ARTICLE pubs.acs.org/JPCC
In Situ Encapsulation and Release Kinetics of pH and Temperature Responsive Nanogels Sasmita Nayak, Sarama Bhattacharjee, and Yatendra S. Chaudhary*,† Colloids and Materials Chemistry Department, Institute of Minerals and Materials Technology (CSIR), Bhubaneswar-751 013, India
bS Supporting Information ABSTRACT: A facile synthesis of pH and temperature responsive poly(N-isopropylacrylamide) (PNIPAM) nanogels is presented. The scanning electron microscope (SEM) and dynamic light scattering (DLS) measurements indicate the formation of nanospheres of the order of 150 ( 20 and 230 ( 30 nm in case of PNIPAM (referred to as NG) and acid functionalized PNIPAM nanogels (referred to as AFNG), respectively, whereas on drug loading, the size increased to 170 ( 20 and 270 ( 20 nm, respectively at pH 7.4. Both the AFNG and amphotericin B (AmB) drug loaded AFNG (AmB-AFNG) show swelling as the pH changed from 3 to 11, but NG does not show any swelling with the change in pH. The AmB-AFNG exhibits better drug release up to ∼94% at pH 11. The better drug release observed in the case of AmB-AFNG is due to (a) swollen hydrated state of nanogel and (b) the acting repulsive forces between acid group of AFNG and AmB drug.
1. INTRODUCTION The ability of polymeric gels to undergo reversible volume phase transition in response to external stimuli such as pH, temperature, and ionic strength of the surrounding medium has generated tremendous interest among researchers. Such polymeric gels are thus being exploited for various applications such as for drug delivery,1,2 sensing,3,4 catalysis,5 template based synthesis of inorganic nanoparticle,6 and pollution control.7 In particular, stimuli responsive polymeric micro/nanogels are being widely explored to accommodate transport and delivery of molecules (for example, genes, beneficial agents and drugs). Poly(N-isopropylacrylamide) (PNIPAM) exhibits the biocompatible and intriguing pH and thermo-sensitive properties. It undergoes rapid and reversible swelling/deswelling transitions at the least critical solution temperature (LCST) that is 32 °C because of the imbalance between repulsive and attractive forces acting on particles. Such behavior exhibited by PNIPAM has made them attractive for their applications in medical and bioengineering fields.810 There has been ever increasing interest to synthesize PNIPAM microgels with desired particle size and the functionalities. Ever since the first synthesis of PNIPAM micro particles/gel reported by Wichterle and Lim in 1954, numerous reports have appeared on its synthesis.1117 Several examples have also been reported on the incorporation of various types of nanoparticles including drug molecules in 3D linked micro/ nanogel porous network to use it as a carrier.1820 However, these methods involve complex synthetic strategies (such as micro emulsion based, etc.) and the particle size has always been larger (of the order of micrometer). It should be noted that, despite the exploration of such polymeric and others (for example, dendrimers, polyelectrolytes, r 2011 American Chemical Society
and inorganic particulates systems) for transport and delivery applications, their effective exploitation has been limited due to poor control over the release. Further, nanocarriers tend to form aggregates due to their high surface reactivity (energy) unless they are stabilized by surface passivation. To surmount such issues, the in-depth understanding of viscoelastic behavior (swellingdeswelling behavior), surface charges/functionalities can offer an insight on how to achieve controlled drug release. Such important aspects have been scarcely addressed in detail so far, to the best of our knowledge. Keeping in view the above-mentioned shortfalls, we have undertaken a detailed study on (i) the synthesis of PNIPAM based nanogels, (ii) functionalization of nanogels to tune the LCST and to introduce/improve responsiveness to other physical variables (pH and ionic strength), (iii) the detailed investigation of the viscoeleastic properties (rheology), surface charges of synthesized nanogels, and (iv) drug release kinetics. We have chosen drug Amphotericin- B (AmB) which is known as a potent antifungal agent. Although a lipid based carrier exist for AmB,21,22 its effectiveness has been found to be low due to low activity within the liposomal carrier.23 Therefore, there is a need to develop an effective carrier for AmB to increase the therapeutic index of the drug. Herein, a novel single step synthetic technique to prepare polymeric nanogels that can effectively encapsulate AmB, detailed results on their swellingdeswelling behavior of drug encapsulated nanogels, and drug release kinetics/mechanism that show Received: May 24, 2011 Revised: November 26, 2011 Published: December 09, 2011 30
dx.doi.org/10.1021/jp209048g | J. Phys. Chem. C 2012, 116, 30–36
The Journal of Physical Chemistry C
ARTICLE
controlled release of the drug in response to external stimuli are discussed in detail.
2. EXPERIMENTAL METHODS 2.1. Materials. The monomer N-isopropylacrylamide (NIPAM), ammonium persulfate, sodiumdodecylsulfate (SDS) and the drug AmB were obtained from Aldrich. The cross-linker N,N-methylenebisacrylamide and dimethylsulfoxide were obtained from Fluka and Merck, respectively. All chemicals were used without any purification. The release experiments were performed in 50 mM citrate (pH 3) and phosphate (pH 7.4 and 11) buffer. 2.2. Nanogel Synthesis. The cross-linked copolymer nanogels were synthesized by free radical precipitation polymerization with some modification of the synthesis reported in the literature.2426 In brief, 1.4 g of monomer (NIPAM), 100 mg of cross-linker N,N-methyl-enebisacrylamide, and 7.3 mg of SDS were dissolved in 150 mL of deionized water and allowed to heat at 70 °C under nitrogen for 1 h. Further initiator ammonium persulfate was added to this reaction mixture to initiate polymerization. Reaction was continued for 6 h. The nanogel was collected by repeated centrifugation and washing with deionized water to ensure the complete removal of any unwanted material. Subsequently, it was subjected to lyophilization. This sample is referred to as NG hereafter in the following text. The acid functionalized nanogel was synthesized following a similar method as mentioned above, except the addition of a calculated amount of 50 mg of acrylic acid was made in the beginning of the reaction. The molar ratio of cross-linker and monomer was maintained at 1:20. This sample is referred to as AFNG in the following text. For drug loading, 5 mg of AmB dissolved in dimethylsulfoxide was added into the solution containing NIPAM, N,N-methylenebisacrylamide, and sodiumdodecylsulfate followed by similar steps mentioned above for NG synthesis. Similarly, the separate synthesis was carried out except for the addition of acrylic acid as mentioned in the AFNG synthesis. AmB drug loaded samples, both nanogel and acrylic acid functionalized nanogel are referred to as AmB-NG and AmB-AFNG, respectively, in the following text. 2.3. Nanogel Characterization. The morphology of as-synthesized samples was studied using a scanning electron microscope (SEM, S-3400N, Hitachi). The samples for SEM were prepared by lypholyzing them at 80 °C. Detailed transmission electron microscopy (TEM) analyses were performed using a FEI Technai G2 20 microscope operated at 200 kV. Surface morphology and topography were characterized by scanning probe microscope (SPM, NANOTEC, Spain). Drug loading in nanogels was investigated using UVvis spectrophotometer (UV-2450, Shimadzu), FTIR spectrometer (Perkin-Elmer, 1000), and thermo-gravimetric analysis (Mettler 51E). Particle size characterization was carried out by dynamic light scattering measurements using a Nanotrac 150 (Microtrac, France) 2.4. Surface Charge Measurement. The specific surface charges of drug, nanogel, and functionalized nanogel were measured with a particle charge detector (PCD; model PCD04-pH, Germany). The working principle of PCD and further experimental details are described elsewhere.27,28 2.5. Rheological Measurement. The phase transition behavior of nanogels was studied by investigating viscoelastic properties using controlled-stress rheometer (MCR 300, Paar-physica). Measurements were performed using parallel plate- plate
Figure 1. SEM image of (a) NG, (b) AF-NG, (c) AmB-NG, (d) AmBAFNG, and (e) AmB-AFNG in citrate buffer pH 3 and (f) AmB-AFNG in phosphate buffer pH 11 and TEM image of (g) NG and (h) AmB-NG; (ae) scale bar corresponds to 200 nm; (g and h) scale bar corresponds to 50 nm.
geometry (diameter, 50 mm; gap between plates, 1 mm). We characterized the linear viscoelastic properties of composites (polymer) by the storage and loss moduli G0 and G00 , respectively, which can be obtained from oscillatory measurements performed at small strain (typically
