J Polym Res (2014) 21:580 DOI 10.1007/s10965-014-0580-7
ORIGINAL PAPER
Synthesis and characterization of thermosensitive poly(N-isopropylacrylamide-co-hydroxyethylacrylamide) microgels as potential carriers for drug delivery Sanda Bucatariu & Gheorghe Fundueanu & Irina Prisacaru & Mihaela Balan & Iuliana Stoica & Valeria Harabagiu & Marieta Constantin
Received: 7 May 2014 / Accepted: 30 September 2014 # Springer Science+Business Media Dordrecht 2014
Abstract Thermoresponsive colloidal microgels were prepared by precipitation copolymerization of N-isopropylacrylamide (NIPAM) and N-hydroxyethylacrylamide (HEAM) with various concentrations of a cross-linker in the presence of an anionic surfactant, sodium dodecylsulphate (SDS). The volume phase transition temperature (VPTT) of the prepared microgels was studied by dynamic light scattering (DLS), ultraviolet–visible spectroscopy (UV–vis) and proton nuclear magnetic resonance (1H-NMR) spectroscopy. In addition, atomic force microscopy (AFM) was used to characterize the polydispersity and morphology of the microgels. Results indicated that poly(NIPAMco-HEAM) microgels are spherical and monodisperse. VPTTs of microgels determined by DLS and UV–vis methods are almost the same and very close to the human body temperature, presenting the microgels as candidates for biomedical application. The temperature at which the phase transition occurred is nearly independent of the cross-linking density, whereas the transition range is deeply influenced by temperature. Also, the SDS concentration was increased to decrease the average hydrodynamic size of the microgels, due to the electrostatic repulsion between the charged particles during the polymerization process. 1H-NMR spectra of the microgels show a decrease in peak intensity with an increased temperature due to a reduction Paper dedicated to the 65th anniversary of “Petru Poni” Institute of Macromolecular Chemistry of the Romanian Academy, Iasi, Romania. Electronic supplementary material The online version of this article (doi:10.1007/s10965-014-0580-7) contains supplementary material available to authorized users. S. Bucatariu : G. Fundueanu : I. Prisacaru : M. Balan : I. Stoica : V. Harabagiu : M. Constantin (*) Department of Natural Polymers, Bioactive and Biocompatible Materials, “Petru Poni” Institute of Macromolecular Chemistry, Gr. Ghica Voda Alley, 41A, 700487 Iasi, Romania e-mail:
[email protected]
in molecular mobility of the polymer segments. Release rates of propranolol from microgels are deeply influenced by temperature; below the VPTT at 25 °C, the drug is rapidly released at a rate comparable to that of a free drug, whereas above the VPTT (37 and 42 °C), a fraction of the drug is mechanically expulsed in the first five min, followed by a prolonged release. Keywords poly(N-isopropylacrylamide) . Microgel . Smart network . Drug delivery
Introduction The field of microgels research is marked by some important milestones, such as the preparation of the first microgel in 1935, represented by poly(divinylbenzene) particles [1], the synthesis of the first thermally sensitive microgel based on poly(N-isopropylacrylamide) (poly(NIPAM)) in 1986 [2], the consequent discovery of the importance of surfactants [3], and copolymerization [4, 5]. Microgels can be defined as cross-linked three-dimensional networks able to absorb a solvent to a high degree without dissolution [6], with the average particle diameter ranging between 50 nm and 100 μm. Microgels show clear unique advantages in comparison with other polymer systems: a fast response rate to external stimuli due to their colloidal particle nature; suitability for subcutaneous administration [7]; good biocompatibility since they contain mostly water in the swollen state [8]; a large surface area for multivalent bioconjugation; an internal network for the incorporation of therapeutic drugs; adjustable chemical and mechanical properties [3]; and soft architecture enabling them to flatten themselves onto vascular surfaces, thus simultaneously anchoring in multiple points [9].
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Environmentally-responsive microgels have attracted great attention due to their potential applications in biosensing [10, 11] drug delivery [12–16], chemical separation [17, 18], catalysis [19, 20] and optical devices [21, 22], etc. The most studied responsive microgels are those based on temperaturesensitive poly(NIPAM) [23–26] because they undergo a largemagnitude volume change around 34 °C (called the volume phase transition temperature, VPTT) [27]. Poly(NIPAM)based microgels swell if the temperature is below the VPTT and shrink if the temperature increases above the VPTT. This process is generally attributed to the reversible formation and breakage of the hydrogen bonding between water molecules and hydrophilic groups, and the hydrophilic/ hydrophobic balance between hydrophilic and hydrophobic groups within poly(NIPAM) polymer chains [28]. However, the biomedical and biological applications of poly(NIPAM) require a sharp phase transition around the physiological temperature of 36.5 °C. The common way to elevate the VPTT of poly(NIPAM) microgels is copolymerization with other hydrophilic co-monomers resulting in alteration of the hydrophilic-hydrophobic balance of the copolymer [29–31]. The hydrophilic comonomers, usually diminish the thermosensitivity; however, if the comonomer is properly selected, the gels preserve the thermosensitive properties [32]. Other chemical approaches for tuning microgel VPTTs are the use of hydrophilic cross-linking agents [33] and the incorporation of nanoparticles [34, 35] in microgel networks during the polymerization process. The copolymer of NIPAM and N-hydroxyethylacrylamide (HEAM) with a molar ratio of 10:2 has a coil-globule transition at around 37 °C in simulated physiological conditions (phosphate buffer at pH=7.4, (PB)) [36]. This composition was used in the present study to prepare a thermosensitive poly(NIPAM-co-HEAM) microgel with a spherical shape and a relatively narrow size distribution via precipitation polymerization. It should be noted that hydroxyl groups of HEAM could help direct selective molecule recognition through physical interaction (e.g., H-bonding) and can easily be modified chemically [37]. Different cross-linker and SDS amounts were used for determining the optimum properties of microgels in terms of size, homogeneity and VPTT. Finally, the potential use of microgels as thermosensitive drug carriers was proved.
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recrystallized from methanol. Potassium persulfate (KPS) and the anionic surfactant sodium dodecyl sulfate (SDS) (Aldrich Chemical Corp., Milwaukee, WI, USA) were used without further purification. Propranolol hydrochloride was supplied from Sigma-Aldrich Co, St. Louis, USA. Methods Preparation of poly(NIPAM-co-HEAM) microgels The poly(NIPAM-co-HEAM) microgels were synthesized by free radical precipitation polymerization in a sealed threenecked round-bottom flask equipped with a condenser, a magnetic stirrer and a temperature-controlled oil bath. In a typical example, NIPAM, HEAM, BIS and SDS were dissolved in 75 mL of ultrapurified water. The solution was filtered to remove any possible precipitates and then transferred into the reaction flask connected to a nitrogen inlet and heated to 70 °C under a gentle stream of nitrogen. After 1 h, 5 mL of KPS aqueous solution was added to initiate the reaction. The reaction was allowed to proceed for 8 h, and then was stopped by cooling the product to room temperature. The resultant microgels were purified by dialysis (molecular weight cut off 12,000–14,000 Da) against water with frequent water change for at least 1 week, and then freeze-dried for 24 hours. Fourier transform infrared spectroscopy (FTIR) The chemical structure of the synthesized copolymers was determined via attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using a FT-IR Vertex 70 spectrophotometer (Bruker, Austria). The copolymers were analyzed in solid (lyophilized) state on a KRS-5 support, within the frequency range of 4,000–400 cm−1. Data processing was done using OPUS 6.5 software (Bruker Optics). Proton nuclear magnetic resonance (1H-NMR) The copolymer composition was determined by 1H-NMR analysis. 1H-NMR spectra of poly(NIPAM-co-HEAM) were recorded in D2O on a 400/Varian VXR 200 spectrometer operating at 400 MHz and at temperatures ranging from 25 to 50 °C.
Materials and methods Atomic force microscopy (AFM) Materials N-isopropylacrylamide (NIPAM) (Aldrich Chemical Corp., Milwaukee, WI, USA) was recrystallized with hexane. Nhydroxyethylacrylamide (HEAM) was purchased from Fluka AG (Buchs, Switzerland). N,N’-methylene-bis-acrylamide (BIS) (Sigma Chemical Co., St. Louis, USA) was
The microgels were characterized by means of a scanning probe microscope (SPM Solver PRO-M AFM, NT-MTD Co. Zelenograd, Moscow, Russia) using a high resolution “Golden” silicon NSG10/Au/50 cantilever with a gold (Au) conductive coating. The samples were prepared by casting four to five drops of the diluted microgel solution (C=
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0.001%, w/v) on the mica surface. After they dried naturally, the AFM images were taken, operating in tapping mode with a driving frequency of 1.01 Hz.
The VPTT of the microgels was determined as the inflexion point of DH variation curves with temperature. Electrophoretic light scattering (ELS)
Volume phase transition temperature (VPTT) The optical density (absorbance) of the microgel aqueous solution was measured at a wavelength of 450 nm (OD450) by an UV–vis Evolution 201 spectrophotometer (Thermo Fisher Scientific Inc., Madison, USA) coupled with a temperature controller, and recorded as a function of temperature from 25 to 60 °C. The microgel solutions (0.1%, w/v) were prepared in ultrapurified water, standard acidic solution (pH 1.2, 64 mM HCl+50 mM KCl) and a standard phosphate buffer solution (pH 7.4, 50 mM Na2HPO4 +NaOH; PB). The heating rate was 2 °C every 5 min. All measurements were conducted in triplicate and the results were expressed as % transmittance plotted against temperature. The VPTT (or cloud point value) of the microgels was determined from the first derivative plot of transmittance versus temperature.
Dynamic light scattering (DLS) DLS analysis was used to determine the hydrodynamic diameter of the microgels (C=0.1%,w/v) and the polydispersity index at different temperatures. The equipment consisted of a Delsa Nano Submicron Particle Size Analyzer (Beckman Coulter Inc., Brea, USA) with a 30 mW laser diode, a wavelength of 658 nm and a size range of 0.6 nm–7 μm. The autocorrelation function is automatically calculated. The measurements were performed at a scattering angle of 165° and temperatures between 25 and 50 °C, with an equilibration time of 300 seconds at each temperature. Calculation of the particle size distribution and distribution averages was performed using CONTIN particle size distribution analysis routines using Delsa Nano 2.31 software. The average histogram peaks from intensity distributions out of 70 accumulations were reported as the average diameter of the particles. All determinations were repeated three times. The swelling behavior of the microgels can be characterized by the de-swelling ratio (α) [38] calculated using the equation (1): α ¼
V V0
¼
DH DH;0
3 ð1Þ
Where DH is the hydrodynamic diameter of the microgels in the collapsed state (at 50 °C) and DH,0 is the hydrodynamic diameter of the microgels in the swollen state (at 25 °C).
The zeta potential (ζ-potential or surface charge) of the aqueous microgel samples (0.1%; w/v) was measured using a Delsa Nano Submicron Particle Size Analyzer (Beckman Coulter Inc., Brea, USA) within a temperature range of 25–45 °C. The zeta potential was calculated from the five electrophoretic mobility measurements using the Smoluchovski approximation of the Henry function. Each value represents the average of three different replicates. Drug loading and release studies Stock solutions in ultrapurified water of microgels (sample M1.9S1.5, 10 mg/mL) and propranolol (2 mg/mL) were prepared. 2.5 mL of each solution were thermostated at 37 °C for 15 min with stirring rate fixed at 350 rpm. Then, the drug solution was dropped on the microgel solution and the stirring was continued at 45 °C for 15 min followed by an additional 15 min at 50 °C. Finally, the microgel dispersion was equilibrated at 4 °C for 1 hour and then centrifuged for 30 minutes at 12,000 rpm and 15 °C. The concentration of propranolol entrapped within microgels was calculated from the difference in the concentrations before and after incubation. The drug concentration in the supernatant was determined by measuring the absorbance at 286 nm using an UV–vis Evolution 201 spectrophotometer. Three replicates were performed and are presented as an average value. The entrapment efficiency (E) was determined using the equation (2):
E ð% Þ ¼
C0 − C1 100 C0
ð2Þ
where C0 is the initial concentration of drug and C1 is the concentration of drug in the supernatant. The in vitro release behavior of propranolol from the poly(NIPAM-co-HEAM) microgels at different temperatures was studied via dialysis tube diffusion [39]. The drug-loaded microgels were suspended in 5 mL PB at room temperature (22±1 °C), then introduced into a dialysis tube (molecular weight cut-off of 12 kDa, Sigma–Aldrich) and dialyzed against the PB solution (95 mL) in a separate beaker. The release was conducted separately, each sample at a given temperature (25, 37 and 42 °C). At fixed time intervals, 1 mL aliquots of the release fluid were taken out and replaced by 1 mL of fresh PB solution. The released propranolol was determined by measuring the absorbance at 286 nm by UV spectrophotometry.
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Results and discussion Synthesis and structural characterization of poly(NIPAM-co-HEAM) microgels The synthesis of poly(NIPAM-co-HEAM) microgels with different amounts of cross-linker and surfactant in the initial reaction mixture (see Scheme 1) was performed by precipitation polymerization [2]. It is already proved that the linear copolymer poly(NIPAM-co-HEAM) obtained at 10:2 molar ratio of the co-monomers in the initial reaction mixture has a lower critical solution temperature (LCST) value close to the temperature of the human body (~37 °C in simulated physiological fluid, PB) [36]. Consequently, this composition of co-monomers was used for microgel synthesis; the samples were coded as MxSy, where x is the molar concentration of BIS and y represents the millimolar concentration of SDS in the initial reaction mixture. The composition of the reaction mixture is listed in Table 1. The chemical structure of poly(NIPAM-co-HEAM) microgels was confirmed by FT-IR and 1 H-NMR spectroscopy. The FT-IR spectrum of the sample M1.9S1.5 in Table 1 (Fig. 1) displays characteristic absorption bands at Scheme 1 Schematic of the formation and thermal behavior in pseudo-physiological conditions (PB at pH=7.4) of poly(NIPAMco-HEAM) microgels
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1,640 cm−1 (C=O stretching of amide I), at 1,540 cm−1 (N– H bending of amide II), at about 1,172 cm−1 (C–N of amide III), and the bands assigned to the isopropyl groups (C–(CH3)2) at 1,387/1,367 cm−1. The absorption bands at 1,459 cm−1 are attributed to C–H bending in –C(CH3)2 and –CH2– groups. The absorption region between 2,870 and 3,000 cm−1 corresponds to C–H stretching vibrations of – CH3 and –CH2– groups. The wide bands in the range from 3,100 to 3,600 cm−1 with clear peaks at 3,285 cm−1 and 3,433 cm−1 are originated from valence vibrations of N-H and O-H groups. The band at 1,065 cm−1 in the spectrum is assigned to the C-O stretching of primary alcohol [40]. In the 1H-NMR spectrum of the microgels (see Fig. S1 in supplementary material), the protons of both co-monomers are well assigned. Characteristic signals of NIPAM appear at 1.15 ppm (−CH(CH3)) and 3.9 ppm (−CH(CH3)). The –CH2and –CH- protons of the backbone are observed in the 0– 2.5 ppm region and the signals from 3.34 to 3.67 ppm belong to the two methylene groups of HEAM. The molar fractions of NIPAM and HEAM in copolymer microgels were determined from the 1H-NMR spectrum by reporting the area of the peak at 3.90 ppm, due to the methine group of NIPAM, and the area of the peak at 3.67 ppm for the methylol group of HEAM (Fig. S1). These values are 82.98 and 17.02 mol % for NIPAM
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Table 1 Composition of the initial reaction mixture and measured values of VPTT, hydrodynamic diameter and zeta potential of poly(NIPAM-coHEAM) microgels Sample code
BIS (g)
SDS (g)
VPTT (°C) in water
25 °C
50 °C
DLS
UV–vis
DH (nm)a
PdI
DH (nm)a
PdI
αb
ξ, 25 °C (mV)c
ξ, 45 °C(mV)c
M1.1 S1.5
0.0136
0.034
39.5
39
366.5
0.111
138.2
0.73
0.054
−0.56
−19.54
M1.9S1.5 M2.75S1.5 M1.9S2.1 M1.9S2.7
0.0238 0.034 0.0238 0.0238
0.034 0.034 0.0484 0.0623
39.2 39.5 39.5 39.8
39 39 39 39
318.4 175.6 309.1 252.2
0.100 0.090 0.157 0.203
154.9 108.3 141.0 102.5
0.023 0.011 0.042 0.045
0.115 0.234 0.095 0.067
−0.71 −7.36 −0.99 −1.32
−13.03 −32.5 −12.45 −4.8
Molar ratio NIPAM:HEAM=10:2; NIPAM-HEAM, 150 mM; KPS, 1.045 mM a
Mean hydrodynamic diameter from the intensity-weighed size distributions measured in water by a Delsa Nano Submicron Particle Size Analyzer (CONTIN analysis)
b
De-swelling ratio calculated with equation (1)
c
Zeta potential (Smoluchovski approximation, five measurements) determined in water by a Delsa Nano Submicron Particle Size Analyzer
and HEAM, respectively, and are close to those obtained using dioxane as a solvent [36]. Thermoresponsive properties of microgels The temperature sensitivity of the poly(NIPAM-co-HEAM) microgels was evaluated comparatively using a wide variety of techniques and is presented below. DLS analysis Figure 2a shows the typical size distribution of the microgels at 25 °C. It can be seen that microgels display a monomodal distribution. The polydispersity index (PdI) values presented in Table 1 were low, around 0.1, typically for relatively monodisperse samples [41]. As expected, the increase in the SDS concentration in the polymerization mixture appears to increase the polydispersity of the final microgels, as can be concluded from the size distributions in Fig. 2a. Fig. 1 FT-IR spectrum of poly(NIPAM-co-HEAM) microgels (sample M1.9S1.5 in Table 1)
By increasing the temperature above the VPTT, the distribution becomes narrower (see Table 1) and no flocculation was observed (Fig. 2b). The plot representing the hydrodynamic diameter (DH) of the microgels, representing a measure of the swelling characteristics, vs. temperature is depicted in Fig. 3. As expected, the synthesized microgels show a decrease in DH values with increasing temperature. The VPTT values determined from these graphs are the same for all investigated samples (~39.5 °C). The microgels characterized by the lowest crosslinking degree display the sharpest phase transition since the resulted three-dimensional polymer network is extremely flexible. On the contrary, with increasing cross-linker content from 1.1 to 2.75 mM, the transition range broadens. Comparable investigations by Inomata et al. [42] on macroscopic poly(NIPAM) gels have shown the temperature at which the phase transition occurred is nearly independent of the crosslinking density; however, the transition range is influenced by the cross-linking density.
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Fig. 3 Temperature-dependent modification of the hydrodynamic diameter of poly(NIPAM-co-HEAM) microgels measured by DLS
Fig. 2 Hydrodynamic diameter distribution of microgels at 25 °C (panel a) and 45 °C (panel b)
Also, it can be observed that by increasing the cross-linker concentration, the microgels size decreases from 366 to 176 nm at a temperature below the VPTT (25 °C). This reduction of particle size might be due to the denser poly(NIPAM-co-HEAM) core. It has been shown [43–45] that the cross-linker distribution within poly(NIPAM) microgels is generally heterogeneous. McPhee et al. [46] demonstrated that the cross-link density decreases from the center to the periphery because of the difference in co-monomer reactivity ratios. McPhee et al. have shown that the reactivity of BIS is greater at the beginning of the synthesis and, hence, the cross-linker is mainly consumed for core generation. The increase in SDS concentration from 1.5 to 2.7 mM decreased the average hydrodynamic size of the microgels by a factor of 1.5 (at 25 °C), due to the electrostatic repulsion between the charged particles during the polymerization process [46]. Also, it can be observed (Fig. 3) that the shrinking
process is more uniform for the particles obtained using higher SDS concentrations, signifying better colloidal stability in these conditions. Also, from the de-swelling ratio α values (Table 1) it is possible to estimate the influence of the structure, crosslinking density and other parameters on final properties of the microgels. Thereby, the most cross-linked sample (M2.75S1.5) shows the highest de-swelling ratio α (0.234) compared to microgels with smaller cross-linker content (0.115 for M1.9S1.5 and 0.054 for M1.1S1.5). This observation is in accordance with previous experiments [47] and shows that the particles become stiffer due to the decreasing polymer chain length between two cross-linker nodes in the threedimensional network; therefore, the swelling capacity decreases. For the same cross-linker content, smaller deswelling ratios (0.095 for M1.9S2.1 and 0.067 for M1.9S2.7) were observed with increasing SDS content. SDS is believed to be associate with the propyl groups of NIPAM and confers additional ionic character to the network. In fact, SDS increases the osmotic pressure within the particle interior and favors swelling [48]. Like most colloids dispersed in an aqueous phase, microgels carry charged groups at the surface, which originate from the initiator fragment (KPS) or from the surfactant (SDS). The larger the absolute value of the zeta potential, the greater the colloidal stability. The variation in the zeta potential of microgels was measured as a function of temperature, the obtained values at 25 and 45 °C being reported in Table 1. For all samples, the zeta potential becomes more negative as the temperature rises above the VPTT. As shown by DLS, the shrinking of microgels means a large decrease in particle volume and, therefore, an increase of surface charge density that induces a much higher mobility in the collapsed state.
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Also, zeta potential measured at 25 °C gets more negative with an increasing cross-linker content. This behavior is related to the structure of the microgels that show a denser polymer network in the outer region with an increasing amount of cross-linker. Our results on poly(NIPAM) microgels are consistent with others [26, 29, 49]. It can be also observed (see Table 1) that at 50 °C, the zeta potential decreases with an increasing concentration of SDS; this decrease is more significant for the sample with 2.7 mM SDS. As other authors demonstrated [50], this behavior is due to more homogeneously structured microgels obtained in a high SDS concentration; thereby, the higher mobility at high temperatures for M1.9S1.5 microgels is related to a mobile surface layer with a polyelectrolyte nature and with a high local LCST [40]. Turbidimetric analysis The plot of % transmittance (deduced by UV–vis spectrometry from the absorbance at 450 nm) of microgel dispersion as a function of temperature is represented in Fig. 4a. It can be seen that transmittance decreases abruptly in a certain temperature rang e, signifying that the microge ls a re h ighly thermosensitive. The VPTT was determined from the first derivative plot of transmittance versus temperature (Fig. 4b). The transition temperatures for all samples are almost equal, illustrating that different synthesis conditions have almost no influence on the VPTT. However, the amplitude of the phase transition decreases with an increasing amount of cross-linker; this behavior can be attributed to topological constraints introduced into the polymer network by an increased number of cross-linking points. For example, between 30 and 50 °C, M1.1S1.5 and M1.9S1.5 microgels undergo a transmittance change of around 35 and 22%, respectively. For microgels with the highest cross-linking density (M 2.75 S 1.5 ), the transition range broadens significantly and determining the VPTT becomes more difficult. These results are consistent with those of Wu et al. [23] and can be explained by the high heterogeneity of the chain lengths of this sample. The amount of SDS used for the synthesis of microgels deeply influences turbidity. As long as the turbidity of the M1.9S2.1 and M1.9S2.7 samples below the VPTT is smaller than that of M1.9 M1.5, it can be concluded that high SDS concentrations preclude formation of highly packed solid particles causing permanent turbidity of the aqueous dispersions. This observation confirms the hypothesis of more homogenous structured microgels and lower surface mobility obtained at a high SDS concentration (see subsection DLS analysis). Fundueanu et al. found a LCST value of 40.4 °C for the poly(NIPAM-co-HEMA) linear copolymer in aqueous solution [36]. This value is slightly higher than the VPTT of M series microgels reported here and can be explained by the
Fig. 4 The relative change of transmittance with temperature (panel a) and the first derivative plot (panel b) of poly(NIPAM-co-HEAM) microgels (the concentration of microgel dispersion=0.1%, w/v)
different composition of the two formulations. M microgels have a smaller concentration of hydrophilic monomers (17.02 mol%) than the linear copolymer (18.70 mol%) synthesized with the same comonomer molar ratio but in a different solvent. The VPTT of microgels determined by DLS and UV–vis are almost the same and very close to the human body temperature, which recommends them as candidates for biomedical application. Consequently, the next step was to study changes in the hydrodynamic diameter (DH) and solution turbidity (OD450) with the changes in temperature in simulated physiological conditions (Fig. 5).
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Fig. 5 Temperature-dependent modification of the hydrodynamic diameter (panel a) and optical density (panel b) for an M1.9S1.5 sample in simulated physiological conditions (standard acidic solution at pH 1.2 and phosphate buffer at pH 7.4)
By analyzing Fig. 5, it can be seen that the VPTT determined by the two methods is lower in the phosphate buffer (pH 7.4) than in acidic conditions (pH 1.2), both values being lower than the VPTT in pure water. Because the microgels do not possess ionizable groups that may be affected by the pH of the solution, the difference between the values of the VPTT is attributed to different ionic strengths of the copolymer solutions. Besides shifting the VPTT to lower or higher temperatures, the presence of electrolytes affects microgel size and stability (Fig. 5a). Below the VPTT, the size of microgels was reduced by about 90% in water due to the salting-out effect [51]. The addition of electrolytes disrupts the hydrogen bonds between amide groups and H2O; thus, attractive inter-chain hydrophobic interactions and hydrogen bonding dominate. Small changes in the size of microgels were detected up to a temperature of 38 °C in pH 7.4 and 39 °C in pH 1.2. A further increase in temperature makes the microgels unstable; they consequently flocculate and the size increases up to 1.6 1.8 μm. These observations are consistent with the turbidity
Fig. 6 1H-NMR spectra of poly (NIPAM-co-HEAM) microgels (sample M1.9S1.5) recorded in D2O at different temperatures (panel a) and the change in f/c peak area ratio vs temperature (panel b)
results (Fig. 5b). At pH 7.4, the microgels dispersion becomes turbid at 35.3 °C (Fig. 5b). At the same time, DLS measurements (Fig. 5a) showed that between 35 and 38 °C, the microgels network collapsed; only aggregates with higher dimensions (1.6 μm) were formed above 38 °C. At pH=1.2, the VPTT shifted to a higher temperature (38.2 °C) (Fig. 5b) and the aggregates have a DH of 1.8 μm at 45 °C (Fig. 5a). Further increases in temperature lead to a decrease in the turbidity of the solution, indicating that hydrophobic and hydrogen bonding interactions occurred between aggregates, with the product sinking to the bottom.
NMR studies Conformational changes of the poly(NIPAM-co-HEAM) macromolecular chains below and above the VPTT were monitored by 1H-NMR spectrometry. 1H-NMR spectra of the microgels (sample M1.9S1.5 in Table 1) show a decrease in peak intensity with an increased temperature (Fig. 6a).
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Fig. 7 Tapping mode height (left column) and amplitude (middle column) AFM images of microgel dispersions on mica surfaces, at C=0.001%, w/v. The right column shows cross-section profiles of the same samples
This behavior is similar to the poly(N-isopropylacrylamideco-triethyleneglycol methacrylate) poly(NIPAM-coTREGMA) [29] and the poly(N-isopropylacrylamide-co-maleic acid) poly(NIPAM-co-MA) [52] microgels, and is due to a decrease in the molecular mobility of the polymer segments with an increased temperature. It can be noted that the peaks start to decrease at 35 °C (Fig. 6b) and at 50 °C they are hardly noticeable. Also, the relative intensity of peak f to c increases when the temperature is augmented, suggesting that the polar -CH2OH groups of HEAM is at the surface (more mobile) covering the isopropyl groups (less mobile) that form the core. The VPTT value determined from 1 H-NMR analysis (38.5 °C) is close to those determined by DLS and turbidimetry. Atomic force microscopy (AFM) The morphology and polydispersity of the synthesized microgels were investigated by AFM measurements against
Fig. 8 Effect of temperature on the kinetics of propranolol release from poly (NIPAM-co-HEAM) microgels (sample M1.9S1.5) under pseudo-physiological conditions. For comparison, the release of free propranolol at 25 °C is given
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air (for illustration, M1.9S1.5, M1.9S2.1 and M1.9S2.7 are presented in Fig. 7). As can be seen, deposition of microgel solutions resulted in nearly monodispersed spherical structures; however for smaller SDS amounts (M1.9S1.5) the microgels were less regular and exhibited an elongated shape (Fig. 7). The increase in SDS concentration in batch synthesis resulted in smaller and more spherical microgels. The diameter of the samples in a dried state, presented in Fig. 7, is found to be around 200, 160 and 110 nm. The decrease in particle size is due to their shrinkage during the drying process for the preparation of AFM samples. In all cases the particle height varied from 4 to 20 nm. As the height of microgels was much smaller than their diameter it can be concluded that the microgels were strongly flattened upon drying. The presence of larger particles or of some circle-like assemblages (M1.9S2.7 sample) may indicate that some interactions occurred upon drying. Drug loading and release studies For loading and release studies, propranolol, an antihypertensive drug, was chosen as a model. The loading/release mechanism of propranolol was based on the swelling/collapsing properties of temperature-sensitive microgels which works as a pump, sucking up a drug solution upon cooling and squeezing it out upon heating [53]. Thereby, the microgel aqueous dispersion (sample M1.9S1.5) was heated above the VPTT and then, after adding the drug solution, the temperature was decreased to 4 °C to suck up the drug within the network. By this approach a 24% entrapment efficiency of propranolol was obtained. “In vitro” release studies provide significant information on the efficiency of a delivery system. Release kinetics were performed under simulated physiological conditions (PB) under and above the VPTT at 25, 37 and 42 °C (Fig. 8). In a thermosensitive drug delivery system, where the entrapped drug is physically dispersed in a polymeric matrix, the major factors that control drug release are the hydrophobic and steric interactions between the drug and the three-dimensional network. Therefore, the highest release rate was observed at 25 °C. At this temperature, the microgels are in a swollen state and no steric interactions occurred. Also, hydrophobic interactions are not favored by low temperatures. The release rate is almost similar with that of a free drug. The release of the drug at 37 °C is governed by some opposite effects: a decrease in pore dimensions (see Fig. 5a) and an increased solubility and diffusion rate of propranolol. Moreover, at this temperature the hydrophobic interactions are favored. Therefore, a large amount of drug is mechanically expulsed in the first 5 min (~29%) due to the sudden collapse of the swollen microgels; then the release rate is reduced because of steric and hydrophobic interactions. When the release is conducted at 42 °C, a high amount of drug is released at the beginning because of the “pumping effect”
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(~35% in the first 5 min), then the microgels become hydrophobic and form aggregates (Fig. 5a). The mean diameter of aggregates is around seven times larger than that of individual microgels; therefore, the drug must traverse by diffusion a greater distance from the interior of the aggregates to the external environment. As a result, the release of propranolol is retarded.
Conclusions Monodispersed and almost spherical thermosensitive poly(NIPAM-co-HEAM) microgels with hydroxyl functional groups were prepared by precipitation polymerization. The thermosensitive properties of microgels were studied by turbidimetric, dynamic light scattering, zeta-potential measurements and 1H-NMR techniques. The VPTT of microgels determined in pseudophysiological conditions are very close to the human body temperature, recommending them as candidates for biomedical application. The microgels were loaded with a model drug, propranolol, and the release studies were performed in vitro below and above the VPTT. It was shown release rates are strongly influenced by temperature. Below the VPTT, the microgels are in a swollen state and no steric and hydrophobic interactions occurred; therefore, the drug is rapidly released. Above the VPTT, the microgels are in a collapsed state and the release rate is reduced. However, a fraction of the drug is mechanically expulsed in the first 5 min due to a “pumping effect.” Acknowledgments The research has received funding from the European Union’s Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 264115 – STREAM. This work was also supported by a grant from the Romanian National Authority for Scientific Research, CNCS—UEFISCDI, project number PN-II-ID-PCCE-2011-2-0028.
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