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

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

Accepted Manuscript Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery Baeckkyoung...

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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*

1

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 <0.05 were considered statistically significant. Data are expressed as mean ± standard error (SE).

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3. Results and Discussion 3.1.

Volume phase transition of microgels to temperature change

The fabricated microgels exhibited a well-characterized spherical morphology and colloidal dispersibility (Fig. 1B). The average diameter of the microgels at swollen state at 22˚C was measured to be 11±5 μm, exhibiting a moderate polydispersity. Single-microparticle observation revealed a temperature-dependent volume change of microgel, which exhibited a monotonic deswelling process as temperature increases (Fig. 2 and Supplementary Movie S1). The polydispersity of the microgels appears not to influence their deswelling characteristics, as evidenced by little correlation between the size of fabricated microgels and the deswelling ratio (Fig. S1). The microgels did not form aggregates even after deswelling in response to temperature increase (data not shown), which has been often the case for microgels with higher content of PNIPAM [40]. However, the re-swelling behaviour during the cooling cycle was not prominent in our fabricated microgels. The mechanism underlying the deswelling behaviour of microgels can be attributed to the shrinkage of the poly(NIPAM-coAAm) chains entrapped in the gelatin network, as evidenced by substantially lower deswelling of pure gelatin microgels to a thermal stimulus than that of gelatin microgels incorporating poly(NIPAM-co-AAm) (Fig. S2). The helix-coil transition of gelatin may in part be responsible for the microgel deswelling even observed in pure gelatin microgels [41].

3.2.

The effect of crosslinking degree on microgel deswelling behaviour

The deswelling and swelling behaviour of microgels with varying crosslinking degree were examined as a function of temperature. For all the examined crosslinking conditions, the extent of microgel deswelling gradually increased in response to increase in temperature (Fig. 3A). At a given increase in temperature from 22˚C to 42˚C, the extent of microgel deswelling increased with decreasing degree of crosslinking and the microgels with higher crosslinking

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degree (e.g. crosslinking time of 120 min) exhibited less deswelling (Fig. 3B). This may be explained by increased elasticity of the gelatin network, which may increase the resistance to thermally-induced deswelling [10]. Thus, our results support that deswelling behavior of the microgels can be tuned by controlling the crosslinking degree of the gelatin matrix. The crosslinking degree-dependent volume phase transition of the microgels can be explained as an interplay between the ternary mixing free energy of gelatin, poly(NIPAMco-AAm) and water within the microgel, to which temperature-dependent coil-globule transition of the entrapped poly(NIPAM-co-AAm) chains contributes, and the elastic free energy of the crosslinked gelatin network that is dependent on the degree of crosslinking [10].

3.3.

The effect of microgel deswelling behavior on the BSA release

We next examined how the volume phase transition behaviour of the microgels influences drug release profiles. The fluorescent TR-BSA molecules were used as a model drug and their release characteristics from microgels were assessed as a function of crosslinking time (Fig. 3C). The temperature increase from 22˚C to 42˚C resulted in an active release of encapsulated BSA from microgels, which is significantly greater than that of passive BSA leakage measured at 22˚C without the temperature increase. However, the extent of active BSA release decreased exponentially with increasing crosslinking time and eventually there was no difference at the crosslinking time of 120 min, which appears to be closely correlated with diminished deswelling behavior of microspheres (Fig. 3B). To further confirm the hypothesis that microgel deswelling behavior positively correlates with the extent of drug release, we examined the effect of poly(NIPAM-co-AAm) concentration on microgel deswelling behavior and assessed its effect on BSA release at a given crosslinking time of 30 min. As shown in Fig. 4, there was a positive correlation between the extent of microgel

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deswelling and the BSA release upon the temperature increase from 22oC to 42oC, both of which increased with increasing poly(NIPAM-co-AAm) concentration in the microgel. The increase of poly(NIPAM-co-AAm) ratio to total polymers from 16.7% to 50% concomitantly increased the extent of BSA release (from 27.2±6.8% to 59.8±8.3%, p<0.05) and the microgel deswelling (i.e., decrease in deswelling ratio from 0.62±0.01 to 0.43±0.02, p<0.05). Taken together, these results support that deswelling of microgels induced by increase in temperature is responsible for the release of encapsulated BSA, in a manner that is highly dependent on poly(NIPAM-co-AAm) concentration in the microgel. However, it may be necessary to carefully determine the optimal ratio of poly(NIPAM-co-AAm) to gelatin, which can achieve desired drug releases while minimizing the potential cytotoxic effect of poly(NIPAM-co-AAm) on cell viability [42].

3.4.

The effect of multiple thermal stimuli on the BSA release

We next evaluated the ability of the microgels to release encapsulated drug upon multiple thermal stimuli by quantifying cumulative BSA releases from the microgels in response to repeated cycles of heating and cooling (22˚C → 42˚C → 22˚C). The cumulative BSA release increased up to 60% after four cycles of heating and cooling, whereas the passive release of BSA maintained at the constant temperature of 22˚C was significantly low at ~10% (Fig. 5A). However, the extent of BSA release decreased over repeated heating cycles, which appears to correlate with decreased microgel deswelling (Fig. 5B). Although the mechanism that results in decreased deswelling capacity of microgels over repeated thermal cycles is not clear, it may be possible that micro-phase separation of collapsed PNIPAM copolymer chains from gelatin network might diminish the deswelling efficacy of microgels [43]. What is the potential mechanism by which volume phase transition in microgel triggers a drug release? The subchains of gelatin network themselves do not go through coil-globule

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phase transition since there is little effect of thermoresponsive deswelling for gelatin microgel in the absence of poly(NIPAM-co-AAm) (Fig. S2). The ternary mixing free energy of gelatin, poly(NIPAM-co-AAm) and water, and the elastic free energy of the gelatin network with certain mesh size may interplay to result in the volume phase transition in response to an increasing temperature [10]. One plausible explanation is that a decrease in the internal pressure due to the shrinkage of poly(NIPAM-co-AAm) to temperature increase may induce the decrease in the volume of an elastic gelatin microgel and thereby trigger convective flows that transport drug (BSA) out of the microgel, as evidenced by our data demonstrating a positive correlation between the deswelling of microgels and BSA release (Fig. 4 and Fig. S3). The convection-driven drug release, often referred as “squashing a sponge” effect, has been also postulated in conventional PNIPAM-based microgels, although some studies mentioned a counter-effect for hindered drug release caused by decreasing mesh size due to the collapse of PNIPAM network [44]. Additionally, due to the hydrophobic nature of deswollen PNIPAM microgels at higher temperatures, they often exhibit aggregations and the phase separation of encapsulated hydrophilic drug molecules [45]. In contrast, our gelatinbased microgels may circumvent the aggregation problems of conventional PNIPAM microgels since the hydrophilicity of our gelatin microgel surface does not change even after the deswelling of microgels. However, we do not rule out the possibility that there may be a small number of hydrophobic domains of poly(NIPAM-co-AAm) within our microgels at high temperature and thereby introduce partitioning effects on the BSA release [46].

3.5.

Biodegradability of the microgels

Lastly, we examined the biodegradability of microgels in the swollen state over days in the presence of collagenase (1 CDU/ml). Although poly(NIPAM-co-AAm) is generally considered to be non-degradable, it was used as a minor component (1.5% w/v, or 16.7%

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ratio to total polymers) in the current study and free chains of poly(NIPAM-co-AAm) could be excreted by the kidney in a random coil or partially-collapsed chain conformation with high flexibility, enough to pass through the renal filtration [47]. The single-microgel observation by DIC and fluorescence imaging revealed that the microgel started to degrade from its surface, while maintaining its original spherical shape (Fig. 6A). The fluorescence images shows that the fluorescence within the degrading gel is intact even after three days of degradation, suggesting that the fluorescent BSA molecules are still encapsulated within the microgel during the degradation process. Furthermore, the degrading microgels in the collagenase solution can still exhibit a deswelling capacity in response to a thermal stimulus from 37˚C to 42˚C (Fig. S4), which implies that the enzymatic erosion has little effect on the internal property of the microgel. It appears that the formation of the crosslinked network structures might confer the gelatin microgels with resistance to the collagenase penetration and thereby resulted in gradual degradation from the gel surface [23]. Nevertheless, the direct evidence on the mechanism of the microgel surface erosion need to be further pursued. It may be necessary to better understand the internal structure of the crosslinked microgel and the conformation of the trapped poly(NIPAM-co-AAm) chains using X-ray or neutron scattering. The kinetic change of the weight of microgels in the swollen state demonstrates that microgels are enzymatically degraded in an exponential-like manner over time (Fig. 6B). More importantly, the rate of degradation by collagenase was significantly attenuated for microgels with a higher crosslinking degree (Fig. 6B). This suggests that the rate of microgel degradation can be tuned by controlling the crosslinking degree of the gelatin matrix. This property can be useful in that it enables an effective tuning of the biodegradation rate by only controlling the crosslinking degree without any modification in the molecular composition or the surface properties of the microgels. Taken together, these results demonstrate that our

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thermoresponsive microgels are biodegradable and the extent of biodegradability can be tuned by controlling the crosslinking degree of the gelatin matrix.

4. Conclusions We have fabricated gelatin-based biodegradable microgel spheres that exhibit tunable thermosensitive volume phase transition as a function of poly(NIPAM-co-AAm) concentration and the crosslinking degree of the gelatin matrix, which is correlated to the release amount of BSA. The development of a microgel-based drug delivery system that achieves both thermoresponsivity and biodegradability has been challenging tasks, since most synthetic thermosensitive microgels normally are not biodegradable in vivo, and biopolymers that

are

enzymatically

thermosensitiveness.

In

degradable this

context,

generally our

do

not

gelatin-based

exhibit

well-characterized

microgels

demonstrating

characteristics of volume phase transition to thermal stimuli as well as biodegradability may offer a promising tool as a stimuli-responsive drug carrier. However, it should be noted that our drug delivery system still has a limitation to be used at body temperature of 37˚C due to a small degree of deswelling characteristic at the temperature, associated with a relatively low value of LCST of 34˚C for poly(NIPAM-co-AAm) polymer used in this study. This necessitates the development or use of thermoresponsive polymer that can exhibit a higher LCST to overcome this issue. Rather, our thermoresponsive microgels still may be used for transdermal drug delivery carrier to the site of skin defect or wounds via local injection for controlled release of therapeutic proteins or growth factors, considering the range of temperature between 32˚C and 34˚C in the subcutaneous layer [48] and capacity for our microgels to deswell to some extent and release drugs at the temperature range (Fig. S5). In addition, the incorporation of magnetic nanoparticles or gold nanoparticles within microgels

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will enable a remotely controlled drug delivery platform, when combined with magnetic field application or near infrared actuation systems, respectively, that can trigger increase in temperature on the surface of nanoparticles [49-51]. This may achieve a smart drug delivery system to release a desired drug concentration at a desired time point remotely. Since there has been a great need for design and synthesis of biocompatible smart microgels that enable the 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.

Acknowledgements This study was supported by startup fund by Kent State University and Farris Family Innovation Award (MK). The authors thank Steven Shaffer (Biological Sciences, Kent State University) and Min-Su Kim (LCI & Chemical Physics Interdisciplinary Program, Kent State University) for their technical assistants.

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Figures Legends Figure 1. The fabrication of gelatin/poly(NIPAM-co-AAm) microgels (1.5% (w/v) or 16.7% of poly(NIPAM-co-AAm) ratio to total polymers) crosslinked by genipin. (A) A schematic for the gelatin-genipin crosslinking reaction. (B) Micrographs (DIC and fluorescence images) of TR-BSA loaded microgels (upper panel). Fabrication process of microgels by standard water-in-oil emulsion (lower left panel). The size distribution of fabricated microgels (lower right panel).

Figure 2. The deswelling and swelling behaviours of single microgel in response to temperature change. Microgels incorporating 16.7% of poly(NIPAM-co-AAm) ratio to total polymers were used in this study. (A) Representative microscopic images of single microgel to temperature change between 22˚C and 42˚C. Scale bar = 5 μm. (B) The deswelling behaviour of microgel during heating and re-heating cycles from 22˚C and 42˚C, in which reheating cycle was applied after temperature was fully cooled down to 22˚C following the first heating cycle. The deswelling ratio (VT/V22˚C) is defined as the ratio of microgel volume at a given temperature (VT) with respect to temperature at 22˚C (V22˚C).

Figure 3. The effect of crosslinking degree on the extent of microgel deswelling and BSA release. Microgels incorporating 16.7% of poly(NIPAM-co-AAm) ratio to total polymers were used in this study. (A) The change in deswelling ratio (VT/V22˚C) of microgels with gradual increase in temperature for varying crosslinking time (t cl). (B) Changes in microgel deswelling ratio in response to increase in temperature from 22˚C to 42˚C (V42˚C/V22˚C). For both graphs, dotted lines were obtained by linearly fitting the measured data. For each crosslinking condition and temperature point, about 20-50 single microgels were tracked. (C)

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Changes in BSA release from microgels in response to increase in temperature from 22˚C to 42˚C as a function of crosslinking time. The thermal stimulation was applied for 30 min. N=5-10 for each crosslinking condition.

Figure 4. Effects of poly(NIPAM-co-AAm) concentrations on the extent of microgels deswelling and BSA release in response to increase in temperature. Responses of microgel deswelling and BSA release were measured for varying concentration of poly(NIPAM-coAAm) at ratio of 16.7%, 30%, and 50% to total polymers. Microgels used in this study were fabricated at crosslinking time of 30 min. The release of BSA from microgels was induced in response to temperature increase from 22˚C to 42˚C and the thermal stimulation was applied for 30 min. About 20 single microgels were tracked for the measurement of microgel deswelling.

N=5-10 samples for BSA release.

Figure 5. The extent of BSA release and microgel deswelling in response to repeated heating cycles. Microgels used in this study were fabricated with 30 min crosslinking time and by incorporating 16.7% of poly(NIPAM-co-AAm) ratio to total polymers. (A) Cumulative release of BSA from microgels (rectangle) in response to repeated thermal stimuli at 42˚C, in which the reheating cycle was applied after temperature was cooled down to 22˚C following the first heating cycle. Each heating cycle was applied for 15 min. For comparison, cumulative release of BSA in the absence of thermal stimuli maintained at 22˚C is given (circle). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 vs. passive leakage at 22˚C. N=10 for each repeated heating cycle. (B) Change of microgel deswelling ratio as a function of heating cycle number, in which all the conditions are the same as in (A). For each condition, 20 microgels were tracked.

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Figure 6. The enzymatic degradation of microgels by collagenase. (A) The representative microscopic images (phase contrast and fluorescence) and the schematic of microgel undergoing enzymatic degradation by collagenase treatment (1 CDU/ml). The fluorescence signals in the image are from encapsulated TR-BSA. (B) The kinetics of enzymatic degradation of microgels by collagenase over days for varying degree of crosslinking. Microgels used in this study were fabricated by incorporating 16.7% of poly(NIPAM-coAAm) ratio to total polymers. N=5 samples for each crosslinking condition.

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Graphical abstract