Injectable Thermoresponsive Hydrogel Formed by Alginate-g-Poly(N

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Injectable Thermoresponsive Hydrogel Formed by Alginateg-Poly(N-isopropylacrylamide) Releasing DoxorubicinEncapsulated Micelles as Smart Drug Delivery System Min Liu, Xia Song, Yuting Wen, Jing-Ling Zhu, and Jun Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12849 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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ACS Applied Materials & Interfaces

Ms. No.: am-2017-12849r 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Injectable Thermoresponsive Hydrogel Formed by Alginate-g-Poly(Nisopropylacrylamide) Releasing Doxorubicin-Encapsulated Micelles as Smart Drug Delivery System Min Liu,†,‡ Xia Song,† Yuting Wen,† Jing-Ling Zhu,† Jun Li*,†,‡ †

Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, 7

Engineering Drive 1, Singapore 117574, Singapore ‡

NUS Graduate School for Integrative Sciences & Engineering (NGS), National University of

Singapore, Centre for Life Sciences, 28 Medical Drive, Singapore 117456, Singapore

Abstract In

this

work,

we

have

synthesized

a

thermoresponsive

copolymer,

alginate-g-poly(N-

isopropylacrylamide) (alginate-g-PNIPAAm), by conjugating PNIPAAm to alginate, where PNIPAAm with different molecular weight and narrow molecular weight distribution was synthesized by atomic transfer radical polymerization (ATRP). The copolymer dissolved in water or PBS buffer solution at room temperature, and formed self-assembled micelles with low critical micellization concentrations when temperature increased to above their critical micellization temperatures. At higher concentration, i.e., 7.4 wt% in water, the copolymer formed solutions at 25 °C, and turned into thermosensitive hydrogels when temperature increased to the body temperature (37 °C). Herein, we hypothesized that the thermoresponsive hydrogels could produce self-assembled micelles with the dissolution of the alginate-g-PNIPAAm hydrogels in a biological fluid or drug release medium. If the drug was hydrophobic, the hydrogel eventually could release and produce drug-encapsulated micelles. In our experiments, we loaded the anti-cancer drug doxorubicin (DOX) into the alginate-g-PNIPAAm hydrogels, and demonstrated that the hydrogels released DOX-encapsulated micelles in a sustained manner. The slowly released DOX-loaded micelles enhanced the cellular uptake of DOX in multidrug resistance AT3B-1 cells, showing effect overcoming the drug resistance and achieving better efficiency for killing the cancer cells. Therefore, the injectable thermoresponsive hydrogels formed by alginate-g-PNIPAAm and loaded with DOX turned into a smart drug delivery system, releasing DOX-encapsulated micelles in a sustained manner, showing great potential for overcoming the drug resistance in cancer therapy.

KEYWORDS: alginate, poly(N-isopropylacrylamide), graft copolymer, hydrogel, micelle, smart drug delivery system

1

ACS Paragon Plus Environment


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1. Introduction Injectable hydrogels have attracted much attention and interest as drug delivery system due to the easy administration without invasive surgical and implantation procedures and patient convenience.1-5 Particularly, “smart” hydrogels that can sense and respond to external stimuli, such as temperature, pH, light and electric fields, are extensively studied.6-7 Among those types of stimuli responsive hydrogels, thermoresponsive hydrogel that is responsive to temperature change is one of the most popular and widely analyzed systems for drug delivery.8-12 Poly(N-isopropylacrylamide) (PNIPAAm) can undergo a reversible phase transition in response to temperature change and is used to form thermoresponsive hydrogels.13-16 PNIPAAm has a lower critical solution temperature (LCST) of 32 °C, at which it undergoes a coil to globule transition.13,17-18 The phase transition in response to changes in temperature is rapid and reversible. PNIPAAm has both hydrophobic isopropylic moiety and hydrophilic amide moiety. PNIPAAm is hydrophilic with the chains expanded and hydrated in water when temperature is below LCST, and becomes hydrophobic with the chains dehydrated and collapsed at a temperature higher than LCST.18-19 The LCST of PNIPAAm could be tuned with the addition of salts, surfactants or copolymerization with various hydrophilic or hydrophobic co-monomers.18,20-21 Copolymerization with hydrophobic monomer would decrease the LCST, while copolymerization with hydrophilic one would increase the LCST.22-23 Sodium alginate is a good choice to cooperate with PNIPAAm to form thermoresponsive hydrogels because it is biocompatible, biodegradable, non-toxic, chelatable, and suitable for chemical modification.24 Alginate is a water-soluble polysaccharide extracted from brown seaweed. It is a liner copolymer of 1–4 linked α-L-guluronic (G) and β-D-mannuronic acid (M). Two G-block aligned regions could form a diamond-shaped hole whose dimensions are ideal for the cooperative binding of divalent cations such as calcium ions to form physically crosslinked hydrogels. In previous studies, hydrogels based on alginate and PNIPAAm are formed by semi-interpenetration network,25-26 addition of calcium ions as physical crosslinker,25-29 addition of glutaraldehyde as chemical crosslinker,24 or grafting PNIPAAm to alginate.25-30 In addition, alginate-g-PNIPAAm could form self-assembled micelles due to temperature change, and doxorubicin (DOX)-encapsulated micelles showed passive accumulation at the tumor site due to the enhanced permeability and retention (EPR) effect.30 While the alginate-g-PNIPAAm micelles showed EPR effect for hydrophobic drugs, the hydrogels formed by alginate-g-PNIPAAm could be a sustained release matrix for drugs, which are also injectable thermoresponsive hydrogels, being solution at temperature below LCST and becoming hydrogel after being injected into the body where the temperature is about 37 °C (above LCST). We hypothesize that the thermoresponsive hydrogels may produce self-assembled micelles with the dissolution of the alginate-g-PNIPAAm hydrogels in a biological fluid or drug release medium. If the drug is hydrophobic, the hydrogel eventually releases and produces drug-encapsulated micelles. 2


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Therefore, this study aims to synthesize alginate-g-PNIPAAm copolymers that can be injected into the body at low temperature and form stable thermoresponsive hydrogels at the body temperature for sustained release of the hydrophobic anti-cancer drug DOX, and then produce DOX-encapsulated micelles with EPR effect, resulting in better cellular uptake to overcome the multidrug resistance (MDR) as we reported in another micellar system based on a supramolecular host-guest interaction system.31-32 Thus, the alginate-g-PNIPAAm injectable thermoresponsive hydrogels form smart drug delivery systems for anti-cancer drugs. Here, we synthesized a series of alginate-g-PNIPAAm copolymers. The PNIPAAm with narrow molecular weight distribution was synthesized by atomic transfer radical polymerization (ATRP). Our alginate-g-PNIPAAm copolymers formed nice self-assembled micelles with uniform sizes and high stability, and efficiently encapsulated DOX. The rheological properties of the alginate-g-PNIPAAm copolymers were investigated to elucidate the thermoresponsive hydrogel formation and the stability and strength of the hydrogels related to the copolymer compositions. The in vitro release study showed that the hydrogels released DOX-encapsulated micelles in a sustained manner, and the micelles further enhanced the cellular uptake of DOX for overcoming the drug resistance and achieving better efficiency to kill the cancer cells.

2. Experimental section 2.1 Materials N-Isopropylacrylamide (NIPAAm, TCI) was purified by recrystallization from hexane and dried at 100 °C under vacuum before use. Copper(I) bromide (CuBr), α-bromoisobutyric acid (BIBA), tris[2(dimethylamino)ethyl]amine (Me6TREN), sodium alginate (M/G = 1.56, Mn = 120k-190k Da), 2-(Nmorpholino)- ethanesulfonic acid (MES), 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS), glycine and deuterium oxide (D2O) were purchased from Sigma-Aldrich. 1-Ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Tokyo Chemical Industry Co. Ltd. Doxorubicin hydrochloride (DOX·HCl, 98.4%) was purchased from Pharmacia and Upjohn. Diethyl ether, tetrahydrofuran (THF), methanol and dichloromethane (DCM) were purchased from QReC.

2.2 Synthesis of Amino-Poly(N-isopropylacrylamide) Polymerization of NIPAAm was conducted in 2-PrOH/water (3:1, v/v) mixed solvent. With target degree of polymerization (DP) of 38 and 50, the molar ratio of NIPAAm:BIBA:CuBr:Me6TREN was = 38:1:1:1 and 50:1:1:1, respectively. NIPAAm, BIBA, Me6TREN were dissolved into the mixed solvent and purged with nitrogen for 1 hour in ice bath. Then, CuBr was added quickly and purged for another half an hour. Then, the reaction continued in ice bath under nitrogen atmosphere for 1 hour before transferring the reaction tube into the 4 °C fridge. When the tube was opened and catalyst was exposed to air, the reaction was stopped. The final green mixture obtained was diluted with THF and 3



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passed through a short Al2O3 column to remove copper catalyst. The resulting eluate solution was concentrated by rotary evaporator and then precipitated in diethyl ether. The white product was collected by centrifugation, and washed with diethyl ether. The product was dried under vacuum overnight. The collected PNIPAAm was dissolved in DCM with EDC and NHS for 1 hour in ice bath. Then, the solution was added into ethylenediamine (EDA) in DCM solution drop-wise in ice bath. The amount of EDC and NHS was determined by the molar ratio of PNIPAAm:EDA:EDC:NHS = 1:10:1:1. The final concentration of NHS was 0.05 mmol/mL. The final product was purified by precipitation in diethyl ether three times and dialysis in dialysis tube (molecular weight cutoff of 2000) for 3 hours and the solution was lyophilized. The synthetic scheme was shown in Figure 1.

Figure 1. Synthetic scheme of alginate-g-PNIPAAm.

2.3 Quantification of Free Amines in Amino-PNIPAAm by TNBS Assay Sodium bicarbonate (0.1 M, pH 8.5) was prepared as reaction buffer. TNBS (0.01%, w/v%) was prepared using the reaction buffer as a diluent. SDS solution (10%, w/v%) was prepared in DI water. The amino-PNIPAAm was dissolved in the reaction buffer at a concentration of 1.2 mg/mL. 0.25 mL of the TNBS solution was added to 0.5 mL of the amino-PNIPAAm solution. The mixture was incubated at 37 °C for two hours. Then, 0.25 mL of SDS solution and 0.125 mL of 1 N HCl was added to the mixture. The absorbance of the final solution was measured at 335 nm. Glycine (7.5 mg) was dissolved in 10 mL reaction buffer. Then, a series of known concentration of glycine solutions were prepared with 200 µM glycine solution and sodium bicarbonate buffer. The series of glycine solutions were then used to obtain a standard curve for the quantitative determination of free amines in amino-PNIPAAm.

4


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2.4 Synthesis of Alginate-g-PNIPAAm The dried amine-ended PNIPAAm was dissolved in MES buffer (10 mg/mL, pH 6.5). The alginate amount was determined by DP and degree of substitution (DS) value. The molar ratio of carboxyl group of alginate:EDC:NHS was = 1:1:1. The synthesis scheme was shown in Figure 1. The final product was purified by precipitation in methanol followed by dialysis in dialysis tube (molecular weight cutoff of 12000 Da) for 4 days. After dialysis, the solution was lyophilized.

2.5 Molecular Characterization 1

H NMR spectra were recorded on a Bruker AV-400 NMR spectrometer at room temperature.

Chemical shift at 4.7 ppm was referred to the solvent peak of D2O. Gel permeation chromatography (GPC) measurement was performed with a Shimadzu SIL-10A and LC-20AD system equipped with two Phenogel 10 µm 100 and 10000 Å columns (size: 300 × 7.8 mm) connected in series and a Shimadzu RID-10A refractive index detector. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 0.6 mL/min at 40 °C. The system was calibrated using monodispersed PEG standards.

2.6 Size and Morphology of Alginate-g-PNIPAAm Micelles Alginate-g-PNIPAAm was dissolved in PBS with concentration of 1, 0.5, and 0.2 mg/mL. The size of the alginate-g-PNIPAAm micelles was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd., MA, USA), with a laser light wavelength of 633 nm at a 173ºscattering angle. The measurements were carried out at 25 °C and 37 °C and 30 minutes equilibration time at each temperature was set before the measurement. The Z-average hydrodynamic diameters of the particles were obtained. The morphology of alginate-g-PNIPAAm micelles was investigated using transmission electron microscopy (TEM). The sample was imaged on a JEOL JEM-3010 FasTEM field emission transmission electron microscope, operated at 300 kV. The sample was incubated at 37 °C for 4 hours for micelle formation. TEM sample was then prepared by directly depositing a drop of sample solution onto a 200 mesh carbon coated copper grid and waited for 10 minutes at 37 °C. Then 0.1 wt% of phosphotungstic acid (PTA) was added onto the copper grid and waited for 8 minutes before removing the rest solution. The prepared sample on the copper grip was kept in a 37 °C incubator overnight before TEM imaging.

2.7 Determination of Critical Micellization Concentration and Critical Micellization Temperature The critical micellization concentration (CMC) values were determined by using the dye solubilization method33-34. The hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH) was dissolved in methanol with a concentration of 0.6 mM. 20 µL of this stock solution was mixed with 2.0 mL of polymer solution in PBS buffer with concentrations ranging from 0.025 to 2 mg/mL and equilibrated at 37 °C for 4 hours. UV-vis spectrophotometer (Shimadzu UV-2600) equipped with a Julabo F12-ED refrigerated/heating circulator was used to obtain the UV-Vis spectra in the range of 330-430 nm at 5



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37 °C. The CMC value at 37 °C was determined by the plot of the difference in absorbance at 378 nm and at 400 nm (A378 – A400) versus logarithmic concentration. The critical micellization temperature (CMT) was determined using the same method. 20 µL of DPH methanol stock solution was mixed with 2.0 mL of polymer solution in PBS buffer with a concentration of 2 mg/mL. The temperature was increased from 25 to 43 °C. The absorbance was measured after equilibrating at each temperature for 30 minutes. The CMT value was determined by the plot of the difference in absorbance at 378 nm and at 400 nm (A378 – A400) versus temperature.

2.8 Sol−Gel Transition Sol−gel transition was determined by a test tube inverting method.35 Each sample of a given concentration was prepared by dissolving the polymer in distilled water in a 4-mL vial. The samplecontaining vial was immersed in a water bath at 37 °C for 30 minutes. The phenomena were recorded when the tube was inverted by 180°.

2.9 Rheological Studies Dynamic rheological measurements were performed on a HAAKE™ MARS III Rotational Rheometer with parallel plate geometry (35 mm diameter) at a gap of 1 mm. The sample was prepared by dissolving 80 mg of copolymer in 1 mL DI water. The sample was carefully loaded onto the measuring geometry and oil was added around the measuring geometry to minimize the effect of water evaporation on the rheology data. The sol-gel transition of the polymer aqueous solution was investigated at a fixed frequency of 1 Hz with constant stress of 1 Pa and heating from 25 °C to 37 °C. Oscillatory stress sweeps were performed by applying increasing shear stress logarithmically from 0.1 Pa at a fixed frequency of 1 Hz at 37 °C, until the hydrogels were destroyed, as evidenced by a G′/G″ crossover, and 100% deformation was reached. The yield stress (τ) was defined as the applied shear stress value at G′/G″ crossover.36

2.10 In Vitro Release Study of Doxorubicin (DOX) from Hydrogels DOX·HCl was dissolved in DI water at a concentration of 1 mg/mL. Polymer (40 mg) was dissolved in the DOX solution to form 0.5 mL solution in a 0.6-mL vial. The solutions (triplicates for each hydrogel) were incubated at 37 °C overnight to form hydrogels before adding to the release medium. Subsequently, the vials were inverted with their opening uncovered, and carefully placed inside 15 mL tubes containing a volume of 3 mL of PBS buffer solution (10 mM, pH 7.4). Bubbles were avoided when placing the vials into the release medium. The sample tubes were shaken at 100 rpm at 37 °C in the dark. At predetermined time intervals, the vials were transferred to tubes containing fresh PBS release buffer. The concentration of DOX in release medium was tested by UV-vis measurement of absorbance at 481 nm with a microplate reader (Infinite M200 PRO, TECAN). DOX solutions of various concentrations in PBS buffer were prepared and absorbance at 481 nm was measured to produce a standard curve. It was calculated to obtain the following equation, where y is the concentration of DOX and x is the absorbance value. The calibration curve under the concentration 6


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range from 6.25 x 10-4 to 0.020 mg/mL (the solubility of DOX in PBS buffer is 0.020 mg/mL)37 is linear with a correlation coefficient of R2 = 0.9995.

y = 0.2337x − 0.0106 The cumulative drug release was calculated from the following equation:

Cumulative release % =

× 100

here, Mt is the amount of DOX released from the hydrogels at time t, and M0 is the amount of DOX loaded into the hydrogel.

2.11 In Vitro Cellular Uptake of DOX-Encapsulated Micelles in Release Medium Briefly, AT3B-1 (MDR+) cells were seeded on cover slips in 24-well plates (8 × 104 cells/well) in 0.4 mL growth medium and allowed to attach and grow for 24 hours. The cells were then incubated with 0.4 mL growth medium and 0.4 mL release medium containing DOX-encapsulated micelles at 37 °C for 24 hours. The cells were washed with PBS and then fixed with 4% paraformaldehyde. The cover slips with fixed cells were mounted onto glass slide with FluoroshieldTM with DAPI, then imaged using an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus, Japan) equipped with a 100 × 1.35 NA oil immersion objective lens. The excitation wavelength for red fluorescence was set at 559 nm with an emission at 619 nm. The excitation wavelength for blue fluorescence was set at 405 nm with an emission at 461 nm. Free DOX solution (20 µg/mL) was used as control.

2.12 In Vitro Cytotoxicity of DOX-Encapsulated Micelles Released from Hydrogel The DOX release medium taken at different pre-determined time points from one of the triplicates prepared above was used directly to test its in vitro cytotoxicity in two cell lines. AT3B-1-N (MDR-) and AT3B-1 (MDR+) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mg penicillin, 100 µg/mL streptomycin, without and with DOX·HCl (1 µg/mL) respectively, at 37 °C and 5% CO2. AT3B-1-N (MDR-) and AT3B-1 (MDR+) cells (2 × 104 cells/well) were seeded in 96-well plates in 0.1 mL DMEM medium and incubated for 24 hours prior to the addition of sample solutions. The cells were incubated with 0.1 mL growth medium and 0.1 mL release medium containing DOXloaded micelles at 37 °C for 24 hours. Subsequently, 10 µL of sterile MTT stock solution (5 mg/mL in PBS) was added to each well. After incubation for 4 h, the unreacted dye was removed by aspiration. The formazan crystals were dissolved in DMSO (150 µL/well), and the absorbance was measured with a microplate reader (Infinite M200 PRO, TECAN) at a wavelength of 570 nm. The cell viability (%) was calculated according to the following equation: Cell viability (%) = OD570(sample)/OD570(control) × 100 where sample is the well treated with release medium containing DOX-encapsulated micelles and control represents the well treated with DMEM medium only. 7



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After the concentration of DOX released from the selected hydrogel replicate at each time point for cytotoxicity was determined, free DOX control was prepared by dissolving DOX·HCl in a 1:1 v/v mixture of PBS and DMEM medium of the same DOX concentration as that of the release medium for cytotoxicity.

3. Results and Discussions 3.1 Synthesis and Characterization of Alginate-g-PNIPAAm Three alginate-g-PNIPAAm copolymers were synthesized (Figure 1), and their molecular characteristics and chemical compositions are listed in Table 1. First, linear PNIPAAm with carboxylic acid terminal was obtained by atom transfer radical polymerization (ATRP) of NIPAAm at low temperature with BIBA as initiator and CuBr as catalyst.38 The linear PNIPAAm samples had a Mn ranging from 3500 to 5200, with a polydispersity index from 1.08 to 1.21 as measured by GPC. The ATRP method allowed for better control of the molecular weight as we as the molecular weight distribution of the linear PNIPAAm, as compared to previously reported free radical polymerization method.28 The 1H NMR spectrum of one PNIPAAm sample (PN48) is shown in Figure 2. The signal at 3.82 ppm is assigned to the proton of the isopropyl group linked to the amide group.27 The broad signals at 1.94 and 1.50 ppm are corresponded to the methine and methylene protons, respectively. The sharp signal at 1.07 ppm is from the six methyl protons of the isopropyl group.25 The absence of signals between 5.0 and 6.0 ppm indicates the absence of vinyl monomers in the purified sample.

Table 1. The molecular characteristics and chemical compositions of alginate-g-PNIPAAm copolymers. Mn of

PDI of

PNIPAAmb

PNIPAAmc

Alg-PN31-77%

3478

Alg-PN48-72% Alg-PN46-81%

Samplea

a

PNIPAAm content

DStheoreticald

DSNMRe

1.08

20

19.7

77

5388

1.13

10

9.5

72

5206

1.21

20

16.4

81

(wt%)

The copolymers are denoted Alg-PNx-y%, where Alg and PN represent alginate and PNIPAAm,

respectively, and x is the degree of polymerization and y% is the weight percentage of PNIPAAm in the copolymer. b

The number average molecular weight of PNIPAAm detected by GPC with THF as mobile phase.

c

The polydispersity index calculated based on Mw/Mn.

d

Theoretical DS value. “DS” was the degree of substitution, the number of conjugated PNIPAAm per

100 saccharide units of alginate. 8


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e

Detected DS value by 1H NMR.

Figure 2. 1H NMR spectra of alginate, PNIPAAm, and alginate-g-PNIPAAm in D2O. Amino group was introduced to the terminal of PNIPAAm-COOH via carbodiimide chemistry with excessive ethylenediamine (EDA). The extra EDA was removed by precipitation in diethyl ether and dialysis. The clearance of EDA was confirmed by thin layer chromatography with acetone as solvent. The terminal amino group was quantified by the TNBS assay.39 Conjugation between alginate and PNIPAAm-NH2 was confirmed by 1H NMR as shown in Figure 2. The physical properties of alginate are typically dependent on the M/G ratio.30 The peaks of alginate were observed at 4.8 ppm (G-1)29-30 and 3.4 - 4.3 ppm (three protons of the alginate ring, i.e., H2, H3, and H4).30, 40 With the successful grafting of PNIPAAm onto alginate, the signals of alginate at 3.4 - 4.3 ppm are overlapped with the characteristic signals of PNIPAAm at around 3.8 ppm.24 The compositions of the copolymers listed in Table 1 were calculated based on the 1H NMR spectra.

3.2 Micelle Formation of Alginate-g-PNIPAAm Alginate-g-PNIPAAm dissolved in PBS buffer at room temperature, and formed self-assembled micelles at 37 °C due to the increased hydrophobicity of PNIPAAm. The critical micellization temperature (CMT) and the critical micellization concentration (CMC) were determined by the hydrophobic dye DPH solubilization method. The absorption coefficient of DPH is higher in a hydrophobic environment than in water.33-34 Thus, it gives an increased absorption when DPH participates into the hydrophobic core of a micelle. The relative absorbance at 378-400 nm was used to determine the CMT and CMC of the copolymer. The point where the absorbance suddenly 9



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increases indicates the conditions at which micelles are formed. The CMT and CMC determination results for Alg-PN46-81% sample are shown in Figure 3 (results for other two Alginate-g-PNIPAAm samples shown in Figures S1 and S2). The CMTs for the alginate-g-PNIPAAm copolymers are in the range from 34 to 36 °C, lower than the body temperature. The CMC values are in the range from 0.36 to 0.56 mg/mL (Table 2). The morphology of the micelles formed by the copolymer measured by TEM is shown in Figure 3(c). The sizes of the micelles were also determined by DLS (Table 2). The micelles formed by the Alginate-g-PNIPAAm copolymers synthesized in this study are smaller than those reported in previous studies.30, 41 This may be due to the narrower molecular weight distribution of PNIPAAm synthesized by ATRP instead of free radical polymerization used previously. The relatively low CMC values and small sizes of the micelles formed by the alginate-g-PNIPAAm copolymers may be advantageous to more efficient passive accumulation of the micelles at the tumor site.42

Figure 3. (a) CMT determination by extrapolation of the difference in absorbance of DPH at 378 and 400 nm for polymer Alg-PN46-81% in PBS buffer at 2.0 mg/mL. (b) CMC determination by extrapolation of the difference in absorbance of DPH at 378 and 400 nm for polymer Alg-PN46-81% in PBS buffer at 37 °C. (c) TEM image of micelles formed by polymer Alg-PN46-81% at 1.0 mg/mL at 37 ºC.

Table 2. Particle sizes, and CMT and CMC values of alginate-g-PNIPAAm in PBS buffer. Sample

Z-Average diameter

CMT (°°C)b

CMC (mg/mL)c

(nm)a

Alg-PN31-77%

116.5 7.4

36.5

0.56

Alg-PN48-72%

124.5 1.6

35.2

0.50

Alg-PN46-81%

98.9 0.1

34.2

0.36

a

Measured by DLS for alginate-g-PNIPAAm at 1 mg/mL in PBS buffer at 37 °C.

b

CMT determined by DPH dye absorption method in PBS buffer at polymer concentration of 2.0

mg/mL. 10


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c

CMC determined by DPH dye absorption method in PBS buffer at 37 °C.

3.3 Hydrogel Formation and Rheological Properties The sol-gel transition of alginate-g-PNIPAAm was determined by a test tube inverting method.35 At room temperature (25 °C), the polymers formed solutions (at 7.4 wt%), which could flow easily in sample vial as shown in Figure 4. When temperature increased to 37 °C, the polymer solutions of Alg-PN48-72% and Alg-PN46-81% became hydrogels, which could not flow even when the sample vials were inverted, while the polymer solution of Alg-PN31-77% became a turbid sol (fluid). It is thought that the gelation is caused by the hydrophobic interaction between PNIPAAm chains at 37 °C, which plays a role of crosslinking the copolymers while alginate segments are still hydrophilic and can withhold water molecules. The PNIPAAm in the copolymer Alg-PN31-77% was not long enough to sustain a gel formation. Moreover, the compositions of PNIPAAm in the copolymer also affected the gel properties. Compared to the firm gel formed by Alg-PN46-81%, the gel formed by Alg-PN48-72% was softer. In addition, there was no hydrogel formation if the concentrations of the copolymers were too low.

Figure 4. Photographs of copolymer solutions at 7.4 wt% at room temperature (25 °C) and those after the temperature increased to 37 °C.

11



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Figure 5. The change of elastic modulus (G′) and viscous modulus (G″) for Alg-PN31-77% (a), AlgPN48-72% (b), and Alg-PN46-81% (c) measured at a constant frequency (1.0 Hz) and fixed stress (1.0 Pa) when the plate temperature was increased from 25 °C to 37 °C. The dotted line indicates the time point at which the plate temperature reached 37 °C. Polymer concentration was 7.4 wt% in DI water.

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The gel formation was also confirmed by rheology testing as shown in Figure 5. When the elastic modulus (G′) is larger than the viscous modulus (G″), a gel is formed. At room temperature, the solutions of alginate-g-PNIPAAm were not very viscous and the elastic modulus was smaller than the viscous modulus (G′ was too small to be detected by the equipment for Alg-PN31-77% at both 25 and 37 °C, no gel formation by this copolymer). However, when the plate temperature was quickly increased to the body temperature (37 °C), both the elastic modulus and viscous modulus began to rise and quickly crossed over for Alg-PN48-72% and Alg-PN46-81% (Figure 5, b and c), indicating the gel formation. The G′ value of the gel formed by Alg-PN46-81% was much higher than that of the gel formed by Alg-PN48-72%, implying the Alg-PN46-81% gel had much higher stiffness. The gelation time was short, which may be advantageous when the copolymers are used as an injectable hydrogel for drug delivery, where a quick gel formation can prevent the flow of the polymer when its solution is injected into the body. Moreover, the shear stress required to destroy the hydrogel network, defined as yield stress σy, is manifested by a G′/ G″ crossover from a solid-like into a liquid-like phase.35 As shown in Figure 6, the yield stress σy value was 117 and 300 Pa for the gels formed by Alg-PN48-72% and Alg-PN46-81%, respectively. The Alg-PN46-81% copolymer formed much stronger hydrogel.

Figure 6. Oscillatory stress sweep measurement of hydrogels formed by Alg-PN48-72% (a) and AlgPN46-81% (b) at 7.4 wt% in DI water. The measurement was carried out at 37 °C with a constant frequency (1.0 Hz).

3.4 In Vitro Release Study of Doxorubicin (DOX) from Hydrogels Doxorubicin (DOX), a hydrophobic anticancer drug, was loaded into the alginate-g-PNIPAAm hydrogel to investigate its sustained release from the hydrogel. Controlled and sustained releases of DOX up to 7 days for the hydrogel formed by Alg-PN48-72% and 20 days for the hydrogel formed by Alg-PN46-81% were achieved as shown in Figure 7. There was no significant burst release observed. The smaller release rate and longer release time for the hydrogel formed by Alg-PN46-81% were 13



consistent with the rheological properties tested above. The stiffer and stronger gel formed by AlgPN46-81% due to the higher content of PNIPAAm gave a longer and sustained release of drug.

100

Cumulative release (%)

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80

60

40

20 Alg-PN48-72% Alg-PN46-81%

0 0

100

200

300

400

500

Time (h)

Figure 7. Cumulative release of doxorubicin from alginate-g-PNIPAAm hydrogels at 37 °C.

In PBS buffer of pH 7.4, DOX·HCl would be deprotonated and the hydrophobic DOX would aggregate and precipitate in the solution. In the alginate-g-PNIPAAm hydrogel, the copolymer used can micellize in response to temperature change. As the hydrogel erodes, the dissolved polymer in PBS release medium can presumably micellize with PNIPAAm as the hydrophobic core and alginate as the hydrophilic shell, encapsulating the deprotonated DOX molecule at the same time. As a result, the DOX-encapsulated micelles can further act as drug carriers for effective drug delivery.

3.5 Characterization of Released Micelles from Hydrogel The drug was released as DOX-encapsulated micelles from the hydrogel into the PBS release medium. The morphology of the micelles in the release medium was observed by TEM as shown in Figure 8. Fine spherical shape was observed for the micelles. The size of the micelles was measured by DLS. Nearly all the sizes measured were below 200 nm (data in Figure S3). It is thought that majority of released DOX was encapsulated in the alginate-g-PNIPAAm micelles, although it was difficult to precisely measure the percentage of the encapsulated DOX in the micelles. DOX is hydrophobic at pH 7.4 in the PBS buffer release medium, and could only be stabilized by the micelles after being released from the hydrogels. Otherwise, precipitates of DOX could be observed in the release medium.

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Figure 8. Morphology of the micelles in the release medium observed by transmission electron microscopy for Alg-PN48-72% at 73 h (left) and Alg-PN46-81% at 109 h (right).

3.6 In Vitro Cellular Uptake of DOX-Encapsulated Micelles in Release Medium The cellular uptake of DOX-encapsulated micelles in the release medium was observed by confocal microscopy (Figure 9). For free DOX control, the cellular uptake in multidrug resistant AT3B-1 (MDR+) cells was minimal. In contrast, much higher uptake of DOX (red) was observed for cells treated with the release medium from the hydrogels. This may be because, unlike free drug molecules, DOX was encapsulated inside the micelles and delivered to the cells by the polymer carrier, evading the recognition and resistant mechanisms by the MDR+ cells. Therefore, the cellular uptake of DOX was enhanced.

3.7 In Vitro Cell Cytotoxicity of DOX-Encapsulated Micelles Released from Hydrogel In vitro cell cytotoxicity study was carried out for the DOX-encapsulated micelles released from the thermoresponsive hydrogel. The cytotoxicity of DOX-encapsulated micelles released from the hydrogel at various time points were tested in AT3B-1-N (MDR-) and AT3B-1 (MDR+) cell lines by directly mixing the release medium with cell culture medium in 96 well plates. The highest DOX concentration in the release medium was 17 µg/mL at 61 h for hydrogel formed by Alg-PN48-72%. After the addition of DMEM medium, the DOX concentration in the well was 8.5 µg/mL. The highest DOX concentration in the release medium was 11 µg/mL at 61 h for hydrogel formed by Alg-PN4681%. After the addition of DMEM medium, the DOX concentration in the well was 5.5 µg/mL. The concentration of free DOX in the 96 well as control experiment was 10 µg/mL, higher than the highest concentrations in the release medium for both hydrogels.

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Figure 9. Confocal microscope images showing cellular distribution of DOX (red) in AT3B-1 (MDR+) cells which were treated with the release medium taken at different time points (61 and 85 hours) at 37 ºC and incubated with cells for 24 hours. Control groups were treated with PBS and free DOX. Cell nuclei were stained with DAPI (blue). Colocalization of DOX and nuclei appeared purple in the merged images. Scale bar is 20 µm.

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The in vitro anti-cancer activities were evaluated using the MTT assays as shown in Figure 10. Free DOX had limited cytotoxicity in the non-resistant AT3B-1-N cells. Moreover, it had almost no cytotoxicity in AT3B-1 cells due to the cells’ multidrug resistance against free drug molecules as shown in Figure 10 and Figure S4. In contrast, the cytotoxicity of the release medium from both hydrogels was significantly higher than that of free DOX, even in the multidrug resistant AT3B-1 cells. These results strongly support that the alginate-g-PNIPAAm copolymers turned into micelles after erosion and dissolution from the hydrogels, encapsulating the released DOX molecule in the micelles. As a result, the micelles further served as a drug carrier for effective drug delivery. Moreover, the sustained release of DOX and the cytotoxicity effect of the release medium were both observed at various time points. Therefore, the alginate-g-PNIPAAm hydrogel system could release micelles continuously as the hydrogel eroded, and the DOX was delivered into cells effectively and continuously trough the micelles. In addition, the pure alginate-g-PNIPAAm copolymers were non-cytotoxic in both cells lines (Figure S5). Therefore, the alginate-g-PNIPAAm hydrogels have great potential as an injectable depot for controlled and sustained drug release, and subsequently as an effective micellar drug carrier for drug delivery, overcoming the multidrug resistance in cancer cells.

Figure 10. Cell viability assay with 100 µL of DMEM medium and 100 µL of release medium taken at different time points for Alg-PN48-72% (a) and Alg-PN46-81% (b) in AT3B-1-N (MDR-) and AT3B-1 (MDR+) cells. The free DOX control has a final concentration of 10 µg/mL. Each value represents the mean value ± SD (* P

0.05, ** P

0.01, *** P

0.001, n = 4).

4. Conclusions We have synthesized three alginate-g-PNIPAAm copolymers, Alg-PN31-77%, Alg-PN48-72%, and Alg-PN46-81%, by conjugating PNIPAAm to alginate, where PNIPAAm with different molecular 17



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weight and narrow molecular weight distribution was synthesized by ATRP. These copolymers dissolved in water or PBS buffer solution at room temperature, and formed self-assembled micelles with low CMCs when temperature increased to above their CMTs. At higher concentration, i.e., 7.4 wt% in water, all three copolymers formed solutions at 25 ºC, while Alg-PN48-72% and Alg-PN46-81% formed thermosensitive hydrogels when temperature increased to the body temperature (37 ºC). However, Alg-PN31-77% at 7.4 wt% could not form hydrogel at 37 ºC due to short PNIPAAm chains. Alg-PN46-81% formed stiffer gel than Alg-PN48-72% due to higher PNIPAAm content. DOX was loaded into Alg-PN48-72% and Alg-PN46-81% hydrogels, and was released from the gels in a slow and sustained manner. Interestingly, the DOX released from the hydrogels is encapsulated in the micelles formed by the copolymers, i.e., the hydrogels released DOX-encapsulated micelles in a sustained manner. Further, it was demonstrated that the slowly released DOX-loaded micelles enhanced the cellular uptake of DOX in multidrug resistance AT3B-1 cells, showing effect overcoming the drug resistance and achieving better efficiency for killing the cancer cells. In addition, the pure alginate-g-PNIPAAm copolymers were non-cytotoxic in the cells lines. Therefore, the injectable thermoresponsive hydrogels have great potential for sustained release and effective delivery of anti-cancer drugs, overcoming the multidrug resistance in cancer treatment.

■ ASSOCIATED CONTENT Supporting Information Additional data for critical micellization temperature (CMT) and critical micellization concentration (CMC), sizes of micelles, cell viability and cytotoxicity. This material is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Author *Tel.: +65 6516 7273. Fax: +65 6872 3069. E-mail: [email protected]. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS We acknowledge the financial support from Ministry of Education, Singapore (Grants Nos. R-397000-188-112 and R-397-000-267-114) and Agency for Science, Technology and Research (A*STAR), Singapore (Grant No. 132 148 0007).

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Table of Contents/Abstract Graphic

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Injectable Thermoresponsive Hydrogel Formed by Alginate-g-Poly(N

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