Accepted Manuscript Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery Baeckkyoung Sung, Chanjoong Kim, Min-Ho Kim PII: DOI: Reference:

S0021-9797(15)00244-1 http://dx.doi.org/10.1016/j.jcis.2015.02.068 YJCIS 20303

To appear in:

Journal of Colloid and Interface Science

Received Date: Accepted Date:

19 December 2014 26 February 2015

Please cite this article as: B. Sung, C. Kim, M-H. Kim, Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery, Journal of Colloid and Interface Science (2015), doi: http://dx.doi.org/10.1016/j.jcis.2015.02.068

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Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery

Baeckkyoung Sung 1,2, Chanjoong Kim1 and Min-Ho Kim2*

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Liquid Crystal Institute and Chemical Physics Interdisciplinary Program, Kent State University, Kent, OH 44242, USA 2

Department of Biological Sciences, Kent State University, Kent, OH 44242, USA

Baeckkyoung Sung, Ph.D. (Email: [email protected]) Chanjoong Kim, Ph.D. (Email: [email protected])

*Corresponding author: Min-Ho Kim, Ph.D Department of Biological Sciences Kent State University Kent, OH 44242, USA Phone: 330-672-1445 Fax: 330-672-3713 E-mail: [email protected]

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Abstract In this study, we present gelatin-based thermoresponsive colloidal microgels that enable the controlled release of drugs by volume phase transition. The microgel was fabricated by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within three-dimensional gelatin networks crosslinked by genipin. We demonstrate that our gelatin-based thermoresponsive microgel exhibits a tunable deswelling to temperature increase, which positively correlated to the

release

of

bovine

serum

albumin (BSA)

as

a

function

of

poly(N-

isopropylacrylamide-co-acrylamide) concentration. The microgel was enzymatically degradable by collagenase treatment. The extent of BSA release and biodegradability were tuned by controlling the crosslinking degree of the gelatin matrix. Meeting a great need for design and synthesis of auto-degenerating smart microgels that enable the controlled release of therapeutic proteins in responsive to external stimuli, our gelatin-based microgels that satisfy both thermoresponsivity and biodegradability have a great potential in tissue engineering applications as a soft microdevice element for drug delivery.

Key words: microgel, colloid, biodegradability, thermoresponsivity, deswelling, phase transition, drug delivery

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1. Introduction Stimuli-responsive polymeric hydrogels have been illuminated as new types of smart functional materials for various biomedical applications including controlled drug delivery. Since the hydrogel is a three-dimensional (3D) network of crosslinked polymers in aqueous media, its sensitivity to external stimuli (e.g. temperature or pH) depends on the corresponding characteristics of its constituting polymer chains in water. Numerous thermoand pH-responsive polymers have been studied for the purpose of stimuli-responsive drug delivery [1-5]. Microscale hydrogels has been widely used for drug delivery application due to their controllable and sustainable drug release profiles as well as tunable chemical and mechanical properties that enable the release mechanisms can be made responsive to external stimuli [1, 2, 5]. Recent studies on the microscale hydrogels for thermoresponsive drug delivery have been focused on using microspheres [6], microellipsoids [7] or micropatterned thin films [8]. The colloidal microgels have advantages for drug delivery application in that they exhibit a rapid responsiveness to external stimuli and suitable injectability to local tissue as aqueous suspensions, compared to their macroscopic counterparts that exhibit relatively slow kinetics and require surgical transplantation. One of the mostly adopted polymers in colloidal microgel for stimuli-responsive drug delivery is thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) or its copolymers [1, 9]. In aqueous solutions, a PNIPAM chain undergoes a well-characterized and reversible coilto-globule transition at its lower critical solution temperature (LCST),

at which the 3D

crosslinked PNIPAM networks exhibit a volume phase transition between swollen and collapsed phases [10]. Most of thermoresponsive microgels have been fabricated based on PNIPAM, its derivatives, or their hybrids [11-16].

Biodegradability is one of the most

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important considerations of scaffolds for tissue engineering. However, the use of PNIPAMbased microgels (including nanogels) as carriers for drug delivery has been limited by their insufficient biocompatibility and biodegradability [17], which may result in accumulation in the body for longer duration that trigger cytotoxicity [18, 19], and hindrance of microcirculation [20]. Furthermore, high aggregation of the PNIPAM microgels in aqueous environments, due to their hydrophobic surfaces at temperature above LCST, has been a major barrier for the drug delivery applications. There have been intensive efforts to fabricate microgels that exhibit thermosensitivity as well as suitable biocompatibility and biodegradability [21]. Frequently used biocompatible and biodegradable polymers include gelatin [22-24], chitosan[25, 26], alginate acid [27, 28], hyaluronic acid [29, 30], poly(lactic acid) [31], and dextran [32]. However they do not exhibit a well-characterized thermoresponsive property in general. To confer the thermoresponsive property to such polymers, several attempts including grafting or copolymerization with PNIPAM have been pursued [3, 33, 34]. More sophisticated synthetic techniques with chemical combinations of polysuccinimide and poly(N-2-hydroxyethyl-DL-aspartamide) [4], or poly(L-glutamic acid-g-2-hydroxyethyl methacrylate) and hydroxypropylcellulose-gacrylic acid [35], have been also introduced. However, these still had limitation in that the thermosensitive volume phase transition mechanism of microgels could not be directly correlated to the drug release profiles. The major objective of this study was to develop a microgel-based drug delivery carrier that is biodegradable and biocompatible and enables a thermally triggered drug release. Here we present colloidal microgel spheres which exhibit tunable volume phase transition characteristics in response to a temperature change, which directly correlate with extent of protein release. The microgel was synthesized by physically entrapping thermoresponsive poly(N-isopropylacrylamide-co-acrylamide) [poly(NIPAM-co-AAm)] chains as a minor

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component within a 3-dimensional gelatin network crosslinked by genipin. Genipin, a naturally derived metabolite crosslinker, has been considered to be cytocompatibile and nontoxic [36, 37]. The gelatin has been shown to be biocompatible and exhibit low immunogenicity compared to other types of hydrogel polymers [38]. We demonstrate that our gelatin-based thermoresponsive microgel exhibits a tunable deswelling ratio and biodegradability as a function of poly(NIPAM-co-AAm) concentration and the crosslinking degree of the gelatin matrix, which is correlated to controllable release amount of bovine serum albumin (BSA).

2. Materials and Methods 2.1.

Preparation of colloidal microgel spheres

The fabrication of gelatin-poly(NIPAM-co-AAm) microgels was performed using water-inoil emulsion method as described previously [24]. Solutions of gelatin type A (porcine skin, Sigma-Aldrich, MO, USA) in sol phase and poly(NIPAM-co-AAm) (Mw = 20,000-25,000, LCST at 34-38˚C, Sigma-Aldrich, MO, USA) were separately prepared in distilled water of equal volumes (215 μl), and thoroughly mixed together. The final concentration of mixtures of polymers comprising gelatin and poly(NIPAM-co-AAm) was maintained at 9% (w/v) within the microgel, by varying poly(NIPAM-co-AAm) concentration from 1.5% (w/v), 2.7% (w/v) and 4.5% (w/v), which corresponds to poly(NIPAM-co-AAm)] ratio of 16.7%, 30%, and 50% to total polymers, respectively. The mixed solution was emulsified in 15 ml silicone oil [polydimethylsiloxane (PDMS)] (viscosity 350 cSt; Sigma-Aldrich, MO, USA) at 32˚C for 30 min through mechanical stirring (900-1000 rpm). The whole emulsion was cooled and stabilized at 4˚C for 1 hour so that the gelatin microdroplets went through the sol-to-gel phase transition. In order to obtain the pellet of the gel microspheres, the emulsion was transferred

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to a tube, vigorously mixed with an excess of a surfactant solution (30 ml) made of 100 ppm poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic® L64; Sigma-Aldrich, MO, USA) in phosphate buffered saline (PBS; pH 7.4), and centrifuged at 4400 rpm (4˚C) for 20 min. This step was repeated two times for the removal of silicone oil and colloidal stabilization of the microspheres. Then, 2 ml of 1% (w/v) genipin (Mw = 226.23; TimTec LLC, DE, USA) solution in PBS was added to the finally obtained pellet. Subsequently, the microspheres were re-dispersed in the genipin solution and the suspension was kept at 23˚C for the crosslinking of gelatin. The crosslinking time (t cl) was varied from 15 min to 120 min. A schematic for the crosslinking reaction is given in Fig. 1A [37]. Following the crosslinking step, the solution of microgels with genipin was centrifuged at 4400 rpm for 20 min, where the sample was kept at 4˚C to stop the crosslinking reaction. Then, the supernatant with unreacted genipin solution was removed and cross-linked microgels were re-suspended in PBS and centrifuged at 4400 rpm (4˚C) for 20 min. This washing step was repeated for 2 times and obtained microgel spheres were suspended in 5 ml PBS for subsequent experiments.

2.2.

Single-microgel observation for deswelling behaviour during temperature change

The microgel suspension was loaded in the space between a slide glass and a cover slip, whose boundary was then sealed with epoxy (Norland Optical Adhesive, Norland Products, Inc, NJ, USA) hardened by UV irradiation (Black-Ray® XX-15BLB UV Bench Lamp, UVP LLC, CA, USA). The specimen was placed in a temperature-controlled chamber and was observed by an optical microscope (BX51, Olympus, Japan) for DIC imaging to observe deswelling and swelling behaviors of microgels during the temperature change in a real time. The temperature change (heating and cooling) was made between 22˚C and 42˚C at a rate of

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0.3-0.4˚C/min. This rate was determined by preliminary observation on deswelling kinetics of the microgels that exhibit a rapid deswelling response that has occurred within a minute.

2.3.

Measurement of BSA release to thermal stimulus

To quantify drug release profiles from microgel, Texas-Red-conjugated bovine serum albumin (TR-BSA) (Life Technologies, NY, USA) was used as a model drug. The encapsulation of TR-BSA within microgels were achieved by adding TR-BSA to the mixture of gelatin and poly(NIPAM-co-AAm) solution and emulsification procedure was performed as described above. The extent of TR-BSA release from microgels to temperature increase was quantified by measuring the ratio of fluorescence intensity of TR-BSA, in the media of microgels solution after thermal stimulus, to total fluorescence intensity of TR-BSA within microgles before thermal stimulus at 22˚C, using a spectrophotometer (Excitation at 584 nm and emission at 612 nm, SpectraMax M4, Molecular Devices, CA, USA). For this, the temperature in the sample well was maintained for 30 min at either 22˚C for measurement of passive leakage or at 42˚C for temperature triggered active release. Then, the microgels solution was centrifuged at 4400 rpm (4˚C) for 20 min following thermal stimulus, supernatants with TR-BSA released from microgels were collected, and the fluorescence intensity of TR-BSA from supernatant was measured. Then, BSA release amount (%) was calculated as follows:

In order to examine the drug release profiles from microgels in response to repeated thermal stimuli, the cumulative release of TR-BSA was measured following each repeating cycles of

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heating (22˚C → 42˚C → 22˚C), in which heat pulse was applied for 15 min duration for each cycle. The quantification of the BSA release amount was performed as described above.

2.4.

In vitro test for enzymatic biodegradation of microgels

The enzymatic degradability of microgels in the swollen state over time was quantified in the presence of collagenase (1 CDU/ml, collagenase from Clostridium histolyticum, SigmaAldrich, MO, USA) by single-particle observation using time-lapse microscopy and weight loss measurements. The concentration of collagenase used in this study was shown to be in the range of collagenase concentration secreted in the mammalian connective tissues [39]. The time-lapse single-particle observation was performed using an optical microscope (IX81, Olympus, Japan) equipped with a cooled CCD camera (C10600 ORCA-R2, Hamamatsu, Japan) while maintaining a temperature at 37˚C with a temperature controller (ThermoPlate, Tokai Hit, Japan) during the entire course of measurements. The extent of microgel degradation by collagenase was quantified by normalizing the weight of remaining microgel after collagenase treatment with respect to initial weight before collagenase treatment. For this, the microgel suspensions in the test tubes were centrifuged (4400 rpm for 20 min) and the weight of the pellet was measured in a swollen state after removing supernatants. This measurement was repeated every day for up to day 12. During the entire course of measurement, the temperature was maintained at 37˚C.

2.5.

Statistical Analysis

Statistical significance between two groups was determined by two-tailed unpaired t tests. P values of