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Journal of Polymer Research

, 19:9979 | Cite as

Synthesis, characterization and drug release properties of thermosensitive poly(N-isopropylacrylamide) microgels

  • Stojanka Petrusic
  • Petar Jovancic
  • Maryline Lewandowski
  • Stéphane Giraud
  • Branko Bugarski
  • Jasna Djonlagic
  • Vladan Koncar
Original Paper

Abstract

This study was aimed at optimizing the structure and properties of poly(N-isopropylacrylamide) (PNIPAAm) microgels prepared by inverse suspension polymerization. The influence of the oil-to-water phase ratio, the concentration of emulsifier, and the monomer-to-crosslinker molar ratio on selected properties of the PNIPAAm microgels was examined. Regularity of the microgels shape was estimated by optical microscopy. Laser diffraction technique was used to determine the microgels mean size and size distribution. Equilibrium swelling ratio was studied gravimetrically. Morphology of microgels was followed by SEM. Volume phase transition temperature of PNIPAAm microgels was determined by differential scanning calorimetry. The results obtained imply that the mean diameter of microgels and their equilibrium swelling ratio highly depend on the concentration of emulsifier in oil phase and the crosslinking degree of PNIPAAm. The crosslinking degree of PNIPAAm has no substantial effect on the volume phase transition temperature that is demonstrated to be around 33 °C even after complete heating-cooling-heating cycle. In addition, it was confirmed that the swollen microgels have a porous, honeycomb-like structure. Release profiles of procaine hydrochloride from the PNIPAAm microgels confirmed their potential to be considered as efficient matrices in drug release applications.

Keywords

Poly(N-isopropylacrylamide) Thermosensitive microgels Inverse suspension polymerization Volume phase transition Controlled drug release 

Introduction

Hydrogels represent water-swollen cross-linked polymeric structures [1]. The ability of hydrogels to absorb water arises from hydrophilic functional groups attached to the polymer backbone and their resistance to dissolution is provided by the crosslinks between the network chains [2]. One of the criteria for classification of hydrogels is based on their sensitivity to environmental changes. Accordingly, we distinguish conventional and stimuli-sensitive hydrogels. The most widely used type of stimuli-sensitive hydrogels is the thermosensitive type due to their unique property to respond to temperature changes by exhibiting volume phase transition. This volume phase transition results from the interactions between the thermosensitive polymer chains in the hydrogel network and the solvent molecules [3]. According to their response to temperature changes, which depends on the type of polymer(s) constituting the hydrogel, thermosensitive hydrogels can be classified into three categories: thermoshrinking type (shrink with increase in temperature), thermoswelling type (swell with increase in temperature), and “convexo” type (shrinking-swelling-shrinking behavior with temperature decrease) [3, 4].

Thermosensitive hydrogels based on poly(N-isopropylacrylamide) (PNIPAAm) are among the most exploited thermoshrinking type of hydrogels in biotechnology and biomedicine [5, 6, 7, 8, 9]. When temperature of aqueous medium is lower than volume phase transition temperature (VPTT) of PNIPAAm hydrogels, they are in swollen state. With increase in temperature above VPTT the hydrogels shrink. Wide application potentials of PNIPAAm hydrogels are partly due to their convenient VPTT that ranges from 30 to 35 °C [10, 11]. These hydrogels could be synthesized in various physical forms, from macro to nano scale. Our group studied macro forms of pure and modified PNIPAAm hydrogels and demonstrated their benefits for application in controlled drug release [12]. However, in this application, micro forms of hydrogels have various benefits over the macro forms. Size of hydrogel microgels impacts their specific surface area and response to temperature changes [13] that are relevant for a control of drug release pattern. Approaches in the synthesis of microgels vary from heterogeneous polymerization [14, 15], over spray drying [16, 17], micromolding and photolithography [18] to extrusion methods [19, 20]. Among techniques of heterogeneous polymerization, inverse suspension polymerization is a good choice for the production of PNIPAAm microgels since the microgels size can be easily controlled by regulation of the reaction conditions [21]. In contrast to emulsion and dispersion polymerization, this technique is suitable for the synthesis of porous microgels [22].

However, in some studies, it was found that formation of thermosensitive microgels based on PNIPAAm by inverse suspension polymerization often resulted in microgels of diameters above 100 μm [13, 23, 24, 25]. Such microgels were mainly intended for use in size-selective separation processes. The leading idea in our research is application of PNIPAAm microgels in the development of a textile-based system for transdermal controlled drug delivery. Therefore, this study deals with optimization of selected formulation parameters in synthesis of PNIPAAm microgels by inverse suspension polymerization and aims to produce regular microgels of diameter close to average fiber diameter (around 20 μm for cotton [26]). In order to choose optimal conditions for synthesis of PNIPAAm microgels, the influence of the oil-to-aqueous phase volume ratio, the concentration of emulsifier, and the monomer-to-crosslinker molar ratio was studied. The optical, thermal, swelling, and morphological properties of prepared PNIPAAm microgels were examined in detail to find out the optimal properties of polymer matrix for subsequent drug release study. A local anesthetic, procaine hydrochloride (procaine HCl), was used in drug release studies from the selected PNIPAAm microgels. The drug release was conducted at two temperatures in Franz diffusion cell, which is a standard for testing transdermal pharmaceutical formulations. Similar works on PNIPAAm microgels synthesized by inverse suspension polymerization have not considered such complete analysis of hydrogel structure that is necessary for the application foreseen.

Experimental

Materials

N-Isopropylacrylamide (NIPAAm, 97 %, Sigma-Aldrich), was purified by recrystallization in n-hexane before use. N,N,N’,N’-tetramethylethylenediamine (TEMED, 99 %), N,N’-methylenebis(acrylamide) (MBAAm, 99 %) and ammonium persulfate (APS) were supplied by Fluka. Procaine hydrochloride (procaine HCl, 99 %) was provided by Sigma-Aldrich. Polyoxyethylene (20) sorbitan monooleate (Tween 80) was purchased from Riedel-de Haen, paraffin oil by Carlo Erba, calcium chloride (CaCl2, 95 %) by Panreac, and acetone by Zorka Pharma Hemija d.o.o.

Synthesis of PNIPAAm microgels

A series of thermosensitive microgels based on PNIPAAm were synthesized by free-radical inverse suspension polymerization according to the procedure reported by Kayaman et al. [25]. The reactions were conducted in a 500-mL three-neck round bottom flask, equipped with a stirrer, reflux condenser, and a nitrogen inlet. The concentration of NIPAAm was kept constant (10 w/v % in a dispersed aqueous phase). The polymerization reactions were conducted in the presence of 2 wt.%-initiator (APS) and 4.6 wt.%-catalyst (TEMED) at 25 °C. The concentrations of APS and TEMED were calculated in ratio to NIPAAm and they remained unchanged in all reactions. The following parameters were varied: the oil-to-aqueous phase volume ratio, the concentration of emulsifier (Tween 80), and the molar ratio of monomer (NIPAAm) to crosslinker (MBAAm), as indicated in Table 1. After the completion of reactions, microgels were separated from the oil phase by multiple washing in a mixture of acetone and water (1:1), and finally with pure water. Each centrifugation cycle was performed at 2,000 rpm in duration of 30 min..
Table 1

Composition of feed mixtures in syntheses of microgels by inverse suspension polymerization

Sample

Oil/aqueous phase (vol/vol)

Emulsifiera, v/v %

NIPAAm/MBAAm (mol/mol)

M-50/1(10/1)–E 0

10/1

0

50/1

M-50/1(10/1)–E 1

10/1

1

50/1

M-50/1(5/1)–E 0.5

5/1

0.5

50/1

M-25/1(5/1)–E 1

5/1

1

25/1

M-50/1(5/1)–E 1

5/1

1

50/1

M-100/1(5/1)–E 1

5/1

1

100/1

aIn ratio to paraffin oil

Characterization methods

Optical microscopy

Microgels were observed under the optical microscope Ergaval (Carl Zeiss-Jena) equipped with TP-1001C Topica CCD camera (Krüss). Image analysis software ImageJ 1.44 g was employed to determine the average diameter of swollen microgels, based on the analysis of 200 microgels per sample.

Laser diffraction

Volume-weighted mean diameter and size distribution of microgels were determined by laser diffraction using a Mastersizer 2000 equipped with a micro unit Hydro μ (Malvern Instruments). The value of δ is a measure of polydispersity and was determined according to the following equation [27]:
$$ \delta = \frac{{{{\mathrm{d}}_{{\left( {0.9} \right)}}} - {{\mathrm{d}}_{{\left( {0.1} \right)}}}}}{{{{\mathrm{d}}_{{\left( {0.5} \right)}}}}} $$
(1)
where d(0.1), d(0.5), and d(0.9) signify 10th, 50th and 90th percentile diameter, respectively. According to a criterion for emulsions, microgels could not be regarded as monodisperse if δ ≥ 0.4 [28].

Differential scanning calorimetry

Thermal analysis of microgels was performed on a Q1000 DSC (TA Instruments) through a heating-cooling-heating cycle. Three consecutive temperature ramps were applied at a heating rate of 3 °C min−1, in the temperature range from 15 to 50 °C, and under a nitrogen flow of 50 ml min−1. All runs were conducted in hermetically sealed aluminum pans, with distilled water used as reference material. Temperature of the endothermic maximum was referred to as VPTT. The scans were performed in triplicates.

Gravimetry

In order to determine equilibrium swelling ratio (ESR) and equilibrium water content (EWC) of microgels at 25 °C, the microgels were incubated in dialysis tubings (Spectra/Por®, Spectrum Laboratories Inc.), closed at both ends. Swollen microgels in sealed membranes were kept in water at 25 °C in a thermostat for 24 h. Prior to weighing and after the removal of closures, the membranes were blotted with filter paper. Finally, the membranes containing microgels were dried until constant weight. ESR and EWC values were calculated as per Eqs. (2) and (3), respectively:
$$ {\mathrm{ESR}} = \frac{{{{\mathrm{W}}_{\mathrm{s}}} - {{\mathrm{W}}_{\mathrm{d}}}}}{{{{\mathrm{W}}_{\mathrm{d}}}}} $$
(2)
$$ {\mathrm{EWC}}\left( \% \right) = \frac{{{{\mathrm{W}}_{\mathrm{s}}} - {{\mathrm{W}}_{\mathrm{d}}}}}{{{{\mathrm{W}}_{\mathrm{s}}}}} \times 100 $$
(3)
where Ws represents the weight of equilibrium swollen microgels and Wd, the weight of the dried microgels. Each ESR and EWC value was obtained as an average of three measurements.

Scanning electron microscopy

The morphology of thermosensitive microgels swollen at 25 °C was analyzed using a JEOL 5800 SEM (JEOL). Samples were previously treated in a freeze-dryer ALPHA 2–4 LD plus (Martin Christ). Freeze-drying was conducted at −70 °C and 80 mbar for 4 h. Freeze-dried microgels were coated with Ag-Pt-Pd alloy by sputtering for 30 s prior to SEM observations. The average pore sizes of the microgels were determined from SEM micrographs using ImageJ 1.44 g software, based on 100 analyzed pores per sample.

Drug release studies

Drug loading

Procaine HCl was loaded into the selected microgels by simple sorption from the concentrated drug solution. Dried microgels (150 mg) were incubated in 30 ml of procaine HCl solution (10 mg ml−1) for 20 h at room temperature. Afterwards, the swollen drug-loaded microgels were removed from the solution and dried until constant weight at 60 °C prior to drug release studies. The amount of incorporated drug was determined spectrophotometrically. Ten milligrams of dried drug-loaded microgels was placed in 5 ml of distilled water and stirred vigorously for 24 h to extract the drug from the microgels.

Drug release

Drug release properties of the microgels were studied using Franz diffusion cell. The cell had jacketed both, donor and receptor chamber, and a diffusional area of ~0.68 cm2. The receptor chamber with capacity 5.65 ml was always maintained at constant temperature of 37 °C. The drug release profiles were obtained at two temperatures of donor chamber (25 °C and 37 °C). Acetate cellulose membrane (0.45 μm mesh size, Fisher Scientific) was used as semi-permeable membrane to mimic a human skin. The drug release was conducted using 0.5 ml of distilled water as a donor fluid and 10 mg of drug-loaded dried microgels. The aliquots of 0.2 ml were periodically withdrawn from receptor chamber and were replaced with the same volume of fresh distilled water. The drug content was also analyzed by UV/Vis spectrophotometer (Carry 100, Agilent Technologies). Drug release experiment was conducted in tiplicate.

An empirical power equation developed by Peppas [29] was applied for analysis of procaine HCl release mechanism from the microgels:
$$ \frac{{{{\mathrm{M}}_{\mathrm{t}}}}}{{{{\mathrm{M}}_{\infty }}}} = {\mathrm{k}}{{\mathrm{t}}^{\mathrm{n}}} $$
(4)
where Mt and M are the absolute cumulative amount of drug released at time t and at infinite time (initially loaded), k is a release rate constant and n is a release exponent.

Results and discussion

Optical microscopy

Influence of the concentration of emulsifier and oil-to-aqueous phase volume ratio

Table 2 summarizes the values of the oil-to-aqueous phase volume ratio and the volume fraction of emulsifier used in initial four syntheses of PNIPAAm microgels, along with calculated values of mean diameters based on optical microscope images. In addition to amount of monomer (NIPAAm), initiator (APS), and catalyst (TEMED), the amount of crosslinker (MBAAm) was also kept constant. The molar ratio of NIPAAm and MBAAm in these reactions was 50/1.
Table 2

Influence of the addition of emulsifier and oil-to-aqueous phase ratio on microgels size

Sample

Oil/aqueous phase (vol/vol)

Emulsifiera, v/v %

Mean microgels diameter, μm

M-50/1(10/1)–E 0

10/1

0

236.8 ± 142.7

M-50/1(10/1)–E 1

10/1

1

46.5 ± 25.0

M-50/1(5/1)–E 1

5/1

1

22.6 ± 14.9

M-50/1(5/1)–E 0.5

5/1

0.5

44.3 ± 23.8

aIn ratio to paraffin oil

At oil-to-aqueous phase ratio of 10/1, two types of microgels were prepared: without emulsifier and with emulsifier at concentration of 1 v/v % (Fig. 1a, b). According to values of mean diameters presented in Table 2, addition of emulsifier in concentration of 1 v/v % drastically reduces the microgels diameter. Obtained microgels are of regular spherical shape in both cases.
Fig. 1

Microgels M-50/1(10/1)–E 0 (a), M-50/1(10/1)–E 1 (b), M-50/1(5/1)-E 1 (c) and M-50/1(5/1)–E 0.5 (d)

The images also show that the oil-to-aqueous phase volume ratio in the presence of 1 v/v %-emulsifier has no significant influence on the microgels shape, i.e. microgels kept the same regular spherical shape when this ratio was 5/1 (Fig. 1c). Furthermore, these microgels have also smaller mean diameter than M-50/1(10/1)–E 1 (Table 2). Therefore, the subsequent syntheses were conducted under smaller oil-to-aqueous phase volume ratio. Additionally, the synthesis of PNIPAAm microgels was performed using 0.5 v/v %-emulsifier (Fig. 1d). This decrease in emulsifier concentration caused prominent formation of agglomerates and less regular shape of microgels as well as their size increase. Thus, the oil-to-aqueous phase volume ratio of 5/1 and the emulsifier concentration of 1 v/v % were confirmed as the optimum conditions for obtaining the microgels of regular shape and adequate mean diameter.

The influence of PNIPAAm crosslinking degree

After establishing the optimum oil-to-aqueous phase volume ratio (5/1) and the concentration of emulsifier (1 v/v %), the amount of crosslinker was varied. The crosslinking degree of PNIPAAm network was optimized by analyzing microgels synthesized under three molar ratios of monomer (NIPAAm) and crosslinker (MBAAm): 25/1, 50/1, and 100/1 (mol/mol). Table 3 contains values of mean diameters obtained from microscope images of microgels with various crosslinking degrees.
Table 3

Influence of crosslinking degree on microgels size

Sample

NIPAAm/MBAAm (mol/mol)

Mean diameter, μm

M-25/1(5/1)–E 1

25/1

32.1 ± 19.9

M-50/1(5/1)–E 1

50/1

22.6 ± 14.9

M-100/1(5/1)–E 1

100/1

27.4 ± 19.6

Values of calculated mean diameters of these microgels do not correspond to the expected trend, i.e. higher crosslinking degree should result in smaller mean microgel diameter. This could be ascribed to the fact that the microgels comprising the agglomerates could have not been analyzed. Microgels with higher crosslinking degree (M-25/1(5/1)–E 1 and M-50/1(5/1)–E 1) are uniform and of regular spherical shape (Fig. 2a, b). In contrast, the microgels synthesized with the lowest amount of crosslinker (M-100/1(5/1)–E 1) are characterized by strong presence of agglomerates composed of irregular microgels as well as the presence of regular spherical microgels, but in lower percentage (Fig. 2c, d). The propensity of these microgels towards the agglomeration could derive from their weak mechanical stability caused by looser PNIPAAm network.
Fig. 2

Microgels M-25/1(5/1)–E 1 (a), M-50/1(5/1)–E 1 (b), and M-100/1(5/1)–E 1 (c and d)

Size and size distribution

Table 4 summarizes results obtained by laser diffraction measurements. Volume-weighted mean diameter (D) was taken as a representative mean value to analyze the influence of selected formulation parameters on the microgels size. The results indicate that volume-weighted mean diameter of prepared microgels ranges from around 20 to 108 μm (excluding the sample prepared without the addition of emulsifier).
Table 4

Values of microgels diameters based on laser diffraction measurements

Sample

d(0.1)

d(0.5)

d(0.9)

D, μm

δ

M-50/1(10/1)–E 0

365.8

531.6

768.1

552.2

0.8

M-50/1(10/1)–E 1

11.9

27.4

70.0

35.9

2.1

M-50/1(5/1)-E 0.5

11.4

22.0

47.0

26.3

1.6

M-25/1(5/1)–E 1

10.3

18.3

33.6

20.7

1.3

M-50/1(5/1)–E 1

9.3

20.2

44.0

23.9

1.7

M-100/1(5/1)–E 1

11.8

27.6

58.3

31.8

1.7

Influence of concentration of emulsifier and the oil-to-aqueous phase ratio

The influence of emulsifier and of the oil-to-aqueous phase ratio on size and size distribution of PNIPAAm microgels is demonstrated in Fig. 3. At oil-to-aqueous phase volume ratio 10/1, and molar ratio of monomer to crosslinker 50/1, mean diameter of microgels obtained in the absence of emulsifier (M-50/1(10/1)–E 0) was 552.2 μm, while those obtained after emulsifier addition at concentration 1 v/v % (M-50/1(10/1)–E 1) was 35.9 μm. Addition of emulsifier reduced the microgels size by 93 % but it caused an increase in microgels polydispersity.
Fig. 3

Influence of emulsifier concentration and oil-to-aqueous phase volume ratio on size and size distribution of microgels

The results of laser diffraction measurements demonstrate that smaller fraction of the oil phase brought about reduction in the microgels size (from 35.9 μm to 23.9 μm). This relationship corresponds well to the observations obtained under the optical microscope and the results of image analysis. The smaller oil-to-aqueous phase volume ratio also resulted in slightly reduced microgels polydispersity (Table 4). These results are not in big contradiction with the empirical relationship since microgels diameter is a complex function of physicochemical properties of the system [30]. Laser diffraction measurements showed that microgels synthesized at emulsifier concentration 0.5 v/v % feature smaller mean diameter than analogous microgels synthesized with 1 v/v %-emulsifier (Table 4) and their size distribution is narrower (Fig. 3). However, it should be underlined that according to the images from optical microscope, the microgels M-50/1(10/1)–E 1 are of more regular spherical shape. Lower concentration of emulsifier caused more pronounced agglomeration, indicating less stable suspension. Hence, it was confirmed that 1 v/v % is optimal concentration of emulsifier in oil phase for obtaining PNIPAAm microgels in the given system.

The influence of PNIPAAm crosslinking degree

The increase in molar ratio of monomer to crosslinker, i.e. formation of looser polymer network, causes increase in the microgels mean diameters from 20.7 to 31.8 μm for M-25/1(5/1)–E 1 and M-100/1(5/1)–E 1, respectively. This is expected since the copolymerization/crosslinking reaction stops earlier when crosslinker content in the feed mixture increases [31]. In addition, the size distribution width is enhanced (Fig. 4), as a result of reduced presence of agglomerates and higher regularity of microgels in comparison with those of lower crosslinking degree. Hence, the polydispersity coefficient is the lowest for the microgels with the highest crosslinking degree (Table 4). General decrease in microgels size with increase in crosslinker concentration is explained by the formation of more compact and dense polymer network.
Fig. 4

Influence of crosslinking degree on size and size distribution of microgels

Thermal characteristics

Thermal characteristics of the selected thermosensitive microgels were analyzed through heating-cooling-heating cycle in the range of 15 to 50 °C. Onset temperatures of the phase transition (Tonset), volume phase transition temperatures (VPTT), and the corresponding enthalpy changes (ΔH) are displayed in Table 5.
Table 5

Thermal results of DSC heating-cooling-heating cycle of microgels

Sample

Heating I

Cooling

Heating II

T onset a (°C)

VPTTa (°C)

ΔHb (J g−1)

Tonset (°C)

VPTT (°C)

ΔH (J g−1)

Tonset (°C)

VPTT (°C)

ΔH (J g−1)

M-25/1(5/1)–E 1

32.7

33.5

1.21

32.3

31.4

1.20

32.5

33.5

1.20

M-50/1(5/1)–E 1

32.1

32.8

1.23

32.3

31.5

1.30

32.2

32.2

1.26

M-100/1(5/1)–E 1

32.6

33.3

1.23

32.1

31.3

1.23

32.6

33.0

1.18

aStandard deviation for Tonset and VPTT values was ±0.2 °C

bStandard deviation for ΔH values was ±0.19 J g−1

VPTT values of the analyzed microgels obtained in the heating steps are around 33 °C, which corresponds to literature data for pure PNIPAAm hydrogels [32]. DSC results show that the crosslinking degree of PNIPAAm has no considerable influence on VPTT of microgels, which is in accordance with other related studies [3]. Furthermore, the corresponding enthalpy changes are almost identical for the microgels of various crosslinking degrees. The main contribution to ΔH values comes from the release (during heating steps) of the structured water molecules that form a shell around the hydrophobic isopropyl groups [33]. Since the amount of NIPAAm in all reaction mixtures was the same, the amount of isopropyl groups in the resulting polymer networks is assumed to be constant. This implies the same energetic effect during heating/cooling over a critical temperature (VPTT). Furthermore, reversibility of the volume phase transition is clearly demonstrated through the complete thermogram of the microgels M-50/1(5/1)–E 1 in Fig. 5. The endothermic peaks of the first and the third thermal step overlap in all cases. These results indicate that after the cooling step hydrogel matrices come into fully swollen state with re-established hydrogen bonds with water molecules. This is of great relevance in controlling “on-off”drug release pattern if microgels are used as drug delivery vehicles.
Fig. 5

DSC thermogram of microgels M-50/1(5/1)–E 1

Fig. 6

Response of dried microgels M-25/1(5/1)–E 1 (a) to water presence after 30 s (b), 2 min (c), and 10 min (d)

Swelling behavior

Equilibrium swelling ratio

The mean values of equilibrium swelling ratio (ESR) and equilibrium water content (EWC) of the microgels are presented in Table 6.
Table 6

Values of equilibrium swelling degree and equilibrium water content of microgels at 25 °C

Sample

ESR

EWC, %

M-50/1(10/1)–E 0

13.0 ± 0.6

92.9 ± 0.3

M-50/1(10/1)–E 1

23.1 ± 1.0

95.8 ± 0.2

M-50/1(5/1)–E 0.5

13.9 ± 0.7

93.2 ± 0.3

M-25/1(5/1)–E 1

15.0 ± 0.8

93.6 ± 0.9

M-50/1(5/1)–E 1

14.8 ± 0.6

93.5 ± 0.1

M-100/1(5/1)–E 1

27.5 ± 1.9

96.5 ± 0.2

Emulsifier-free PNIPAAm microgels (M-50/1(10/1)–E 0) exhibited almost two-fold lower ESR value compared to the analogue microgels prepared in the presence of 1 vol %-emulsifier. This difference in ESR values is probably caused by more compact structure and larger size of emulsifier-free microgels. When oil-to-aqueous phase volume ratio was decreased, swelling capacity of PNIPAAm microgels was reduced. Explanation for this behavior could be found in mean size of the microgels (see Table 4). Microgels M-50/1(10/1)–E 1 have more than 30 % higher mean diameter than M-50/1(5/1)–E 1 and hence, a lower surface area that indicates more efficient purification from the remaining emulsifier on microgels surface. Thus, the hydrophilic groups in PNIPAAm network are more available for the interaction with water molecules causing enhanced swelling of hydrogel.

In a series of microgels with various crosslinking degrees, i.e. molar ratios NIPAAm/MBAAm varying from 25/1 to 100/1, the ESR values were in the range from 15 to 23. When considering these results as a whole, the trend of ESR increase with decrease in crosslinking degree is noticeable, as found in another similar study [34]. More crosslinked structures have weaker water absorption abilities due to the formation of denser and tighter polymer network that limits the water absorption. This dependence is valid for both microgels as well as for bulk hydrogels [31, 35]. Since there is reverse relation between the preparation temperature and ESR of the microgels prepared by inverse suspension polymerization [25], further optimization preparation procedure could refer to increase in temperature while keeping high monomer/crosslinker ratio. High EWC values of above 90 % demonstrate strong water absorption capacity of the microgels that has an important role in procedure of drug loading into the microgels [36]. In addition, high water content in the microgels affects easier diffusion of active agents from the hydrogel matrix [37].

Response of dried microgels to water presence

Swelling rate of the selected microgels from dried state was investigated by optical microscope at room temperature (23 °C). A drop of microgels suspension was dried on a glass slide for 2 h at 50 °C. Afterwards, the slide was positioned on a microscopic stage and finally a drop of water was carefully placed on the dried microgels fixed on a glass slide. From that moment on, snapshots were taken at short time intervals. Figure 6 presents M-25/1(5/1)–E 1 microgels in dried state and after 30 s, 2 min, and 10 min from the initial contact with water, i.e. start of swelling. These microscope images show that dried microgels swell almost instantly. Such fast swelling of pure PNIPAAm microgels was also reported by Suárez et al. [38]. According to the given images, no significant differences in microgels size after 30 s and 10 min from the start of swelling were found. These results serve as a useful reference for a design of drug release studies and prediction of microgels drug release behavior.

Morphology

SEM micrographs of the freeze-dried microgels demonstrate that synthesized thermosensitive microgels possess highly porous structure with interconnected pores (Fig. 7). This property is in particular favorable for drug loading/release applications of the microgels. Open pores are also evidence of the successful removal of paraffin oil from the reaction mixture, i.e. complete washing steps [39]. Calculated values of mean pore size and mean diameter of the microgels are given in Table 7.
Fig. 7

SEM micrographs of microgels with various amounts of emulsifier and volume ratio of oil to aqueous phase: M-50/1(10/1)–E 0 (a), M-25/1(5/1)–E 1 (b), M-50/1(5/1)–E 1 (c), and M-100/1(5/1)-E 1 (d)

Table 7

Values of mean pore sizes and mean diameter of freeze-dried microgels determined from the SEM micrographs at 25 °C

Sample

Mean pore size, μm

Mean microgels diameter, μm

M-50/1(10/1)–E 0

M-50/1(10/1)–E 1

4.4 ± 1.7

49.2 ± 23.5

M-50/1(5/1)–E 0.5

5.8 ± 1.2

38.2 ± 18.2

M-25/1(5/1)–E 1

4.4 ± 0.9

28.9 ± 13.0

M-100/1(5/1)–E 1

3.9 ± 1.5

40.3 ± 15.4

It is clearly seen from Fig. 7a that the microgels prepared without emulsifier (M-50/1(10/1)–E 0) have flower-like porous structure. Such structure was reported by Cheng et al. who studied pure PNIPAAm microgels prepared by inverse emulsion polymerization [40]. Other types of microgels feature honey-comb like structure that allowed easier calculation of their mean pore sizes by image analysis. Obtained values of mean pore sizes of analyzed microspheres are in the range of 4.2 to 7.9 μm and have proportional dependence on microgels diameters determined by laser diffraction (Table 4). Values of pore sizes of pure PNIPAAm microgels were in accordance with study of other authors [40]. Furthermore, SEM analysis confirmed the impact of oil-to-aqueous phase volume ratio in synthesis of microgels on their ultimate size. Likewise, analysis by optical microscopy showed that microgels M-50/1(10/1)-E 0.5, obtained at lower amount of emulsifier, have larger diameters and are more polydisperse than analogous microgels obtained with larger amount of emulsifier. Figure 7b, c, d also displays the influence of crosslinking degree on microgels morphology and size. Image analysis showed the crosslinker concentration has insignificant impact on the pore size of PNIPAAm microgels. The same dependence was found in the study of Park and Hoffman who characterized microgels prepared by inverse suspension polymerization, having diameters in the range from 200 μm to 400 μm [41].

Drug release profiles

Detection of drug incorporated in selected PNIPAAm microgels (M-100/1) showed that total drug uptake was 115.4 mg of drug/g of dry microgel, which is close to values reported in similar system [42]. Release of procaine HCl from the microgels had temperature-positive pattern in major part of the studied period (Fig. 8). During the initial 90 min, amount of drug released was around 10 % higher at 37 °C than at 25 °C. Faster drug release at higher temperature that was primarily pronounced in first 3 h could be partly ascribed to temperature differences that affect faster diffusion of drug molecules distributed on the surface of the microbeads. According to Arrhenius relationship, diffusivity of drug molecule increases with increase in temperature [43]. Afterwards, the structure of microgels probably becomes predominant in drug release process. Contribution of smaller microgel mesh size at temperature above VPTT becomes the leading factor in rate of drug release. Release at lower temperature becomes even slightly higher in the sixth hour. At that point, around 36 % of initially incorporated drug was released at 25 °C. Relatively high amount of unreleased drug is partly related to possible hydrophobic interactions between the isopropyl groups of PNIPAAm and the aromatic ring of procaine HCl. After the primary burst release due to drug molecules distributed on the surface of microgels and fast initial swelling, subsequent slower release could indicate possibility of overall long-lasting drug release pattern that is of great relevance for specific biomedical applications. In any case, further optimization of the process parameters of synthesis as well as the formulation of reaction mixture in order to obtain proper microgel structures for more efficient drug release should be additionally explored.
Fig. 8

Release profiles of procaine HCl from the microgels M-100/1 at 25 and 37 °C

Drug release mechanism from the microgels M-100/1 was estimated by Peppas’ model (Eq. 4). Values of correlation exponents (R2 = 0.964 for data at 37 °C and R2 = 0.992 for data at 25 °C) show that fit of experimental data using the applied model is satisfying. According to calculated values of release exponents (0.61 and 0.64), release of procaine HCl from the microgels M-100/1 at both temperatures could be classified under non-Fickian or anomalous transport since 0.43 < n < 0.85 [44]. Hence, drug diffusion and the polymer relaxation are comparable, i.e. drug release is both, diffusion- and swelling-controlled. Therefore, drug release profile could be modified by changing the microgels structure and swelling behavior, which is a leading idea in design of efficient and controllable drug release systems.

Conclusions

A series of thermosensitive PNIPAAm microgels were synthesized by inverse suspension polymerization. The oil-to-aqueous phase volume ratio, the concentration of emulsifier, and the crosslinking degree were varied. The optimization of formulation parameters showed that a decrease in the oil-to-aqueous phase volume ratio, the presence of emulsifier, and higher fraction of crosslinker cause a decrease in mean diameter. The most regular and the smallest PNIPAAm microgels of 20 μm in diameter were obtained at 5/1 oil-to-aqueous phase volume ratio, at 1 v/v % of emulsifier (in oil phase), and at 25/1 NIPAAm/MBAAm molar ratio. VPTT of PNIPAAm microgels was around 33 °C. The microgels of less crosslinked network feature higher swelling capacity than those of more crosslinked network. The joint feature of all synthesized microgels in the presence of emulsifier is porous, honeycomb-like structure with the pore size of around 4 μm. Large open pores indicate existence of interconnected channels making favorable structure for drug release applications. Studies in the Franz diffusion cell showed that release of procaine HCl from the PNIPAAm microgels was temperature-dependent and its mechanism was diffusion- and swelling-controlled. The thermosensitive microgels with regular spherical shape obtained by inverse suspension polymerization have the potential to be considered as promising matrices in controlled drug release application: the intended aim is to develop a thermosensitive textile-based system for transdermal drug release.

Notes

Acknowledgments

The financial support for this research work has been provided by the project ARCUS 2006 – Nord-Pas-de-Calais/Bulgarie – Roumanie – Serbie, granted by the French Ministry of Foreign Affairs and the Region Nord-Pas-De-Calais. The research is also supported in part by the project number III46010, granted by the Ministry of Education and Science of Republic of Serbia. The authors would like to thank Dr. Smilja Markovic from the Institute of Technical Sciences of the Serbian Academy of Sciences and Arts from Belgrade for her valuable help in laser diffraction analysis.

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Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Stojanka Petrusic
    • 1
    • 2
    • 3
  • Petar Jovancic
    • 1
  • Maryline Lewandowski
    • 2
    • 3
  • Stéphane Giraud
    • 2
    • 3
  • Branko Bugarski
    • 1
  • Jasna Djonlagic
    • 1
  • Vladan Koncar
    • 2
    • 3
  1. 1.Faculty of Technology and MetallurgyUniversity of BelgradeBelgradeSerbia
  2. 2.Université Lille Nord de FranceLilleFrance
  3. 3.Ecole Nationale Supérieure des Arts et Industries Textile, GEMTEXRoubaixFrance

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