Lignin inspired PEG hydrogels for drug delivery

Accepted Manuscript Title: Lignin inspired PEG hydrogels for drug delivery Author: Gema Marcelo Mar L´opez-Gonz´alez Isabel Trabado M. Melia Rodrigo Mercedes Valiente Francisco Mendicuti PII: DOI: Reference:

S2352-4928(16)30032-0 http://dx.doi.org/doi:10.1016/j.mtcomm.2016.04.004 MTCOMM 97

To appear in: Received date: Accepted date:

5-4-2016 11-4-2016

Please cite this article as: Gema Marcelo, Mar L´opez-Gonz´alez, Isabel Trabado, M.Melia Rodrigo, Mercedes Valiente, Francisco Mendicuti, Lignin inspired PEG hydrogels for drug delivery, Materials Today Communications http://dx.doi.org/10.1016/j.mtcomm.2016.04.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Lignin inspired PEG hydrogels for drug delivery Gema Marcelo*a, Mar López-Gonzálezb, Isabel Trabadoc, M. Melia Rodrigoa, Mercedes Valientea and Francisco Mendicutia a

Departamento de Química Analítica, Química Física e Ingeniería Química.

Universidad de Alcalá, Alcalá de Henares, 28871, Madrid, Spain. E-mail: [email protected] b

Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), C/Juan de la Cierva 3,

28006-Madrid, Spain c

CAI Medicina Biología. Universidad de Alcalá, Alcalá de Henares, 28871, Madrid,

Spain.

1

Graphical Abstract

. AIBN ∆

CHAIN EXTENSION

. Phenoxy Radical Coupling

CHAIN CROSSLINKING

2

=

=

Highlights







A novel methodology for the preparation of robust PEG hydrogels which is inspired in the aryloxy radical coupling mechanism that leads to lignin biosynthesis is reported. These lignin-mimicking hydrogels are used as a platform for the preparation of a more advanced multifunctional material by incorporating magnetite nanoparticles and cyclodextrin macrorings. They show high potential as a controlled drug delivery system.

3

In the lignin biosynthesis aryloxy groups act like radical propagating species during a radical polymerization. We take advantage of this special chemistry to use catechol as the cross-linking species in a radical polymerization to form robust polyethylene glycol (PEG) hydrogels. Hydrogels are prepared by the radical co-polymerization of poly(ethylene glycol) methyl ether methacrylate and a methacrylamide catechol derivative monomer. The crosslinking results from the catechol groups, through the generation of aryloxy radicals that react with the C-C monomer double bonds. These lignin-mimicking hydrogels display a high swelling degree (near 2400 %), good mechanical properties in their swollen state (up to 390 Pa) and free catechol groups that can be used for further functionalization. These hydrogels can therefore be used as a platform for the preparation of a more advanced multifunctional material by incorporating magnetite nanoparticles and cyclodextrin macrorings (CD). The magnetic-CD- functionalized PEG hydrogel exhibits a high water-swelling capability, robust mechanical properties (G´1900 Pa in the swollen state), magnetic behavior, a great Doxorubicin loading capacity (ca. 0.6 mg Doxorubicin per gram of dried gel) and highly controlled temporal drug release. As a result, this hydrogel shows high potential as an advanced drug delivery system.

1. Introduction

4

Polymeric hydrogel materials are 3D networks which have the capacity to swell with a large amount of water. These materials play a very important role in many different areas such as sensing,1 catalysis,2 and particularly in biomedicine3 as drug delivery systems4,5 and cell culture platforms6 among others. Currently, much research is being done on new hydrogel formulations and gelation strategies6-9, which will make it possible for them to be designed to contain different complex features such as a specific functionality,6 sensitivity towards external stimuli,10 a similarity with the extracellular matrix,11 tunable mechanical properties,12 adhesion behavior,13 etc. With regard to the use of hydrogels as drug delivery systems, the presence of a porous structure in their polymeric network would allow drugs to be placed inside thus protecting them from the environment. However, one of the main limitations of the hydrogels is their limited capability for loading the drugs and controlling their subsequent release. To overcome this downside, hydrogels are designed in such a way that specific sites interact with the drug and smartly control its release.14,15 This can be done by either chemical bonds or electrostatic interactions, for instance, which respond to an external stimulus, establishing sites that permit the enzymatic degradation of the gel or by its modification with macrocycles, which act as hosts for the drugs, among others. Last but not least, the mechanical property of the hydrogel is a challenging aspect to take into account and may limit the application of the hydrogel. To prepare hydrogels with adequate mechanical behavior, the combination of the hydrogel with inorganic nanoparticles has been reported as a strategy that overcomes said limitation.12,15 Among the different polymeric matrixes, PEG hydrogels are important platforms in the development of systems for biomedical applications because of their properties such as good biocompatibility, nonimmunogenity, and resistance to protein adsorption.16 They

5

are mainly prepared by a polymerization reaction in the presence of a chemical crosslinker or by other conjugation strategies such as thiol-ene photoclick reactions, Michael-type additions, thiol-norbornene reactions, oxime chemistry, Diels–Alder cycloadditions, cyclooctyne–1,2-quinone cycloaddition and strain-promoted azidealkyne cycloadditions and catechol chemistry.17, 18 The last one is inspired by the adhesion chemistry of mussels to almost any kind of surfaces. A high content of catechol, such as the catecholic amino acid 3,4dihydroxyphenyl-L-alanine (dopa), is found in the mpf-1 protein they segregate. The mechanism of protein solidification and adhesion entails the interaction of dopa residues with transition metals, mainly iron. In this approach the catechol acts as a cross-linker through the formation of coordinative complexes with Fe3+, to form robust gels at pH  8.5. 18,19 This mimicking has been a main focus in the advance of polymeric gels with healing capabilities for tissue engineering.20 Additionally, this chemistry has inspired the discovery of dopamine self-polymerization in basic or oxidative media, 21 which has marked great progress in surface modification chemistry and has been the starting point for the development of new gel formulations. In contrast, the chemistry of a catechol group in a radical polymerization reaction is very limited due to its known behavior as a radical scavenger.

22,23

Nevertheless, the generation of aryloxy radicals and its coupling chemistry are essential in the biosynthesis of lignin,24,25 The mechanism involves an oxidative process initiated by an oxidases bounded cell wall (peroxidase and laccase) that generates free radicals, which leads to a polymerization reaction by the coupling of phenoxy radicals in the alcohol species with the double bonds of the different lignin alcohol building blocks. Moreover, the reaction between catechol groups and propagating radicals in the

6

polymerization reaction between dopamine methacrylamide and 2-methoxyethyl methacrylate leading to the chain crosslinking has also been reported recently. 26 In the present work we make use of the catechol ability of acting as radical propagating species during a radical polymerization, with a similar mechanism to that of lignin biosynthesis, to use catechol as cross-linker specie in a radical polymerization reaction for the preparation of robust polyethylene glycol (PEG) hydrogels aimed at drug delivery. This synthetic approach establishes that during the radical co-polymerization of poly(ethylene glycol) methyl ether methacrylate and a catechol derived methacrylamide monomer, the catechol groups, far from acting as radical scavengers, are the crosslinking species. Reaction occurs through the generation of aryloxy radicals in the catechol groups that couple with the monomer C-C double bonds, which in turn leads to the formation of the polymeric 3D network. See Scheme 1. Moreover, this mechanism permits free catechol groups to be introduced in the network for further modifications. Another important advantage of this approach is the widening of the network functionality by introducing other functional monomers during the radical polymerization, for instance a methacrylamide monomer bearing -cyclodextrins (CD). The CD functionalization of the hydrogel would therefore lead to the development of materials containing specific and controlled sites to interact with drugs.4 See Scheme 2. At a more advanced stage, we use the free catechol group affinity towards Fe3+, mimicking the mussel adhesion strategy to functionalize the hydrogel network with magnetite nanoparticles (MNPs). It makes it possible to combine MNPs and CDs in the PEG hydrogel, which render great potential to these advanced systems for drug delivery.

7

.

AIBN ∆

CHAIN EXTENSION

. Phenoxy Radical Coupling

GEL FORMATION

CHAIN CROSSLINKING

Scheme 1. Polymerization mechanism leading to catechol modified PEG hydrogels.

2. Experimental Section 2.1 Synthesis of N-(3,4-dihydroxyphenethyl) methacrylamide (DOMA). The synthesis and NMR characterization (Figure S1 of the Supporting Information) of a methacrylamide monomer functionalized with a catechol group were performed according to what was described elsewhere and included in the Supporting Information.27 2.2 Synthesis of -Cyclodextrinmethacrylamide (CDMA). The preparation and NMR characterization (Figure S2 of the Supporting Information) of a methacrylamide monomer bearing a pendant -Cyclodextrin (CDMA) were achieved according to what was reported. A brief description is in the Supporting Information.9 2.3 Synthesis of the hydrogels. Poly(ethylene glycol) methyl ether methacrylate (before polymerization, the inhibitor was removed by passing the monomer through a basic alumina column) and DOMA were dissolved in DMSO at different weight percentages, Table 1. The solutions were degassed by bubbling N2 directly into the solution for at least 30 min. AIBN was added 8

to the solutions and the temperature was increased to 84 ºC. The reaction was left to proceed overnight, after which the hydrogels were swollen and cleaned each day with a large amount of deionized water (1L) during ten days. The swelling degree was defined as: SD (%) = (Ws-Wd/Wd)  100, where Ws and Wd is the weight of the swollen and dry gel respectively.

Table 1. Experimental synthesis conditions for the different gel preparations: amount of monomers and solvent volume. Gel

PEGMA

DOMA

CDMA

DMSO

(g)/ mol %

mol %

mol %

(mL)

G1

5.14/ 97.2

2.8

_

10.3

G2

6.48/ 93.7

6.7

_

13.8

G3

4.80/ 89.1

10.9

_

10.4

GCD

4.9/ 92.0

5.8

2.20

10.3

2.4 Synthesis of hydrogel functionalized with CD moieties. Different amounts of poly(ethylene glycol) methyl ether methacrylate, DOMA and CDMA, collected in Table 1, were dissolved in DMSO. Polymerization was carried out according to what was described above. 2.5 Hydrogel functionalization with Fe3+. Incorporation of iron (III) in the hydrogel structure was performed by immersing a swollen portion of each hydrogel (ca. 0.5 g) in 25 mL of aqueous solution of FeCl3 (11 mg in 25 mL of water) during 8 hours. After that, the hydrogels were cleaned in deionized water and their rheological properties were studied. The medium pH was then adjusted to 10 by adding some drops of a concentrated NaOH solution in order to study the influence of the change of the complex structure in the mechanical properties of the 9

hydrogels. The hydrogels were left in this basic solution for two hours before studying their rheological behavior. 2.6 Functionalization of hydrogels with magnetic nanoparticles. The functionalization with magnetic particles (MNPs) was carried out in several steps as follows: (1) the hydrogel (5.5 g previously swollen in water) was immersed in 30 mL of water containing FeCl3 (302.4 mg) and FeCl2 (123.5 mg) for 12 hours; (2) the hydrogel was cleaned with deionized water and put in 100 mL of water; after that, an Ar stream was bubbled in water in order to remove oxygen and (3) 15 mL of NH4OH were added. The mixture was first heated at 60 ºC for 30 min and subsequently at 80 ºC for 1.5 hours. 2.7 Interaction of the hydrogels with Doxorubicin (Dox). A piece of dried gel, ca. 0.2 g, was immersed in a PBS solution containing Dox in a 210-5 M concentration for 48 hours. The amount of Dox loaded in the gel was determined by measuring the absorbance of the solution when the gel was completely swollen and removed from the solution, Dox(490 nm) =11,500 cm-1M-1.28 2.8 The kinetics of Dox release from the hydrogels. Approximately 0.15 g of each Dox loaded gel was placed at the bottom of a 10 mm path quartz cuvette containing 2 mL of PBS solution. The amount of released Dox was evaluated by measuring the fluorescence intensity at 590 nm (or concentrations, obtained by calibration using standard Dox buffer solutions) of the supernatant liquid at regular time steps. The intensities at infinite time (i.e., equilibria values) were obtained by the Kezdy Swinbourne method.

[29]

Results were reasonably adjusted to first order

kinetics in all cases. Rate constants at 37ºC were calculated. 2.9 Cell growth and Interaction with Dox loaded hydrogel.

10

NIH-3T3 cells were kindly provided by the cells culture unit of UAH. Cells were cultured with a DMEM medium containing 10% fetal bovine serum (FBS, Sigma Ref. F7524) and 10% antibiotic antymicotic solution (Sigma, Ref. A5955) at 37 °C in a 5% CO2, 95% air-humidified atmosphere. The culture media was changed every 2 days. After that, NIH-3T3 cells were seeded into μ-Dish 35mm, high glass bottom (Ibidi, Ref. 81158) at an initial cell density of 2×104 cells/dish with 2 mL of medium. After 48h, a piece of swollen hydrogel (0.215 g, containing 3.210-8 moles of Dox) was immersed in the cell medium. The intensity of fluorescence of the PBS solution was then tracked at 40 and 210 min.

3. Results and Discussion 3.1 Hydrogel synthesis and gel formation mechanism The classical radical co-polymerization reaction of a highly concentrated mixture of monomers, ca. 490 g/L, composed of a catechol derived methacrylamide monomer (DOMA) and polyethyleneglycolmethylether methacrylate (PEGMA), was carried out in the presence of AIBN in DMSO at 84ºC without any cross-linker. See Scheme 2 (top). Gel formation was observed after 6 hours of reaction. Three gels (named Gx, where x increases with the catechol content) were prepared with different amounts of DOMA in the range from 2.8 to 10.9 mol % (see experimental section). The possibility of the gel formation by hydrogen bonding interaction among catechol groups30 was excluded by thermal treatment of the gels with 5 M aqueous sodium thiocyanate solution, a hydrogen bond breaking agent.31 Upon stirring at 90 ºC during 3 h the gel was not dissolved. We hypothesize that the mechanism for the formation of this PEG network is closely related to that taking place in lignin biosynthesis, i.e. the generation of phenoxy radicals

11

in the catechol group and their coupling reaction with double C-C bonds of the monomers. From a chemical point of view this mechanism, see Scheme 1, is quite surprising as catechols are well-known to act as radical scavengers.22,23 However, a similar reactivity of catechol groups leading to the branching of a linear polymer has recently been reported, although the mechanism was not identified.26

AIBN DMSO/84 º C

PEGMA

DOMA content: G1: 2.8 mol % G2: 6.7 mol% G3: 10. 9 mol%

DOMA

AIBN DMSO/84 º C

DOMA content: 5.8 mol % CDMA content: 2.2 mol %

CDMA

Scheme 2. Monomers and hydrogel systems of this work.

Raman spectroscopy was used to shed light on the polymerization mechanism. The Raman spectra for PEGMA, DOMA and G3 are shown in Figures S5, S6 and S7 (Supporting Information).The proposed mechanism for the chain crosslinking would entail the modification of the aromatic ring with an alkoxy group. An alkoxy on an aromatic ring usually gives rise to two correlated Raman bands in the 1210-1310 cm-1 and 1010-1050 cm-1 ranges.32,33 The appearance of these bands in the gel Raman spectrum was therefore investigated. Figure 1 shows the Raman spectra for the PEGMA, DOMA and G3 in both spectrum regions. Firstly, the band in the 1050-1010 12

cm-1 range for the gel and the monomers was analyzed; the fitting parameters are given in Figures S8, S9 and S10 (Supporting Information). For PEGMA, the band was decomposed into four Gaussian components whose peaks (intensities) were 1005 cm-1 (12642), 1024 cm-1 (2929), 1032 cm-1 (3162) and 1044 cm-1 (14937). The intensity ratio (R) of the bands placed at 1044 and at 1032 cm-1 (1032/1044) was 0.2. The band for the catechol derived monomer (DOMA) was decomposed into three components, located at approximately 1014, 1044 and 1062 cm-1. The band at 1044 cm-1 was not representative because of its scarce contribution and the peak at 1062 cm-1 was not considered because it was not within the studied range. The band for G3 was decomposed into three components at 1029 cm-1 (16386), 1042 cm-1 (9889) and 1063 cm-1 (4149). It is remarkable that the ratio between the 1042 and 1029 cm-1 band (1029/1042) is rather different from the one for the PEGMA bands, since R was 1.6. The increase in the R value for the gel was attributed to the appearance of a new band near 1030 cm-1 in the spectrum. Thus, this underlying band could be ascribed to the presence of an alkoxy group on an aromatic ring. It would therefore reinforce our suggested mechanism for gel formation. R was determined for all gels (1-3) and it was found that it increases with the DOMA content (Figures S11 and S12, Supporting Information). Therefore, the higher the catechol content the greater the alkoxy group on an aromatic ring formation during polymerization. On the other hand, the Raman spectrum from 1210 to 1350 cm-1, in Figure 1B, could not be studied because of the great number of bands resulting from the deconvolution for both the monomers and the gel (Figures S13, S14 and S15, Supporting Information). Moreover, in this region the catechol contribution could not be neglected. It was therefore not possible to determine the appearance of a new band in this spectrum range with certainty.

13

Intensity (a.u.)

B

Intensity (a.u.)

A

1000 1020 1040 1060 1080 1100

1200

Wavelength(cm-1)

1250

1300

1350

Wavelength (cm-1)

Figure 1. Raman spectra for the G3 (), PEGMA () and DOMA () in the spectrum regions: A) 1000-1100 cm-1 and B) 1210-1350 cm-1.

The

lignin-bioinspired

polymerization

mechanism

constitutes

a

novel

methodology for the preparation of PEG gels, in which catechol groups play an important role as they not only allow for the crosslinking during polymerization but also permit functionalization of the network with free catechol moieties. See Scheme 1. It therefore opens an alternative route to achieving further network modification. The possibility of widening the functionalization of the gel network was demonstrated by introducing a methacrylamide monomer bearing a -cyclodextrin macroring (CD) in the polymerization reaction, see bottom of Scheme 2. The resulting hydrogel was named GCD. The CD mol content was roughly estimated to be 1.9 % by 1

H NMR (Figure S3, Supporting Information). 14

3.2 Hydrogel characterization All the gels were characterized in terms of their water swelling behavior, as well as their thermal and mechanical properties. All the gels presented great capacity to swell in water with a swelling degree (SD) value that slightly increased with the catechol % molar content from approximately 1700 to 2400%, SD values are collected in Table 2. It could be explained considering that the higher the catechol content the greater the alkoxy group on an aromatic formation during polymerization. So the degree of branching is greater, but it does not imply a higher cross-linking degree. The GCD presented a swelling degree (near 1800%) rather similar to that of hydrogel that did not contain CD.

Table 2. Values of: DOMA mol %, swelling degree (SD), thermal degradation temperature (Td), storage modulus (G´) and magnetite weight percentage.

Gel

DOMA mol %

SD (%)

Td (ºC)

G´ (Pa)

G1

2.8

1728

344

384

G2

6.7

2072

347

356

G3

10.9

2368

397

244

GCD

5.8

1816

392

808

G1Mag

2.8

397

1020

G2Mag

6.7

G3Mag

10.9

GCDMag

5.8

MNP content (wt. %)

3.2 2.4

870

15

407

610

1.7

404

1877

4.9

The thermal stability of the gel was evaluated by thermal gravimetric analysis (TGA). The weight loss and the derived weight change for 1-3 gels with temperature are shown in Figure S4 (Supporting Information). The values of the degradation temperature (Td) are collected in Table 1. It is observed a greater thermal stability when the catechol content is increased. The Td value for the gel with the lower catechol content (G1) is 344 ºC whereas for the gel with the highest catechol content (G3) it is near 400 ºC. According to Raman spectroscopy study, with the increase of catechol content there are fewer free pendant catechol groups that are more feasible to undergo a thermal degradation process. Therefore, the thermal stability would be enhanced. TGA for GCD, shown in S4 (Supporting Information), indicates that the presence of CD shifts degradation to higher temperatures, from 347 ºC for G2 to 392 ºC for GCD. It could be explained taking into account that the degradation of the CD molecule stars at a temperature (ca. 330 ºC)34 near the Td of G2. Therefore, the incorporation of CD in the gel would improve the thermal stability. The mechanical properties of the gels in the swollen state were characterized by oscillatory rheology experiments in order to determine the variation of storage (G´) and loss (G´´) moduli as a function of frequency for the different hydrogels at 20ºC. Results are shown in Figure 2. All gels presented an elastic behavior with the absence of changes in the G´ with the frequency and a G’/G’’ ratio higher than 10. It was greater than the loss modulus over the entire range of frequencies, which is consistent with the mechanical behavior of a cross-linked network. In addition, the catechol mole content seems to have a small influence on their final mechanical properties. The values of G´ were also collected in Table 1. Gels G1-3 presented a G´ in the 244-384 Pa range, which indicates that they presented good mechanical properties and could be suitable for 16

biomedical applications, because of their similarity with the elastic modulus of soft mammalian tissues. Tissue moduli ranged from 100 Pa for the softest ones such as the brain, to around 100 kPa for cartilages.35 Modification of the gel structure with CD (GCD) led to a strong impact on the elastic behavior. The G´ value was increased up to 808 Pa, which was more than twice the value for G2. The steric effect of CD has been reported to play a role in polymer network flexibility because of CD rigidity and big volume which reduces the expansion of network.36 We think that this effect could be present in our gels, which would explain the increase in the G´ value.

1000

900

A

B

800

GCD

G´(Pa)

G´, G´´ (Pa)

700

100

600 500 400 300

10

-3

10

-2

10

-1

10

0

10

200

1

10

2

4

6

8

10

12

catechol mole percent (%)

frequency (Hz)

Figure 2. A) G´ (filled symbols) and G´´ (open symbols) moduli dependency with the frequency for G1 (square), Gel2 (circle), G3 (triangle) and GCD (rhombus). B) G´ modulus versus the catechol mole percentage used in the gel preparations.

3.3 Hydrogel interaction with ferric ions. As mentioned above, the functionalization of the hydrogel network with free catechol groups considerably expands the versatility of these materials. Catechol moieties form stable complexes with Fe3+ whose stoichiometry depends strongly on the medium pH.18 As we expected, our gels were able to incorporate a large amount of Fe3+ 17

inside the network. G1 was chosen to prove this concept. TGA for G1 after exposure to Fe3+ (G1Fe), shown in Figure S4 (Supporting information), reveals a 4.2 wt. % of Fe3+. The influence of the catechol:Fe3+ complexation chemistry on the properties of the hydrogel was also rationalized through rheological experiments, see Figure S16 (Supporting information). The G´ modulus increased from 346 up to 548 Pa when G1 was loaded with Fe3+ at a neutral pH. Great reinforcement of the mechanical properties of the network was, however, observed after interaction with Fe3+ at pH 9. The G´ value was 724 Pa as a consequence of the Fe3+ complex formation at this pH with two or three catechol groups of the network. Next, because of the great affinity of the gels towards Fe3+, hydrogels could be used as a reactor for the preparation of magnetic iron oxide nanoparticles inside the network.37 Thus, the immersion of the gels in a water solution containing Fe2+ and Fe3+ in molar ratio 1:2, the subsequent addition of NH4OH and temperature treatment in the Ar atmosphere led to the formation of magnetic nanoparticles (MNPs) in the network.38 Gels with the most extreme compositions in catechol, G1 and G3, as well as GCD, were selected for the modification with MNPs. The resulting magnetic gels were named G1Mag, G3Mag and GCDMag. The XRD study corroborated the presence of magnetic nanoparticles. The XRD difractrogram for the GCDMag as a representative example is shown in Figure S17 (Supporting Information). All of the diffraction peaks were consistent with the database in JCPDS file No. 19-629 and could be indexed according to the inverse spinel structure of magnetite. The weight percentage of MNPs in the hydrogels was determined by TGA and the values are collected in Table 1. Values ranged from 1.7 to 4.9 % but we could not correlate them with the catechol content. The presence of MNPs in the structure

18

enhanced the thermal stability of the gel. Figure S4 (Supporting Information) depicts the TGA of G1 and G1Mag as an example. The modification of G1 with MNPs increased the degradation temperature (Td) by around 50 ºC. The Td values for the studied magnetic hydrogels are summarized in Table 1. The incorporation of inorganic materials in a polymer network has been reported as an important strategy to reinforce the mechanical properties of polymeric materials.12 This effect is significantly observed upon incorporation of MNPs in our gel structures. For instance, in the case of G1, the G´ increased from 384 up to 1020 Pa when a 3.2 wt. % of MNPs was introduced in the network, see Figure S16 (Supporting Information). Table 1 collects the values of G´ for G1-G3 and GCD after their modification with magnetic nanoparticles. It is also remarkable that the G’ for the gel containing CD increased from approximately 800 Pa to nearly 1900 Pa upon magnetite incorporation. Apart from tuning up the mechanical properties of the hydrogel, the presence of MNPs endows another new feature to the hydrogel, magnetic behavior. The magnetic behavior of GCDMag was determined at 25ºC, it is shown in Figure S17 (Supporting Information). The magnetization curve showed that GCDMag has a superparamagnetic behavior at room temperature. The saturation magnetization value was 0.3 emu/g. This is an important result which opens the door to the study of the heating ability of GCDMag under an external oscillating magnetic field to be used in the magnetic hyperthermia treatment.

3.4 Study of hydrogels for Dox drug delivery Hydrogels are widely being studied as platforms for drug delivery.4 However, the main limitation of these systems comes from their scarce interaction with the drug. If there are no specific interactions between the gel and the drug, the drug loading is too poor to

19

achieve therapeutic levels. Even with a high loading of the drug, the release is too fast and it cannot be controlled. It has been reported that the presence of CDs is key to controlling the release of the drug.41,42 They form inclusion complexes mainly with hydrophobic drugs. The modification of the hydrogel with CDs creates specific sites to interact with the drug, forming the well-known inclusion complexes, which permit a great drug load to be incorporated. These complexes are a balance between the free drug and the complexed drug. The release of the drug from the hydrogel that first the balance shifts towards the free drug form and that the drug is subsequently diffused out of the hydrogel.5 The release of the drug is therefore more controlled. In this section the interaction of the gels with Doxorubicin (Dox) was studied. Dox was selected because it is a chemotherapeutic drug used in the treatment of a variety of tumors. In addition, it forms stable complexes with CDs, particularly with the one containing seven glucopyranose units (CD).43,44 Dox spectroscopy properties (absorption and fluorescence) also allow interaction with the CDs and therefore with the hydrogels to be tracked. G2, GCD, G2Mag and GCDMag were selected for the study of the interaction with Dox. We chose G2 and GCD to evaluate the influence of the CD presence in the drug loading and release. G2 was selected as the blank because it was the most similar in catechol composition to GCD. G2Mag and GCDMag were chosen to study the influence of MNPs in the carrier ability. The loading of Dox in the different gels was carried out by making them swell in a high concentrated solution of Dox in an aqueous phosphate buffered saline solution (PBS) at pH 7.4. The amount in mg of Dox per gram of dried hydrogel was collected in Table 3. In general, the presence of CDs considerably increased the Dox loading capacity of the gels. From the values of 0.063 and 0.35 mg/g for G2 and G2Mag gels in the absence of

20

CDs to loading capacities of 0.17 and 0.57 mg/g for GCD and GCDMag were reached. Apart from the great influence of CDs, another outstanding factor is that the presence of magnetite nanoparticles also increased the amount of Dox loaded in the hydrogels. We think that Dox interacts with the magnetite surface through a non-covalent interaction. Dox is a molecule with possible functional groups (amine and hydroxyl groups) to interact with the magnetite surface.45 Remarkably, a synergy was found when the gel contained both CD macrocycles and magnetite nanoparticles (GCDMag) which led to the highest Dox loading capacity per gram of dried hydrogel. The releasing of Dox from the four hydrogels at 37ºC was studied in a phosphate buffered saline (PBS) medium by tracking the increase in the fluorescence intensity (at 590 nm) with time at the previously described experimental conditions (see Experimental section). The data were fitted to a first order rate kinetics model46 (Figure S18, Supporting Information). Figure 3 also shows the percentage of Dox released with time for the different systems. The values of rate constants (k) and the percentage of drug release at the equilibrium are collected in Table 2. By comparing the systems with and without magnetite, we can note that the values of the rate constants (k) decrease to half when there is CD in the gel structure, from 20.110-3 min-1 for G2 to 8.910-3 min-1 for GCD. Hence, a great degree of control is achieved in the release of the drug in the presence of CD. On the other hand, the modification of the hydrogel with MNPs led to an even slower rate kinetics of Dox release. For instance, the k value for GCD was 8.910-3 min-1 whereas for GCDMag it was 4.010-3 min-1. Therefore, GCDMag presented the slowest kinetics for drug release together with the highest drug loading capacity. Finally, the time for the complete release of Dox was estimated from the initial rate, v 0. The values are collected in last column of Table 3. This value could be interpreted as

21

the time to achieve, with a constant fluid exchange, the complete release of Dox from the hydrogel. The longest time was near 2 months for releasing the total drug charged per gram of GCDMag hydrogel.

Dox released (%)

60 50 40 30 20 10 0 0

50

100

150

200

250

300

time (min) Figure 3. Percentage of Dox released from the different types of gels immersed in a 2 mL PBS buffer solution at 37ºC. Symbols are: () G2; () GCD; () G2Mag and () GCDMag. Dashed lines are a guide for the eye.

Table 3. Values of the total amount of Dox loaded per gram of dried gel, first order kinetics constant (k), the percentage of Dox released at the equilibrium at 37 ºC (%), initial releasing rate (v0) and time for the total releasing of the charged Dox (per gram of dried gel). Dox /gel -1

Gel

(mg g )

103k (min-1)

%

109 ×v0

released

-1

total releasing -1

(molL min )

time (days)

G2

0.063

20.1  1.8

52.7

1.6  0.2

25  3

GCD

0.174

8.9  0.6

44.2

2.4  0.3

47  5

22

G2Mag

0.349

9.0  0.3

38.1

9.7  0.3

23  1

GCDMag

0.573

4.0  0.1

19.4

6.3  0.6

58  5

The release of Dox from the GCDMag hydrogel was studied in a fibroblast medium which simulates the conditions studied in the kinetics experiments. NIH 3T3 murine fibroblasts were cultured in 2 mL of a DMEM medium at 37 ºC. A portion of a GCDMag:Dox hydrogel containing 3.210-8 moles of Dox was placed in the fibroblast medium. The study of the Dox release and subsequent Dox cell internalization was monitored by confocal fluorescence microscopy due to the fluorescence of Dox (exc = 514 nm) at two times, 40 and 210 min. Fibroblasts were also incubated with only Dox as a blank. The Dox number of moles was 110-8. This amount represents 31% of the Dox moles loaded in the hydrogel. Figure 4 shows the images obtained with the confocal laser scanning microscope at the selected times. In agreement with the kinetics study, the Dox is slowly liberated from the hydrogel and captured by the cells. The intensity of Dox fluorescence inside the cells increases with time. At 40 min Dox fluorescence inside the cells is very scarce, but at 210 min Dox fluorescence intensity inside the cells is quite notable. However, it can be seen that after an exposure of 210 min with the hydrogel, the Dox fluorescence intensity in the cells is less strong than the Dox intensity presented by the cells incubated only with Dox. According to the kinetics study, after 210 min only 11 % of Dox is freed, which would correspond to 0.35 10-8 moles of Dox.

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40 min Gel:Dox

210 min Gel:Dox

210 min Dox

Figure 4. First column: bright field images, second column: fluorescence intensity images upon excitation at 514 nm and the resulting merged images in the last column for the NIH 3T3 cell after interacting with the GCDMag:Dox hydrogel for 40 (first row) and 210 min (second row) and also with only Dox for 210 min (third row).

It has been demonstrated in vitro that the GCDMag hydrogel can be considered an excellent platform for the loading of a great amount of therapeutic drugs and to control the release of Dox, avoiding the extremely fast drug diffusion process during drug releasing that occurred in most hydrogels without specific sites for drug interaction. This was also corroborated by experiments in vitro. 24

4. Conclusions The aryloxy radical coupling process that takes place in lignin biosynthesis was mimicked to describe a novel methodology for the preparation of robust polyethylene glycol (PEG) hydrogels. The hydrogels were prepared by the radical polymerization of poly(ethylene glycol) methyl ether methacrylate and a catechol derived methacrylamide monomer. The catechol groups seem to be the species responsible for the crosslinking process, through the generation of aryloxy radicals that react with the monomer C-C double bonds. These hydrogels were characterized for a high swelling degree (17002400%) and good mechanical properties (G´ ranging from 244 to 384 Pa and they were used as a platform for the preparation of a more advanced multifunctional material aimed at drug delivery.

The hydrogel functionalization with CDs and magnetic

nanoparticles led to a hydrogel with great mechanical properties, G´ of 1877 Pa (swollen state), magnetic behavior, high loading capacity for Doxorubicin (ca. 0.6mg of Dox per gram of dried gel) and good temporal control for its release. These results were corroborated in vitro in a fibroblast cell medium.

Acknowledgements M. López-González would like to thank MINECO (Spain) for financial support (MAT2014-57429-R). We wish to express our thanks to M.L. Heijnen for assistance with the preparation of the manuscript.

Supporting Information

25

Supplementary information includes synthesis conditions, NMR characterizations, spectral data, rheological characterization and kinetic study of Dox release.

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