductive surgery, Obstet. Gynecol. 77 (1) (1991) 48–52. [18] H. Yu, et al., Distraction osteogenesis combined with tissue-engineered cartilage in the reconstruction of condylar osteochondral defect, J. Oral Maxillofac. Surg. 69 (12) (2011) 558–564.

286

M. Farrugia et al. / Materials Science and Engineering C 34 (2014) 280–286

[19] M.J. Song, D.S. Lee, J.H. Ahn, D.J. Kim, S.C. Kim, Dielectric behaviour during sol–gel transition of PEO–PPO–PEO triblock copolymer aqueous solution, Polym. Bull. 43 (2000) 497–504. [20] M. Cespi, G. Bonacucina, G. Mencarelli, S. Pucciarelli, G. Giorgioni, G.F. Palmieri, Monitoring the aggregation behaviour of self-assembling polymers through high-resolution ultrasonic spectroscopy, Int. J. Pharm. 388 (2010) 274–279. [21] J. Rassing, D. Attwood, Ultrasonic velocity and light-scattering studies on the polyoxyethylene–polyoxypropylene copolymer Pluronic F127 in aqueous solution, Int. J. Pharm. 13 (1) (1982) 47–55. [22] K. Takeda, T. Norisuye, Q. Tran-Cong-Miyata, Origin of the anomalous decrease in the apparent density of polymer gels observed by multi-echo reflection ultrasound spectroscopy, Ultrasonics 53 (5) (2013) 973–978. [23] M.L. Mather, A.K. Whittaker, C. Baldock, Ultrasound evaluation of polymer gel dosimeters, Phys. Med. Biol. 47 (9) (2002) 1449–1458. [24] C.L. Moore, J.A. Copel, Point-of-care ultrasonography, N. Engl. J. Med. 364 (2011) 749–757. [25] O. Glatter, G. Scherf, K. Schillen, W. Brown, Characterization of a poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (EO27–PO39–EO27) in aqueous solution, Macromolecules 27 (1994) 6046–6054. [26] M. Bohorquez, C. Koch, T. Trygstad, N. Pandit, A study of the temperature-dependent micellization of Pluronic F127, J. Colloid Interface Sci. 216 (1) (1999) 34–40. [27] P. Chandar, P. Somasundaran, N.J. Turro, Fluorescence probe investigation of anionic polymer–cationic surfactant interactions, Macromolecules 21 (4) (1988) 950–953. [28] I.R. Schmolka, Artificial skin I. Preparation and properties of pluronic F-127 gels for treatment of burns, J. Biomed. Mater. Res. 6 (6) (1972) 571–582.

[29] P. Alexandridis, T. Alan Hatton, Poly(ethylene oxide)-poly(propylene oxide)poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling, Colloids Surf. A Physicochem. Eng. Asp. 96 (1–2) (1995) 1–46. [30] A. Yekta, et al., A fluorescent probe study of micelle-like cluster formation in aqueous solutions of hydrophobically modified poly(ethylene oxide), Macromolecules 26 (8) (1993) 1829–1836. [31] J.B. Birks, L.G. Christophorou, Excimer fluorescence spectra of pyrene derivatives, Spectrochim. Acta 19 (2) (1963) 401–410. [32] T. Nivaggioli, et al., Fluorescence probe studies of Pluronic copolymer solutions as a function of temperature, Langmuir 11 (3) (1995) 730–737. [33] J. Krautkrämer, H. Krautkrämer, Ultrasonic Testing of Materials, Springer-Verlag, 1983. [34] P. Alexandridis, J.F. Holzwarth, T. Alan, Micellization of Poly(ethylene oxide)-Poly(propylene oxide)-Poly(ethylene oxide) Triblock Copolymers in Aqueous Solutions: thermodynamics of Copolymer Association, Macromolecules 27 (9) (1994) 2414–2425. [35] N.J. Turro, P.L. Kuo, Pyrene excimer formations in micelles of nonionic detergents and of water-soluble polymers, Langmuir 2 (4) (1986) 438–442. [36] S.A.B.A. Karim, N. Yahya, Seabed logging data curve fitting using cubic splines, Appl. Math. Sci. 7 (81) (2013) 4015–4026. [37] L.E. Kinsler, Fundamentals of Acoustics, Wiley, 2000. [38] J.G. Álvarez-Ramírez, et al., Phase behavior of the Pluronic P103/water system in the dilute and semi-dilute regimes, J. Colloid Interface Sci. 333 (2) (2009) 655–662. [39] J. Phillips, The energetics of micelle formation, Trans. Faraday Soc. 51 (1955) 561–569.