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Master in physics and physical technologies Master Thesis Remote drug release study using ferrogels under AC fields Ignacio J. Bruvera Supervisor:Dr.Gerardo F. Goya Rosetti 24 June 2012 CONTENTS 1 Contents 1 Introduction 2 1.1 Magnetic nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 Hydrogels ................................... 4 1.3 Ferrogels:State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 Experimental 7 2.1 Samplesynthesis ............................... 7 2.2 Sample characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2.1 VSM ................................. 7 2.2.2 SEM ................................. 7 2.2.3 TEM ................................. 7 2.2.4 RFresponse ............................. 8 2.2.5 Test drug. B12 vitamin: . . . . . . . . . . . . . . . . . . . . . . . 8 2.3 Devicedesign ................................. 9 2.3.1 Water circuit and pump: . . . . . . . . . . . . . . . . . . . . . . 9 2.3.2 RF field generator . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.3.3 Dewar ................................. 12 2.3.4 Photometer .............................. 12 2.4 Releaseexperiments ............................. 14 2.4.1 Blank ................................. 14 2.4.2 Ferrogel ................................ 14 3 Results 15 3.1 Sample characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.1.1 VSM ................................. 15 3.1.2 SEM ................................. 15 3.1.3 TEM ................................. 15 3.1.4 RFresponse ............................. 16 3.2 Photometer calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.3 Releaseexperiments ............................. 19 3.3.1 Blank ................................. 19 3.3.2 Ferrogel ................................ 19 4 Discussion and conclusions 24 4.1 Sample characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.2 Deviceperformance.............................. 25 5 Acknowledgements (in Spanish) 25 6 References 26 Remote drug release study using ferrogels under AC fields Ignacio Javier Bruvera E-mail: [email protected] Director:Gerardo F. Goya Rossetti Instituto de Nanociencia de Arag´on (INA). Universidad de Zaragoza Ed. I+D - Calle Mariano Esquillor s/n Campus Rio Ebro 50018-Zaragoza Phone: (34) 976 76 2777 (ext 2987) Fax: (34) 976 76 2776 web: http://www.unizar.es/gfgoya Abstract. A dedicated device was built with the purpose of studing the remotely controlled drug release of a temperature sensitive hydrogel in a water flow. Samples of the hydrogel (semi-interpenetrating polymer networks constituted by alginate and PNIPAAm) with in situ synthesized magnetite nanoparticles (average size 10nm) were characterised by TEM, STEM/EDS, SEM, VSM and power dissipation measurements. These results were used to design and construct a system able to measure the drug release of the samples in a water flow when exposed to an external RF field. The device was calibrated to obtain absolute values of drug concentration in the water flow in real time by a built-in photometer. Vitamin B12 was used as model drug. First results indicate a good response of both the sample and the device. Field induced drug liberation events were clearly recorded. A liberated B12 mass in the order of 10ng was calculated for each event. Further experiments are necessary for a complete characterization of the drug release process. 1. Introduction 1.1. Magnetic nanoparticles Magnetic nanoparticles (MNP) represent a very interesting field of study, both in the basic research as in applied science. In particular, its uses in biomedicine are numerous and with a intense development in the past decade [1]. One of the most commonly exploited characteristic of the MNP is its capacity of Remote drug release study using ferrogels under AC fields 3 Figure 1. Basic shape of the Magnetization vs. Field plots for ferromagnetic (FM) big and small particles and superparamagnetic (SPM) particles (from [2]). absorbing power from an AC electromagnetic field and then release it in the surrounding medium as heat. An important application of this phenomena is the Magnetic Fluid Hyperthermia (MFH) therapy used in cancer therapy. This technique consists in the injection of a suspension of MNP, called ferrofluid (FF), in the body of the patient and the exposure of the affected region to a AC field in order to increase its temperature. Tumour cells, more sensitive to high temperatures than healthy ones, then decrease their viability[3]. The MNP can be functionalized to interact preferentially with tumour cells and concentrate in the affected zone [6][7]. The size of the MNP is determinant for its magnetic response. Particles above 1µm are multidomain, they contain multiple regions of coordinated magnetic alignment separated by domain walls which require some energy to be moved. When a external field is applied, the domains with a magnetization parallel to the field direction grow at the expense of the rest. This can be seen in a magnetization vs. field measurement as a narrow hysteresis loop compared with the response of a bulk sample of the same material which has bigger domain walls. For smaller particles, the domain walls turn too expensive in energy since the surface to volume ratio increases. So they present a single domain state with a wider hysteresis loop since the whole particle must change its magnetization during the process. In particles under 100µm size, the magnetic moment is free to fluctuate due to thermal agitation and the remanence of the system disappear. This is called superparamagnetism (SPM)[2](Fig. 1). The field power to heat conversion occurs in the SPM particles by two principal mechanism. Due the interaction between the MNP magnetic moment and the external field, the MNP can change the direction of its total magnetization or, if is allowed, rotate to reach the minimum energy orientation. In the first case, called N´eel relaxation, a uniaxial MNP dissipates power when crossing the potential barrier between its two easy magnetization directions. In the second case, called Brown relaxation, the dissipation occurs due the viscous interaction with the medium(Fig.2). When this mechanism compete the fastest prevails. Remote drug release study using ferrogels under AC fields 4 Figure 2. Schematic of the two relaxation mechanisms present when a FF of SPM particles is exposed to a RF field of amplitude Hcand frequency f. In N´eel mechanism the magnetic moment orientation switches between the two easy axis jumping the anisotropy energy barrier ∆E. In Brown relaxation the MNP dissipate power when rotate in a medium with viscosity η[5]. 1.2. Hydrogels Hydrogels are crosslinked polymeric networks that absorb and retain large amounts of water[8]. The characteristic network structure of hydrogels is responsible for their unique ability to undergo abrupt volume changes in response to environmental stimuli such as change in pH, temperature or ionic strength[9]. It is known that the degree of swelling of gel is controlled by the free energy changes associated to both (i) the mixing of the polymer and water and (ii) the network elasticity. Related to that, there exist two general types of responsive microgels: (i) LCST-type (lower critical solution temperature) microgels such as the widely studied poly(N-isopropylacrylamide) and (ii) UCST-type (upper critical solution temperature) microgel such as poly(acrylamideacrylic acid) first reported by Bouillot and Vincent. Systems that show LCST-type volume phase transition temperature present a negative change of enthalpy and entropy. As the temperature increases, the entropic contribution to the energy grows leading to the phase separation and, thus, the shrinkage of microgel; therefore, LCST-type systems collapse upon heating. On the contrary, UCST-type microgel exhibits a positive swelling response related to the positive change of the enthalpy and entropy. The swelling behavior, and hence volume phase transition, is driven by hydrogen bonding that causes the microgel to shrink at temperatures below the UCST and swell at temperatures above the UCST[10]. 1.3. Ferrogels:State of the Art A method to obtain ferrogels with response to temperature and magnetic fields has been recently described. Iron oxide magnetic nanoparticles (MNP) synthesis is carried out through coprecipitation of iron salts in alkaline solutions inside semiinterpenetrating (semiIPN) polymer networks constituted of alginate and poly(N- isopropylacrylamide)(PNIPAAm)[11]. These ferrogels exhibit an improved deswelling rate with respect to pure PNIPAAm. In addition, the synthesis in situ of MNP inside Remote drug release study using ferrogels under AC fields 5 Figure 3. Schematic showing the effect of ONOFF cycles of a RF field on the magnetic nanocomposites of NIPAAm. It shows the field triggered collapse and resultant burst release due to squeezing effect.LCST=lower critical solution temperature [4] . Alg-PNIPAAm semi-IPNs allows controlling the polydispersity of the particles when compared to the reaction carried out in an alginate solution. The polymeric gel acts as a spatial framework to control the MNP size distribution [12]. Results on magnetic properties and heating experiments on hydrogels derived from polysaccharides (chitosan and alginate) have been reported showing good response to external radiofrequency (RF) magnetic fields, opening the possibility of using them as smart drug delivery materials for bioapplications[13]. The general motivation for this work is the study of magnetic hybrid poly(acrylamideacrylic acid) thermoresponsive random copolymer hydrogel samples as a remote controlled drug release device. The hydrogel presents a tunable temperature triggered face transition that can be driven by an external RF field thanks to the MNP contained in the sample. In this way, a portion of gel hydrated in a water soluble drug solution can be use as a carrier and controlled releaser of this drug in vivo. (Fig.3) Static release experiments have been made but not published yet by Hern´andez et al[14]. In this experiments, the liofilised hydrogel samples were first immersed in a PBS solution (12.5 g/l vitamin B12) for 48 h. The amount of B12 taken by the gel was determined by spectrophotometric measurements of small volumes of the solution: Remote drug release study using ferrogels under AC fields 6 a calibration was made first by measuring the absorbance at 361nm of B12 solutions of known concentration in a UV-Vis spectrophotometer. The samples from the gel bath were diluted and then measured in the same way. For the release experiment the gels were transferred into 10 ml of PBS at 37oC. Samples of 0.2 ml of the bath were taken at certain times and replaced by the same volume of PBS so that the total volume remained constant (10 ml). Each aliquot was diluted to 3 ml to measure absorbance at 361nm. The experiment was repeated with the PBS bath at 25oC. The comparison of the two experiments shows important differences between them: the hydrogel at 37oC completely released the loaded vitamin B12 and the release occurs faster and in larger extension due to the collapse of the hydrogel. The natural next step in the direction of an in vivo application is the study of the dynamic release of the gel under a RF field in a water flow. With that propose, an ad hoc device must be constructed. This device must: •Circulates a controlled water flow around the sample. •Exposes the sample to a controlled RF field. •Be adiabatic. •Measures water and sample temperatures. •Measures B12 concentration in the water flow in function of time. Remote drug release study using ferrogels under AC fields 7 2. Experimental 2.1. Sample synthesis Semi-interpenetrating polymer networks constituted by alginate and PNIPAAm were obtained as reported elsewhere[11],[12]. The synthesis of iron oxide nanoparticles inside Alg-PNIPAAm semi-IPN hydrogels involves two steps. As a first step, gels are immersed in an acid solution containing Fe2+ and Fe3+. As a second step, the gels are carefully immersed in an alkaline solution to oxidize the iron cations to iron oxide nanoparticles[13]. The mass of iron oxide nanoparticles in the ferrogels (WNP) was obtained from thermogravimetric analysis of dried ferrogels [11],[15]. The total water content, Wt, was calculated from the weight of the hydrated gels (Ms) and the weight of the dried gels (Mdry) as Wt=Mdry −Ms/Mdry −MNP (I) The samples were shaped as cylinders with 1cm diameter 4cm long and liofilized for a better manipulation. 2.2. Sample characterization 2.2.1. VSM : Small dry portions of the ferrogel (6.5(1)mg) and blank (14.2(1)mg) samples were measured in a Vibrating Sample Magnetometer (LakeShore 7304). A magnetization cycle in the range [-1.9,1.9]T was performed for each sample with a different point to point step for each field region (B∈[0,10]mT →∆B= 0.5mT, B ∈ (10,100]mT →∆B= 10mT and B∈(100,1900]mT →∆B= 100mT). 2.2.2. SEM : Low vacuum SEM mesurements were performed in a environmental SEM-FEG (FEI Quanta 250). Ferrogel and blank samples in fully hydrated state were observed under different temperature/pressure/humidity conditions. 2.2.3. TEM : Conventional TEM images of dried ferrogel samples were taken by Hern´andez et al [13]. Additionally, samples were prepared by plunge-freezing in liquid ethane. Then, thin slices (less than 100nm) of the material were cut with the ultra-cryomicrotome, deposited on a copper grid and transferred under liquid nitrogen atmosphere (T=100K aprox.) to the TEM. The low temperature preserves the original state of the material; also, quick vitrification of the samples avoids the formation of crystalline ice, whose diffraction would interfere with the TEM observation. Samples were then observed at liquid nitrogen temperature in a Tecnai T20 (FEI) equipped with a Lab6 filament and operated at 200KV. Bright field images were collected in a CCD camera with an acquisition time of 1s. The same samples were also observed in a Tecnai F30 (FEI) equipped with a Field Emission Gun (FEG) operated at 300KV in Scanning Transmission (STEM) mode. A Remote drug release study using ferrogels under AC fields 8 Figure 4. Heating curve of the hydrogel under RF field.t=0 in II indicates the moment of turning on the field. narrow probe (1nm aprox in diameter) was formed and scanned over the surface of the sample. Thus, a High Angle Annular Dark Field (HAADF) image and a an Electron Dispersive X-Ray spectrum (EDS) were collected simultaneously for each pixel[17]. 2.2.4. RF response : Power dissipation measurements were performed using a commercial AC applicator (EasyHeat)working at 360 kHz and field amplitudes up to 45kA/m equipped with an adiabatic sample space (∼0.5 ml) for measurements in liquid phase. Temperature data were taken using a fiber optic temperature probe (Reflex, Neoptix) immune to radio frequency environments. The power dissipated by the gel under the RF field PRF is determined from the initial slope ∂T ∂t (0) of the T vs.t plot of the experiment (Fig.4) with the expression PRF =cGmG ∂T ∂t (0) (II) where cGand mGare the specific heat an mass of the gel sample. 2.2.5. Test drug. B12 vitamin: The test drug for the experiment must have two principal characteristics: •Water solubility: the gel will be loaded with the drug by submerging it in an aqueous solution and will deswell in a water flow. •Adequate optical activity: The determination of the drug concentration in the water flow will be made by spectrophotometric method. Therefore the test drug must have a well known absorption peak in an accessible wavelength range. There are more things to consider. The drug must be affordable since a big quantity Remote drug release study using ferrogels under AC fields 15 3. Results 3.1. Sample characterization 3.1.1. VSM : Figure 14 shows the results for the magnetization measurements of ferrogel and blank. The blank response is a diamagnetic curve with a slope of -3.52Am2 Tkg which is typical for organic materials like the components of the hydrogel. The ferrogel response is clearly SPM with a saturation magnetization of 13.23(1)Am2 Tkg and negligible remanence of 0.10(1)Am2 Tkg and coercive field of 1.3(1)G. Figure 14. Magnetization(magnetic moment per sample mass) vs. field plot for ferrogel(a) and blank(b). The response of the blank is clearly diamagnetic. The ferrogel curve corresponds to a practically SPM response with very small remanence and coercive field(magnification inserted). Offset due off-centered sample. 3.1.2. SEM : Figures 15, 16 and 17 show the topology of the gels. Besides of the expected pore structure([20,100]µm pore size),small clusters of micron size can be seen in all images of blank and ferrogel samples. The amount of clusters tend to increase when temperature rises and humidity decrease. Although the measurements were made in low vacuum conditions ([540, 760]Pa), the initially hydrated samples lose all its water in the process ending the experiment completely dried. Changes in the structure of the pores can be notice between the beginning and the end of the measurements. 3.1.3. TEM : From the conventional TEM result shown in figure 18, a MNP average size of approximately 10nm can be observed. The MNP appear agglomerated in clusters inside the gel. The measurements of the vitrificated samples was difficult. The quantity of material attached to the grid was very little and, even at liquid nitrogen temperature, the electron beam deteriorates the sample which reacts with violent movements preventing to take long exposition images. Even so, a net structure three orders of magnitude smaller that the one observed by SEM can be notice in Fig. 19. Remote drug release study using ferrogels under AC fields 16 Figure 15. SEM images from the blank sample taken at 760Pa where the pore structure of the gel can be notice. At low temperature and high humidity (A,B) small clusters can be seen. At high temperature and low humidity (C,D)the number of cluster increase. Trying to obtain an image of the MNP inside the gel, the EDS analysis was performed in all the available material in the grid(Fig. 20). Although the presence of Fe was confirmed, no MNP could be observed. Instead, multiple materials contamination was detected in form of micro and nanoparticles like the one shown in figure 20. 3.1.4. RF response : Figure 2 shows a representative temperature vs. time measurement of a sample of ferrogel while exposed to the RF field. After a short transition time, the curve turns linear so the process can be taken as adiabatic. A linear function fit of the adiabatic section of the curve was used to obtain the slope ∂T ∂t (0) for the power dissipation calculation II. The final value of 1.73(1)W for a field amplitude of 45kA/m was the result of a three determinations average. With this information, a Remote drug release study using ferrogels under AC fields 17 Figure 16. SEM images from the ferrogel taken at 760Pa, 3oC and 100% RH. Cluster structures similar to those in the blank appear. maximum caudal of 0.41ml/s was calculated to maintain a 1K difference between the extremes of the sample. 3.2. Photometer calibration From the voltage vs. time curve obtained directly from the diodes (Fig.21) a calibration curve was made (Fig. 22) which shows a typical Lambert-Beer exponential dependence between the diode voltage and the B12 concentration in the water flow: V(c) = Ae−c/τ +V0(V) Where cis the concentration, A+V0is the value of the signal whit pure MiliQ water circulating (c= 0) and τis the constant where the optic path and the molar extinction coefficient are contained. While the value A+V0depends on the light intensity emitted Remote drug release study using ferrogels under AC fields 18 Figure 17. SEM images from the same section of a ferrogel sample in the initial measurement conditions: 3oC, 760Pa, 100%RH (left) and final measurement conditions: 40oC, 580Pa, 21.6%RH(right). Differentiated surface and lateral section can be observed. Structural modifications can be notice. Figure 18. Representative TEM images of the MNP inside the gel. The particles appear agglomerated with a individual average size of 10nm (image from [13]). by the lamp, τmust be the same for all the calibrations if the absorbent substance and the geometry of the photometer remains unaltered. The exponential fit confirms the linear relation between the light intensity and the diodes signal since the later maintain the same Lambert-Beer dependence with the concentration than the first should follow in the conditions of the experiment [16]. Six closed tubes filled with B12 solutions at different concentrations were assembled to perform periodic calibrations. The tubes have exactly the same geometry and Remote drug release study using ferrogels under AC fields 19 Figure 19. TEM images of the vitrificated ferrogel sample. A net structure with 50nm holes can be noticed. constitution of the one in the photometer and can be exchanged with these one. This system allows to perform the calibration protocol in less than 10 minutes. 3.3. Release experiments 3.3.1. Blank : The voltage readings from the diodes in function of time for the three stages of the experiment show the response of the photometer to the change in light intensity due the variations in B12 concentration. The system was able to detect even the bubbles circulating trough the circuit (Fig.23). No effect of the field exposure was detected. 3.3.2. Ferrogel : Figure 24 shows the data from the field induce liberation experiment described in 2.4.2. In A, the complete record is shown: the initial signal of 2V corresponds to the circulating B12 solution used to load the gel for 24h (time scale was restarted). Around t=20mn the B12 is replaced by pure MiliQ water to wash the whole device and the sample so the only B12 left is that inside the gel. After 100mn the signal was stable in the c=0 equivalent value. Magnification of the three liberation events induced by the field application are shown in B,Cand D. The correlation between liberation and field application is clear since the effect of the field can be seen in the data as electromagnetic noise before every concentration peak. The amount of vitamin released by the gel is smaller each time. From the acquired data is possible to determine the absolute mass mof B12 vitamin expelled each time. Converting the voltage values V(t) to concentration c(t) by the equation V, the amount of B12 liberated is equal to the peak area ,which can be Remote drug release study using ferrogels under AC fields 20 Figure 20. STEM images an EDS analysis of the ferrogel sample. Small peaks correspondig to Fe presence can be notice but no MNP was could be detected. A micron size particle of Ca is shown in B has example of the contamination. estimated by a numerical integration, multiplied by the caudal Q: m=QZtf t0 c(t)dt (VI) where t0and tfdetermine the duration of the liberation event. Using VI with the first peak, a liberated B12 mass of 25.3(2.6)ng is obtained. Remote drug release study using ferrogels under AC fields 21 Figure 21. Voltage vs. time plot of the calibration experiment. Starting with pure water (maximum signal), small volumes of concentrated B12 solution were added every certain time. Each addition rises the B12 concentration in the circuit provoking a decrease in the light intensity reaching the diodes and therefore, a decrease in the signal. Figure 22. Voltage vs.concentration plot from the calibration experiment. The voltage is proportional to the light intensity in the range [450-600]nm. The average values from every plateau in the voltage vs. time plot were plotted vs. the calculated concentration. A typical Lambert-Beer exponential dependence can be notice. Remote drug release study using ferrogels under AC fields 22 Figure 23. Diode voltage vs. time plot corresponding to the first experiment where B12 vitamin is added to the circulating water. A very significant drop in the light intensity can be seen when the diluted B12 reaches the photometer (t∼75mn). The signal stays steady when the B12 is equally distributed in the whole circuit.Insert: Magnification of the red square. Light intensity fluctuations caused by air bubbles in the circuit Remote drug release study using ferrogels under AC fields 23 Figure 24. Results of the field induced liberation experiment with the ferrogel. The Y axis voltage (Ch1) is proportional to the light intensity reaching the diodes and so, it can be translated to B12 concentration through expression [16]. Ashows the complete experiment: the initial signal of 2V corresponds to the B12 solution circulating. Then, around t=20mn, the solution is replaced by pure MiliQ water and the signal rise to 7.4V. In t={120mn, 200mn, 274mn}the peaks of concentration produced by the field induced liberation can be seen. Magnifications of the peaks shown in B,Cand D. The interference before every peak is caused by the RF field. Remote drug release study using ferrogels under AC fields 24 4. Discussion and conclusions The are two aspects to speak about in this work: the sample characterization and the device design, construction and performance. Although the original motivation was only the first, problems with the RF generator prevented from continuing with the experiments and achieve a complete characterization of the drug release process. Also, the design, construction and calibration of the device proved to be a complex and interesting objective on his own. 4.1. Sample characterization The morphology of the gel was studied by low vacuum SEM and cryo-TEM. Since the conventional TEM measurements were done by the collaboration that synthesized the samples, the intention was to exploit INAs facilities and study the gels in his native state i.e. hydrated. This proved to be very difficult due to the extreme sensibility of the samples to the environmental conditions. The aim of the environmental SEM experiment was to try to observe the structural face transition of the sample. Although this device is designed to perform the measurements in a more ”biological samples friendly” conditions than the conventional SEM, it wasn’t enough for the hydrogels. The 760Pa pressure is still much smaller than the atmospheric value and the initially hydrated gel lose his water content very fast. However, the general pore structure of the gel was observed with good definition at the micron scale. Also, a difference between the aspect of the sample at 3oC and 40oC was observed. The ferrogel sample was study first so the micron sized clusters were expected to be made of MNP, but the posterior observation of the same objects in the blank sample eliminate that possibility. This finding indicates that the cluster are made of the same material of the gel. The irreversible proliferation of the clusters with the temperature rising suggests that they are the product of some kind of degradation process in the material. The cryo-TEM measurements were less productive. In the same direction of preserving the native structure of the sample, the conventional fixation process for soft specimens was avoided. The sample reacted not so well to the vitrification process and a very small quantity ended in the copper observation grid. Also, as already stated in 3.1.3, the electron beam provoked the small pieces of gel to jump out of the grid. With these conditions it was impossible to obtain even one image of a MNP. The STEM-EDS measurements were able to confirm the existence of Fe in the sample (although this was already evident from the magnetization and calorimetric experiments commented ahead) but also failed to provide a MNP visualization. Instead, a lot of particles of a priori unexpected compositions appeared. This contamination couldn’t be explained yet. The principal conclusion from this results is that a better technique to preserve the native state of the sample during TEM observation must be found. The magnetic and power dissipation responses of the samples were the expected one: a clearly SPM response typical for magnetite MNP of that size in the ferrogel, and