Long-term doxorubicin release from multiple stimuli-responsive hydrogels based on α-amino-acid residues

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

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European Journal of Pharmaceutics and Biopharmaceutics

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Research paper

Long-term doxorubicin release from multiple stimuli-responsivehydrogels based on a-amino-acid residues

http://dx.doi.org/10.1016/j.ejpb.2014.06.0050939-6411/� 2014 Elsevier B.V. All rights reserved.

Abbreviations: NPs, nanoparticles; DOXO, doxorubicin; AMF, alternating mag-netic field; c.c.c., cell copper condenser; c.c.h., copper coil honeycomb; FT-IR,Fourier transform infrared; HVa, N-acryloyl-L-valine; PHE, N-acryloyl-L-phenylala-nine; NIP, N-isopropylacrylamide; pNIP, poly(N-isopropylacrylamide); APS, ammo-nium peroxodisulfate; TEA, triethylamine; EBA, N,N0-ethylene-bis-acrylamide;HVa2, hydrogel poly(N-acryloyl-L-valine) cross-linked with 2 mol% EBA; Phe-NIP1,hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) with 0.2 mLof magnetic NPs; Phe-NIP2, hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopro-pylacrylamide) with 0.6 mL of magnetic NPs; Phe-NIP3, hydrogel poly(N-acryloyl-L-phenylalanine-co-N-isopropylacrylamide) with 2.0 mL of magnetic NPs; DS, degreeof swelling; EDS, equilibrium degree of swelling; PARP, poly(ADP-ribose) polymer-ase; LCST, lower critical solution temperature.⇑ Corresponding author. Department of Biotechnology, Chemistry and Pharmacy,

University of Siena, I-53100 Siena, Italy. Tel.: +39 0577 234388.E-mail address: [email protected] (M. Casolaro).

Please cite this article in press as: M. Casolaro et al., Long-term doxorubicin release from multiple stimuli-responsive hydrogels based on a-amiresidues, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.005

Mario Casolaro a,⇑, Ilaria Casolaro b, Severino Bottari c, Barbara Del Bello d,e, Emilia Maellaro d,e,Konstantinos D. Demadis f

a Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italyb Graduate Student in Psychiatry at the University of Siena, Italyc Department of Physical Sciences, Earth and Environmental, University of Siena, Siena, Italyd Istituto Toscano Tumori (ITT), Firenze, Italye Department of Molecular and Developmental Medicine, University of Siena, Siena, Italyf Department of Chemistry, University of Crete, Heraklion, Greece

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 March 2014Accepted in revised form 4 June 2014Available online xxxx

Keywords:Stimuli-responsive hydrogelsMagnetic nanoparticlesDoxorubicin releaseAlternating magnetic fieldBasicity constantsHeLa cells

We have developed a series of pH- and temperature-stimuli-sensitive vinyl hydrogels, bearing a-aminoacid residues (L-phenylalanine, L-valine) and incorporating magnetic nanoparticles of different chemicalcompositions (CoFe2O4 and Fe3O4). The goal was to study the potential applications of these nanocom-posites in the controlled release of doxorubicin (DOXO), a potent anticancer drug. The strength of theelectrostatic interaction between the protonated nitrogen of the DOXO molecule and the ionized carbox-ylic groups of the hydrogel allowed effective control of the drug release rate in saline solutions. Theembedded magnetic nanoparticles were an additional remote control of the drug release under the stim-ulus of an appropriate external alternating magnetic field (AMF). Data showed that the controlled releaseof DOXO proceeded for months and followed a diffusion-controlled release mechanism, while maintain-ing the amount of released drug within acceptable therapeutic windows. The amount of the releasedDOXO was found in all cases substantially higher than the ‘‘control’’ because the application of theAMF augments in stimulating the nanoparticles within the DOXO-loaded hydrogel. In vitro experimentshave shown that the released DOXO is able to induce cell death to cervix adenocarcinoma cells (HeLacells).

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Chemotherapy is a major conventional approach for the treat-ment of a wide range of cancers. Unfortunately, although this

approach may effectively combat cancerous cells, it also leads toseveral collateral side effects on healthy tissues [1]. To mitigatethis problematic issue, intense research efforts have focused onthe design and implementation of carrier systems for targeteddelivery of the therapeutic agent [2,3]. In recent years, ‘‘smartmaterials’’ have found new and promising applications as drug car-riers for delivery of new therapeutic agents [4]. In the field of smartpolymers, hydrogels have been extensively investigated as drugdelivery carriers in biomedical applications, because they can beenvisioned as models of human soft tissues [5].

Hydrogels are cross-linked polymer networks that easily absorblarge amounts of water. Water absorption is accompanied by gelswelling. The amount of absorbed water is strongly dependenton the cross-linking degree and on the hydrophilic character ofthe polymeric structure. Polyelectrolytes bearing ionizable groupsusually enhance the swelling properties giving rise to stimuli-responsive hydrogels of multiphase mixtures including fixed

no-acid

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charges, mobile ionic species and interstitial fluid phases [6]. Thus,the polymeric network improves the ability to control the incorpo-rated drug loading, while maintaining the drug bioactivity intact.

Recently, we reported a promising approach to complex/coordi-nate some commonly used metal-based chemotherapeutics (plati-num and ruthenium coordination compounds) in multiple-stimuliresponsive hydrogels [7–10]. These systems are of great interest, asevidenced by their use in clinical trials to fight tumor cells, princi-pally because of two reasons: (a) the drug efficacy is improved, and(b) the toxic side effects of these drugs are reduced. According tothis strategy the metal center was stoichiometrically complexedby fixed charges present on a-aminoacid residues (L-phenylala-nine, L-valine, L-histidine) and was by design inserted into cross-linked polymeric networks [7,8,11].

In some cases, a chemically-controlled release, coupled withthe diffusion-controlled mechanism, improves the pseudo zero-order release kinetics [8]. It is well established that the metal-polyelectrolyte complex principally depends, among other factors,on the strength of the coordinating groups. A strong coordinatinggroup leads to formation of a more stable complex, as can be evi-denced by the numerical stability constant value [12]. Likewise,metal-based drugs, or other ‘‘uncomplexed’’ drug molecules, bear-ing fixed charges may interact with an ionized group of thehydrogel, thus providing a suitable electrostatic coupling, withthe goal of achieving controlled release of the bioactive species.This is a general phenomenon and we recently reported therelease of pilocarpine from vinyl hydrogels based on L-valine res-idues [13]. The latter was found to be a suitable polyelectrolytehydrogel that provides pH- and temperature-responsiveness,due to its structural similarity to poly(N-isopropylacrylamide)(pNIP), the latter being a well-known temperature-responsivepolymer [14].

Our goal in this paper was to achieve controlled release of anti-cancer drugs at the desired therapeutic levels and to maintainthese for extended time periods, while reducing systemic sideeffects. Specifically, we report doxorubicin (DOXO) release fromnanocomposite hydrogels based on L-phenylalanine and L-valineresidues (see Scheme 1). Various doxorubicin carrier systems havebeen reported in the literature [15–22].

Although DOXO is one of the most potent and commonly usedanticancer drugs, it shows some undesirable side effects on healthytissue. Doxorubicin hydrochloride (adriamycin) is a valuable drugin the treatment of a variety of solid and hematologic malignancies[23]. Its usefulness is hampered, however, by its cardiotoxicity, anadverse effect that can preclude its use in certain patients and,thus, limit the therapy duration in many others [24]. DOXO is apositively charged molecule having a pKa of �8 [24,25]. TheDOXO-hydrogel coupling process can be envisioned to occur viaelectrostatic interactions with the ionized carboxyl group(pKa = 5) of the hydrogel [18,26]. Some observations support thisclaim. Addition of a dilute DOXO hydrochloride solution to a swol-len hydrogel led to a dramatic collapse of the network structure,due to charge neutralization. The 1:1 stoichiometric ratio betweenthe DOXO molecule (containing a primary amine, –NH2) and the

H2C CH

HNCHCH

Scheme 1. Structure of the doxorubicin and of the monomers N-acrylo

Please cite this article in press as: M. Casolaro et al., Long-term doxorubicin reresidues, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014

hydrogel (containing ionized –COO� groups) may conceptuallylead to preparation of hydrogels loaded with the proper amountof DOXO, which can in turn be released at the desired therapeuticlevels for prolonged time periods.

Release of DOXO can occur ‘‘on-demand’’ if an extra stimulus ispresent in the hydrogel. For example, magnetic nanoparticles (NPs)embedded in the hydrogel matrix allow an additional remote-control mechanism to trigger the drug release by a non-contactforce, which is superior to traditional stimuli such as pH or temper-ature [27]. Hence, the magnetic carriers loaded together withDOXO can be guided to the site-specific target and then providea sustained drug release under the application of an externalalternating magnetic field (AMF). Another important point is thatthe application of external AMF may increase the temperature ofthe magnetic NPs and their local microenvironment, which couldbe beneficial for creating local hyperthermia suitable for cancertherapy [28].

The results reported in this work show that the sustainedrelease of DOXO from hydrogels is ensured for several months,while preserving the drug’s bioactivity. The amount of the releaseddrug can be controlled by fine-tuning the DOXO-hydrogel acid-base chemistry, and by external ‘‘triggers’’ such as temperatureand/or strength of the external applied magnetic field. This allowsthe design of viable injectable nanocomposite systems with greatpotential for the treatment of cancer. Lastly, we have carried outin vitro experiments on cervix adenocarcinoma cells (HeLa cells).These have demonstrated that the DOXO released from DOXO-loaded hydrogels induces cell death at a rate that is proportionalto the amount of the hydrogel utilized.

2. Experimental section

2.1. Instrumentation and materials

FT-IR spectra were recorded on a Thermo-Electron Nicolet 6700FTIR optical spectrometer. A TitraLab 90 titration system (Radiom-eter Analytical) was used to perform potentiometric titrations at25 �C. Alternating magnetic field (AMF) measurements wereobtained by two home-built AMF apparatus. The first wasequipped with two solenoids connected to two ferrite bars at thecenter of which was placed the 1 cm quartz cuvette containingthe hydrogel sample. The 40 kHz RF generator provides a magneticfield of 1.1 mT within the two solenoids facing each other [29]. Inthe second AMF apparatus, the model AG 1006 amplifier/generator(T&C Power Conversion, Inc.) provides the required control andmonitoring functions (operation frequency: 0.02–14 MHz). Twocurved plates of copper (6 cm height and 5.5 cm internal diameter)were connected to the generator to perform drug release measure-ments in a 50 mL container (see Panel A of Fig. 1, left side). The AG1006 generator applies an electric field of 50 V at 20 kHz to the cellcopper condenser (c.c.c.) and to the solenoid winding with honey-comb (copper coil honeycomb, c.c.h.: 4 cm height and 6 cm

O

2

COOHH2C CH

HNCH

H3C CH3

O

COOH

yl-L-phenylalanine and N-acryloyl-L-valine used for the hydrogels.

lease from multiple stimuli-responsive hydrogels based on a-amino-acid.06.005


Fig. 1. Panel A: General view of the AG 1006 RF generator with the cell copper condenser (c.c.c., left), and the copper coil honeycomb (c.c.h., right). Panel B: Principle diagramsof the home-built AMF apparatus: solenoids (a); copper coil honeycomb, c.c.h. (b); cell copper condenser, c.c.c. (c), with the magnetic field and electric field. In parallel to thegenerator (G) is connected a resistor 50 ohm load as adapter. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)

M. Casolaro et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx 3

internal diameter (see Panel A of Fig. 1, right side). The three prin-ciple diagrams are presented in the Panel B of Fig. 1.

UV-visible measurements were carried out with a Specord 210spectrophotometer (Analytikjena) equipped with 10 mm quartzcuvettes. The two monomers, N-acryloyl-L-valine (HVa) andN-acryloyl-L-phenylalanine (PHE), were synthesized as previouslyreported [30,31]. The monomer N-isopropylacrylamide (NIP), aswell as the doxorubicin hydrochloride (adriamicyn) and some ofthe chemicals and solvents used in this research were purchasedfrom Sigma-Aldrich. Ammonium peroxodisulfate (APS, 98%), tri-ethylamine (TEA, 99.5%), and N,N0-ethylene-bis-acrylamide (EBA,98%) were purchased from Fluka Co. A commercial aqueousdispersion of cobalt ferrite (CoFe2O4) magnetic NPs was providedby Colorobbia, Italy [32]. Iron oxide NPs (magnetite, Fe3O4) wereembedded into the hydrogel by co-precipitation of iron ions, sup-plied either by ammonium iron(II) sulfate hexahydrate or iron(III)chloride hexahydrate salts, inside the swollen polymeric networkin ammonia solution [33,34].

2.2. Synthesis of hydrogels

The homopolymeric hydrogel HVa2 containing the L-valine res-idues and cross-linked with 2 mol% of EBA was obtained as reportedin a previous paper [8]. Magnetic NPs were embedded following adescribed procedure to prepare Fe3O4 from Fe(II) and Fe(III) saltsin the presence of NH3 [33,34]. Briefly, a dry sample (83.4 mg) ofHVa2 was swollen in slightly alkaline (with NaOH) water. The swol-len hydrogel was washed with water and then immersed inan aqueous solution (50 mL) containing dissolved salts of Fe(III)(FeCl3�6H2O, 1.36 g, 5.03 mmol) and Fe(II) [(NH4)2Fe(SO4)2�6H2O,1.00 g, 2.55 mmol] in a 2:1 molar ratio. The hydrogel color turnedto brownish and was left under a stream of nitrogen for about 4 huntil it collapsed. It was then placed in a solution of 0.5 M NH3

(50 mL), turning darker in color. It was allowed to react for �3 h,after which the product was filtered. After repeated washings itwas left to air-dry. Yield: 100.0 mg. Three copolymeric hydrogelsincorporating N-acryloyl-L-phenylalanine and N-isopropylacrylam-ide, with a PHE/NIP molar ratio of 1:1, were cross-linked with2 mol% of EBA. During the polymerization procedure differentamounts of magnetic CoFe2O4 NPs dispersion were used for theirembedding into the hydrogels. Table 1 summarizes the amountsof reagents used for the synthesis of hydrogels.

All reagents were dissolved in doubly-distilled water in a glasstube reactor under nitrogen atmosphere, except the monomer PHE,which was dissolved in a slightly alkaline solution. To each tubethe same measured amount of TEA was added, together withdifferent measured amounts of magnetic NPs as an aqueous dis-persion supplied by Colorobbia [32]. Each reaction mixture wasdegassed under vacuum for 30 min and flushed with nitrogen.


Subsequently, a freshly prepared and degassed aqueous solutionof APS was added to each reaction tube. Although gelationoccurred after a few minutes, the reaction mixture was allowedto proceed at ambient conditions for 24 h. Afterward, the gelsnamed Phe-NIP1, Phe-NIP2, and Phe-NIP3 (see Table 1) wereslightly acidified with a 2 M HCl solution (pH > 3) and gentlyremoved from the mother liquor. They were washed daily withdoubly-distilled water (400 mL each washing) for 1 week, thencut in small discs (�1 cm diameter), and dried at room tempera-ture to a constant weight.

2.3. Characterization of hydrogels

Potentiometric titrations were carried out at 25 �C in aqueoussolutions containing the three Phe-NIP1, Phe-NIP2, andPhe-NIP3 hydrogels by using a TitraLab 90 titration system, as pre-viously described [35]. Titrations of the hydrogels were performedin a thermostated glass cell filled with 50 mL of a NaCl solution(0.15 M or 0.01 M) in which a weighed and finely crushed solidmaterial (20–34 mg) was dispersed under stirring, together witha known excess amount of standard sodium hydroxide solution,under a presaturated N2 stream. When the hydrogel was com-pletely swollen and the equilibrium pH reached, the titration (withstandard 0.06 M HCl solution) was initiated. An equilibration timeof 600 s was allowed to pass after each portion of titrant (0.04 mL)was added. The basicity constants were evaluated with the ApparKprogram [36]. Swelling measurements of the hydrogels wereperformed in a thermostated glass cell connected to a Haake DSthermostat and a temperature probe controlled by the TimTalk 9software [35]. Swelling kinetics was measured in PBS at pH 7.40.The effect of pH was studied in 0.01 M acetate or phosphate buf-fers, in 0.15 M NaCl solutions. A hydrogel sample (10–40 mg) indry slab form, was weighed in a Strainer cell (70 lm pore size)and immersed in 50 mL of the bathing medium and kept at thedesired conditions for 24 h. Thus, the equilibrium degree of swell-ing (EDS, wt/wt) was evaluated. The conditions can be changed inthe same set-up, such as pH, temperature, addition of salt. In allcases the hydrogel samples immersed in the Strainer cell wereremoved at specific intervals, blotted with tissue paper to removeany surface water, and weighed (wet weight, Wwet). The EDS valuewas calculated by using the following equation

EDS ¼ ðWwet �WdryÞ=Wdry

where Wdry is the weight of the dry hydrogel before swelling.

2.4. Loading and in vitro release of doxorubicin

Doxorubicin hydrochloride (16–150 mg) was loaded on a pre-weighed sample of the selected hydrogel (45–73 mg of Phe-NIP3



Table 1Feed composition for the synthesis of nanocomposite Phe-NIP hydrogels.

Hydrogel Monomersa/mmol (g) lL mL NPsb mLc mLd Yield/g

PHE NIP EBA TEA CoFe2O4 APS Volume

Phe-NIP1 4.56 (1.00) 4.71 (0.55) 0.18 (0.030) 130 0.20 1.00 11.20 1.44Phe-NIP2 4.56 (1.00) 4.71 (0.55) 0.18 (0.030) 130 0.60 1.00 11.60 1.42Phe-NIP3 4.56 (1.00) 4.71 (0.55) 0.18 (0.030) 130 2.00 1.00 13.00 1.45

a Weighed amount of monomers.b Measured amount of a solution dispersion of magnetic NPs (Colorobbia, Italy).c Amount of APS aqueous solution (5.0 mg/mL).d Total volume of mixture.


and 106 mg of HVa2), previously swollen in a slightly alkalineenvironment, by soaking in an aqueous solution of known concen-tration (2–4 mg/mL). DOXO loading was carried out in the dark atroom temperature for 1 week and with occasional stirring. This ledto hydrogel collapse yielding a dark red solid, which was filteredand repeatedly washed with doubly-distilled water. The solidwas then dried in air and then under vacuum to a constant weight.The dry solid was brittle and easily pulverized. It showed an appre-ciable weight increase. In vitro release of DOXO was carried out inPBS solution (pH 7.4) starting from dry DOXO-loaded hydrogelsamples (0.5–8.3 mg), immersed in a specific solvent volume(3.20 mL in the cuvette, 50 mL in the container). In all experiments,with application of an alternating magnetic field (AMF), therelease of the drug was conducted in parallel on two differentDOXO-loaded hydrogel samples of approximately the sameweight: the first was subjected to the AMF, and the second wasused as control. In all cases the temperature was kept constant at25 �C except a variable temperature experiment with hydrogelPhe-NIP3. For the first 24 h the release of DOXO was analyzed atspecific time intervals by spectrophotometric measurements at500 nm. For longer periods DOXO was measured every 24 h. How-ever, after each ‘‘long-term’’ measurement the hydrogel wasquickly washed with doubly-distilled water, and transferred to afresh release medium in order to maintain infinite sink conditions[37]. The cumulative amount of DOXO released from the hydrogelwas determined using a calibration curve.

2.5. In vitro cytotoxicity

HeLa cells (derived from a human cervix adenocarcinoma) wereused in this study. Cells were cultured in standard conditions:RPMI 1640 medium (Sigma), supplemented with 10% heat-inacti-vated fetal calf serum (Invitrogen), 2 mM L-glutamine (Sigma),and 50 mg/L gentamycin, at 37 �C in a humidified atmosphere con-taining 5% CO2. For routine reseeding and for experiments, cellswere harvested with phosphate-buffered saline (PBS)–EDTA(0.2 g/L). For experiments, cells were seeded in 5 cm2 plastic dishes(Sarstedt) at density of 7 � 105 cells and left overnight, whereuponthe medium was replaced with 2 mL of fresh medium, and cellswere treated for 18 h or 24 h with native doxorubicin (2 mM, inDMSO 0.01%, final concentration) or with different amounts ofthe DOXO-loaded hydrogel Phe-NIP3, the latter floating on themedium surface. In selected experiments, HeLa cells were treatedwith the higher amounts of the hydrogel with no loaded doxorubi-cin to rule out any possible toxic effect afforded by the hydrogelitself. At the end of the experiments, aliquots of detached and stilladhering cells (gently harvested by PBS-EDTA) were counted in aBürker chamber. The percentage of floating on total cells was usedas a first quantitative indication of cell injury. The apoptotic modeof cell death was evaluated in cell lysates of adhering plus floatingcells by means of two typical parameters: the caspase 3/7 activity,by a fluorogenic assay which detects the caspase-dependentproteolytic cleavage of a synthetic substrate [38], and the caspase


3/7-dependent proteolysis of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), by Western blot analysis [39].

3. Results and discussion

3.1. Syntheses and characterization of hydrogels

The three Phe-NIP hydrogels studied herein incorporate vary-ing amounts of CoFe2O4 magnetic NPs. They were obtained by rad-ical copolymerization of the two vinyl monomers, thecommercially-available N-isopropylacrylamide (NIP) and the syn-thetic N-acryloyl-L-phenylalanine (PHE) in a NIP/PHE molar ratioof 1:1. The EBA cross-linking agent was used throughout in a2 mol% concentration. Unlike similar hydrogels previouslyreported, which exhibit mechanical consistency and transparency,the presence of magnetic NPs makes these hydrogels brittle anddark, especially in the dry state (11,40) The morphology of thesamples is fairly uniform, both on the surface and in the bulk.TEM micrographs of the DOXO-loaded hydrogel Phe-NIP3 showedthat the nanoparticles are uniformly distributed throughout thenetwork with a mean particle diameter of �5 nm (see Supplemen-tary Material, Fig. S-1). Infrared analysis supported the interactionbetween the DOXO molecule and the hydrogel (see below). In fact,FT-IR spectra of the hydrogel Phe-NIP3 (in the protonated form)clearly show bands assigned to the –COOH group (1720 cm�1),amide I (1623 cm�1) and amide II (1516 cm�1) (see SupplementaryMaterial, Fig. S-2). Complexation of DOXO by the hydrogel isevidenced by the presence of bands which are a mixture of thoseoriginating from the hydrogel and the DOXO molecules. TheFT-IR spectrum still shows the presence of –COOH, as evidencedby the band at 1730 cm�1, as a result of the proton transfer fromthe DOXO to the –COO� group of the hydrogel, despite the lowerpKa of the latter. This band becomes negligible after a long-termDOXO release (one month), while the bands assigned to dN-H(1618 cm�1 and 1521 cm�1) of DOXO are still present and shiftedto higher frequencies [41].

3.2. Basicity constants

Potentiometric titrations of the three Phe-NIP hydrogelsshowed a significant amount of titratable carboxylic acid groups(59 wt% in 0.15 M NaCl, and 62 wt% in 0.01 M NaCl), as evidencedby the end-point analysis of the titration curves. The result is inagreement with the feed composition (63 wt%). A similar patternof log K in relation to the degree of protonation (alpha) wasobtained for the three hydrogels (Fig. 2). This reveals a polyelectro-lyte behavior of the charged Phe residues within the cross-linkedpolymer that is similar to that already noted for the ‘‘free’’ polymeranalogs [31]. Fig. 2 reveals two characteristic features. At the sameionic strength, the three hydrogels show virtually the same poly-electrolyte behavior that is independent of the different amountsof magnetic NPs present. As the ionic strength becomes lower,



Fig. 2. Relationship between the basicity constants (log K) and the degree of protonation (alpha) of the –COO� groups present in the three hydrogels Phe-NIP containingmagnetic NPs at 25 �C. The ionic strength was 0.15 M NaCl or 0.01 M NaCl and the amount of finely dispersed gel was 30–34 mg or 20–22 mg, respectively. (For interpretationof the references to color in this figure legend, the reader is referred to the web version of this article.)


the log K value increases. This is a general trend observed in thepolyelectrolyte domain, where electrostatic effects are dominant.

During protonation, the log K value decreases with increasingalpha up to �0.3 and 0.5 for 0.15 M and 0.01 M NaCl, respectively.In these low-range alpha values the electrostatic effect is predom-inant and the hydrogel contracts (de-swells) upon protonation ofthe –COO� groups. Above the critical alpha values, the electrostaticforces interfere with the hydrophobic forces, due to the presence ofisopropyl and phenyl groups located into the hydrogel. This effect,as already reported for the ‘‘free’’ polymer analogues, results in ahigher log K value upon protonation, due to the abundant exposureof the remaining –COO� groups to incoming protons [31,42]. Eval-uation of the log K value of a polymer is necessary prior to its use asa complexant of an oppositely-charged drug. Appropriate log K val-ues allow a considerable loading of DOXO molecules into the poly-mer matrix. In fact, the higher is the basicity of the –COO� group,the greater the degree of protonation at neutral pH, and, thus, thecomplexing ability of the drug molecule is enhanced. At neutral pH,both the DOXO molecule (pKa 8.3) and the –COO� group (pKa 5) ofthe hydrogel are found completely ionized. This is the basis of fine-tuning the electrostatic binding between the drug and the polymerchain of the hydrogel. Thus, the loading of DOXO into the hydrogelwas carried out at pH 7.

3.3. Hydrogel swelling

The swelling kinetics of the three hydrogels Phe-NIP loadedwith magnetic NPs is shown in Fig. 3 (Panel A). The DS/time plotshows a different swelling behavior under the same experimentalconditions of temperature, pH and solvent. In contrast to thehydrogel Phe-NIP3 that reached its equilibrium degree of swell-ing (EDS) in about 1 week, the other two hydrogels Phe-NIP1and Phe-NIP2 reached their EDS values in a shorter time (2 days)at the same temperature (25 �C) and solvent (PBS, pH 7.40). Thelarger amount of NPs dispersed into the network of Phe-NIP3can explain this difference, by providing a higher resistance tothe solvent entering the core. This phenomenon is related to theslow ionization of the –COOH groups and yields longer equilibra-tion times for the swelling in PBS.

Faster swelling kinetics is observed in the alkaline pH region. Infact, the hydrogel Phe-NIP3 showed an initial DS/time slope of


about 30 times greater in doubly-distilled water at pH 10. Its swell-ing sharply increased reaching an EDS value of about 700, provingits sensitivity to pH.

The effect of NaCl concentration on EDS is also worth-examin-ing. Fig. 3 (Panel B) shows a decreasing pattern of the EDS of hydro-gel Phe-NIP3 as the concentration of NaCl increases. The fasterdecrease is observed in a limited concentration-range of NaCladded (0–0.15 mol/L). Subsequently, the EDS smoothly decreasesfor NaCl concentrations up to 2.5 mol/L, without any appreciablefurther gel-collapsing phenomena, as also observed in relatedhydrogels containing a-aminoacid residues [31,43].

Furthermore, the EDS behavior for the hydrogel Phe-NIP3 wasevaluated at different pH and temperatures. Fig. 4 (Panel A) showthe effect of pH on the swelling in 0.15 M NaCl and at 25 �C. Theswelling behavior of the hydrogel HVa2 is also plotted in the sameFig. 4, for comparison. In both cases, the hydrogels show greaterswelling at pH > pKa of the corresponding –COOH group. As thepH approaches 5 for Phe-NIP3 and 4 for HVa2 the hydrogel col-lapses because hydrophobic interactions prevail over the electro-static repulsion forces above the critical degree of protonation(alpha) value of the COO� groups [31,42].

For the hydrogel Phe-NIP3, the swelling behavior at differenttemperatures was studied in buffered solutions at the pH regime >5.Thus, the EDS values for the swelling-collapsing process are shownin Fig. 4 (Panel B). In these experiments the temperature was reg-ularly changed every 24 h. As the temperature increased, a smoothand virtually identical decrease of the swelling was observed for allpH values >5 studied. Only at pH = 5.0 a weak inflection point wasobserved around 33 �C. This means that the hydrogel, even in thepartially ionized form, maintains its hydrophilicity over a widerange of temperatures, despite the presence of NIP units that arestrongly sensitive to the release of water molecules in the sametemperature-range. The random copolymerization with Phe unitshampers the hydrophobic interaction of the isopropyl side groups.Any hydrophobic interaction with the phenyl residues can beexcluded because similar hydrogels with L-valine residues clearlyshowed inflection points, as reported before [11,40]. In the lattercase, any decrease in pH values shifts the hydrogel de-swellingto lower temperatures. This is accompanied by a sharp inflectionpoint. In the case of the hydrogel Phe-NIP3 the hydrophilic-



Fig. 3. Panel A: Time-dependence of the degree of swelling (DS, wt/wt) for the hydrogels Phe-NIP1 (black triangles), Phe-NIP2 (red circles), and Phe-NIP3 (blue squares) inPBS buffer (pH 7.4) at 25 �C. Green squares represent the DS of Phe-NIP3 in doubly-distilled water at pH 10. Panel B: Dependence of the equilibrium degree of swelling (EDS,wt/wt) on NaCl concentration (mol/L) of the hydrogel Phe-NIP3 at 25 �C in water. The error refers to at least two measurements averaged. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Panel A: Equilibrium degree of swelling (EDS, wt/wt) in relation to pH for the hydrogels Phe-NIP3 (blue squares) and HVa2 (red circles) at 25 �C and ionic strength0.15 M NaCl. Panel B: Dependence of EDS on temperature (�C) for the hydrogel Phe-NIP3 at different pH values: 7.4 (PBS, blue circles); 5.5 (acetate buffer in 0.15 M NaCl, pinkdiamonts); 5.1 (acetate buffer in 0.15 M NaCl, green triangles); 5.0 (acetate buffer in 0.15 M NaCl, red squares). The error refers to two measurements averaged. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


hydrophobic forces become competitive, and the sensitivity of thehydrogel to temperature increases, but only at pH values >5.

3.4. Loading and in vitro release of doxorubicin

The first phase of the study, the focus was on the loading andrelease of DOXO of two hydrogels, namely Phe-NIP3 and HVa2.The goal was to compare the long-term drug release and, ideally,correlate it to the different drug-hydrogel interactions. It is extre-mely important to know the quantity of DOXO present in biologicalfluids for in vivo application. Loading of Doxorubicin hydrochloridewas accomplished by soaking a pre-swollen hydrogel network inits ionized form in a freshly prepared aqueous solution of the drug.In this way, electrostatic interactions are optimized. For the hydro-gel Phe-NIP3 two different Phe/DOXO molar ratios (4:1 and 2:1)


were tested, while for the HVa2 hydrogel the HVa2/DOXO molarratio was 2:1. The choice of the aforementioned molar ratios allowsan excess of free carboxyl groups, which provide gel swelling dur-ing the course of the drug release. It is worth-noting that excessiveloading of DOXO onto the gel via complexation renders the gelundesirably compact and difficult to hydrate in the subsequentrelease phase. However, in all cases a rapid collapse of the gelwas observed after addition of the DOXO solution [44]. The intensered color of the solution disappeared within a few minutes withsimultaneous dark red-staining of the collapsed gel (see Supple-mentary Material, Fig. S-3). To ensure complete drug complexationat equilibrium, the system was maintained for 1 week in the darkwith occasional stirring. The final product showed an increase indry weight consistent with the amounts of reactants initiallyweighed. The amount of DOXO on two Phe-NIP3 gel samples




was 26 wt% and 48 wt%, while that on the HVa2 gel was 56 wt%, inagreement with the initial feeds.

The in vitro doxorubicin release from hydrogels was carried outin PBS at pH 7.4 and 25 �C, with and without AMF application. Insome cases, the temperature was increased to 37 �C in order toevaluate the response of a possible temperature trigger of thesetemperature-sensitive hydrogels. Furthermore, in this study ‘‘infi-nite sink conditions’’ were simulated. Hence, a pre-weighedamount of DOXO-loaded hydrogel in a Strainer cell was suspendedin 3 mL and/or 50 mL of the release medium, while the mediumwas regularly replaced every 24 h [37]. Fig. 5 shows the compari-son of the cumulative DOXO release profiles from hydrogelPhe-NIP3 loaded with different amounts of the drug (26 wt% and48 wt%). The DOXO release profile from gel HVa2 with 56 wt%loading is also shown for comparison.

It is evident that, for Phe-NIP-based hydrogels, increased drugloading causes an increased drug release at fixed time intervals.Importantly, electrostatic DOXO-hydrogel interactions play a keyrole in controlling the amount of free drug available for therapeuticpurposes. This is corroborated by the DOXO release profile fromhydrogel HVa2, loaded with a considerably higher quantity ofDOXO, compared to hydrogel Phe-NIP3. DOXO is a positivelycharged molecule with a pKa 8.3 [24,25,44]. It can therefore partic-ipate in electrostatic interactions with the ionized carboxyl groupsof the hydrogel at pH 7.40. Complexation takes place with theexpected 1:1 stoichiometry. The intensity of this drug-polymerinteraction increases as the pKa of the –COOH moiety of the gel-forming polymer increases. Hydrogel HVa2 mainly consists ofpolymer chains of acrylic units with residues of L-valine, slightlycross-linked with EBA. Furthermore, pKa values for the –COOHgroups are higher for the L-valine units, especially at low degreesof protonation and at low ionic strength [31,42,44]. This suggeststhat hydrogel HVa2 may form electrostatically more stable com-plex species with the DOXO molecule [18].

To trigger DOXO release from the hydrogels, two different formsof triggers were used: a temperature stimulus and an AMF stimulusof low frequencies. Fig. 6 shows the release profiles from the DOXO-loaded Phe-NIP3 hydrogel (26 wt% DOXO) (Panel A), and from theDOXO-loaded HVa2 hydrogel (56 wt% DOXO (Panel B), in PBS.

A two-phase release process can be seen when incubated at25 �C. An initial burst-like release is followed by a stable diffusioncontrolled release profile. This may be attributed to the initial gelswelling from the dry state. Once hydrated, the hydrogel does

Fig. 5. Cumulative release of DOXO (mg/g) from different DOXO-loaded hydrogels: Ph(56 wt% loading, red circles). Release experiments were performed in PBS, pH 7.4 at 25 �Creferences to color in this figure legend, the reader is referred to the web version of thi


not undergo large changes in swelling as the complexed DOXOmolecules prevent fast ionization of the carboxyl groups. The lowswelling of the hydrogel, even at longer times, follows the slowkinetics of release of the complexed drug. When temperatureincreases from 25 �C to 37 �C, a higher release rate is observed(Fig. 6, Panel A). This enhancement in release rate may be attrib-uted to the collapsing process of the hydrogel, because it is temper-ature-sensitive due to the presence of the NIP units. It isworth-noting that the effect of temperature on hydrogel collapseis pronounced in this case, in contrast to the native hydrogel(Fig. 4, Panel B). This is due to the fact that only a limited numberof carboxyl groups are in the uncomplexed state, because of thepresence of the complexed DOXO (about 30%). The latter, by par-tially neutralizing the polymer system, induces a lowering of theLCST (Lower Critical Solution Temperature). The process of thehydrogel collapse is exhausted in a few days, as evidenced by thesubsequent decrease of DOXO released during a week. At 25 �C,the release mode restores to a slow profile, becoming comparableto that of the control, for more than a week. In the next cycle oftemperature rise from 25 �C to 37 �C, and after 24 days, theenhancement in release rate is much more limited. In this situa-tion, since the release of DOXO has reached the double value(60%), the shrinking stops and the hydrogel undergoes changesonly due to temperature, similar to the native gel.

When the second AMF trigger was applied, the rate of DOXOrelease was enhanced for extended time. Panel A of Fig. 6 shows thatDOXO release is significantly accelerated under an applied AMF oflow frequency (40 kHz). Exposure of nanocomposite systems to highmagnetic fields usually causes a temperature rise in the surroundingenvironment [27]. Therefore, we limited our AMF stimulation tomild conditions in order to avoid heat shock responses [45]. Duringthe application of AMF for the entire duration of the release curve, notemperature increase was observed in the release media (PBS main-tained at 25 �C). Despite this, the release curve is systematicallyhigher than the control. This increase yields a release profile that isalmost identical to that described above due to temperatureincrease. An undetected temperature increase inside the hydrogelmay be due to the presence of magnetic NPs, stimulated by theapplied AMF. This can lead to enhancement of the diffusion rate ofthe loaded DOXO. Moreover, the energy induced by AMF can causeoscillation or vibration of the network-embedded CoFe2O4 magneticnanoparticles [29]. This in turn may cause twisting and/ordisplacement of the polymeric chains, resulting in an enhancement

e-NIP3 (48 wt% loading, blue squares; 26 wt% loading, green triangles) and HVa2. The error refers to at least two measurements averaged. (For interpretation of the

s article.)



Fig. 6. Panel A: Cumulative DOXO release (%) from DOXO-loaded hydrogel Phe-NIP3, in PBS pH 7.40 at 25 �C: under AMF stimulation (5 V and 40 kHz, blue triangles); control(25 �C, black circles with error bars obtained from two parallel experiments); effect of the 37 �C temperature (red filled squares). Panel B: Cumulative doxorubicin release (%)from DOXO-loaded hydrogel HVa2 under different AMF stimulation (PBS pH 7.40 at 25 �C): control experiment (black circles with error bars obtained from three parallelexperiments: 2.81 mg in 3.20 mL PBS; 5.6 mg in 50 mL PBS; 8.3 mg in 50 mL PBS); stimulated by 5 V and 40 kHz (green triangles: 3.71 mg in 3.20 mL PBS); stimulated by 50 Vand 20 kHz with cell copper condenser (c.c.c., red squares: 5.1 mg in 50 mL PBS); stimulated by 50 V and 20 kHz with copper coil honeycomb (c.c.h., violet squares: 4.9 mg in50 mL PBS). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


of the diffusion process. It is believed that the magnetically-induceddeformation of the hydrogel as a result of the oscillation or vibrationof the embedded magnetic nanoparticles is elastic. This elasticdeformation ensures a long-term, reliable controlled release of thedrug. This permeability control mechanism has also been addressedby other researchers [46–48].

In a similar manner we proceeded to evaluate the long-termrelease of DOXO from DOXO-loaded hydrogel HVa2. The Panel Bof Fig. 6 summarizes the cumulative DOXO release (%) under differ-ent AMF conditions and, even at the same AMF conditions, withdifferent stimulation systems (a cell copper condenser, c.c.c., anda copper coil honeycomb, c.c.h.). Control experiments for eachrelease curve are also shown under appropriate stimulation ofAMF. These parallel experiments were done with preciselyweighed amounts of hydrogel samples in the same volume of thereleasing PBS medium. The release of DOXO from DOXO-loadedhydrogel HVa2 is enhanced with the application of the sameAMF stimulation (5 V and 40 kHz), although to a lesser extent com-pared to similar experiment with the Phe-NIP3 hydrogel. This maybe ascribed to the lower amount of free DOXO in equilibrium withthe strongly hydrogel-complexed DOXO. The magnetic stimulationproduces a vibrational state of the polymer network by increasingthe diffusion of the drug. Increase in the applied voltage (50 V) anddecrease in the frequency (20 kHz) enhance DOXO release. The useof the copper coil honeycomb strongly improves DOXO releasekinetics compared to the cell copper condenser. The curves relatedto the experiment with the generator AG 1006 and with the coppercoil honeycomb confirm the greater significance of the magneticfield effects compared to those of the electric field exerted by thecell capacitor. In the first case mechanical stimulation of the mag-netic NPs occurs throughout the whole sample mass. This is due tothe vibrationally-induced effect with consequent increase of thetemperature (about 3 �C) of the bathing medium. In the secondcase, the enhanced DOXO release compared to the control curve(without stimulation) can be attributed only to small currentsinduced on the surface of the hydrogel sample without affectingthe hydrogel inner volume. Indeed, the effects of low and mediumfrequency electric fields on temperature are limited on the surfacesof the exposed bodies. Research is underway to assess and betterunderstand the mechanism involved, also in view of the diversityof magnetic nanoparticles used (Fe3O4).


In the absence of any external stimulus, as well as under theAMF application, the DOXO release profiles for both hydrogelsappear to obey the empirical Peppas’s power law expression,

Mt=M� ¼ ktn

where Mt and M� are the cumulative drug releases at time t and infi-nite time, respectively; k is the rate constant relative to the proper-ties of the matrix and the drug, and the n value is the diffusionexponent characteristic of the release mechanism [49,50]. In thecase of pure Fickian release, the n has a limiting values of 0.50 fromslabs, whereas values of 0.5 < n < 1 indicate a non-Fickian (anoma-lous) diffusion mechanism.

The results of our analysis, conducted on the hydrogel systems,are reported in Table 2. It can be seen that, for the initial time ofrelease (1 week) the diffusion exponent n for both hydrogels isbetween 0.58 and 0.91, in the presence and in absence of AMF.

The analysis for the successive time period (late release) showsthat the n value is less than 0.5 in all cases. Thus, unlike the initialanomalous diffusion mechanism most likely due to the swelling ofthe dry sample, the release of the DOXO follows a Fickian diffusionmechanism for the period >8 days. These results confirm that themain release mechanism is diffusion, as expected considering theelectrostatic hydrogel-DOXO interactions. Furthermore, it is inter-esting to note that the value of k, though different for the two typesof hydrogels, significantly increases with the application of anexternal stimulus (AMF). This observation may be related to thegreater permeability and mobility inside the hydrogel and favoredFickian release of DOXO inside the network [44,51,52].

3.5. Cytotoxicity of doxorubicin released from DOXO-loaded hydrogelin HeLa cells

Experiments were performed on HeLa cells to compare thecytotoxic effects between native DOXO and DOXO released fromthe loaded hydrogel Phe-NIP3. The selected concentration fornative DOXO was 2 mM, which was able to induce approximately50% cell death in our experimental conditions. In an initial set ofexperiments, HeLa cells were treated with different amounts ofDOXO-loaded hydrogel, ranging from 90 to 740 lg. As shown inFig. 7 (Panel A), similarly to the native drug, doxorubicin releasedfrom the gel induced a remarkable extent of cell death, as



Table 2Values of the rate constant (k) and exponent (n) of the empirical Peppas’s power law expression (Mt/M� = ktn) for DOXO release from nanocomposite hydrogels (in PBS pH 7.40and at 25 �C).

Hydrogel Amount of DOXO loaded (wt%) AMF stimulation Initial release (0-7 days) Late release (8 to final days)

n k R2 a n k R2a

Phe-NIP3 26 No 0.58 0.090 0.998 0.33 0.184 0.98548 No 0.91 0.042 0.999 0.34 0.192 0.99348 5 V/40 kHz 0.87 0.049 0.999 0.34 0.213 0.995

HVa2 56 No 0.72 0.026 0.999 0.48 0.038 0.99956 5 V/40 kHz 0.78 0.029 0.999 0.49 0.044 0.99856 50 V/20 kHzb 0.80 0.038 0.999 0.49 0.057 0.99456 50 V/20 kHzc 0.70 0.102 0.996 0.36 0.150 0.999

a Correlation coefficient.b Cell copper condenser.c Copper coil honeycomb.

Fig. 7. Panel A: HeLa cells were exposed for 18 h to 2 mM native doxorubicin (pink square) or to different amounts of DOXO-loaded Phe-NIP3 hydrogel (blue circles). Cellinjury was evaluated as percentage of floating cells on total cells. For native doxorubicin, results are presented as mean ± S.E. of four independent experiments. For DOXO-loaded hydrogel, each point of the plot represents the mean value (±S.E.) of two to four very similar amounts of the sample used in several independent experiments. Panel B:HeLa cells were exposed for 24 h to 2 mM native doxorubicin (Dx) or two amounts of DOXO-loaded hydrogel (GD). Apoptotic cell death was assessed as cell detachment (% offloating cells on total cells), as caspase 3/7 activity (Arbitrary Units of Fluorescence (AUFs)/min/mg protein), and as PARP proteolytic cleavage evaluated in Western blot.b-actin was also revealed and used as loading control. A typical experiment out of three is shown. C = untreated, control cells. (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)


demonstrated by the high percentage of injured cells detachedfrom the monolayer.

As expected, the extent of cell death has a good positive corre-lation with the DOXO-loaded hydrogel amounts. Similarly to thenative drug, DOXO released from the gel stimulates apoptosis inHeLa cells, as demonstrated by two hallmarks of this cell deathmode (Fig. 7, Panel B): the notable increase of caspase 3/7 activity,and the caspase-dependent proteolysis of PARP protein. Thedecrease of the full-length protein, along with the concomitantappearance of the cleavage product, strictly mirrors the extent ofapoptosis and, as expected, matches with the levels of cell detach-ment and caspase-3/-7 activity.

4. Conclusions

Hydrogels containing ionizable –COOH groups and hydrophobicmoieties present an attractive family of gel systems because oftheir ability to form variable-stability complexes with oppositelycharged drugs, while they are responsive to external stimuli, suchas pH and temperature. The studies presented herein represent aninitial preliminary attempt for potential applications for implantsor injections to the human body.

In this study we explored the loading and release capabilities ofDOXO entrapped within novel nanocomposite hydrogels. Thesehydrogels utilize L-phenylalanine and/or L-valine residues, and arealso embedded with magnetic nanoparticles for remote control ofdrug release when stimulated by an external AMF. DOXO can beefficiently loaded onto negatively charged hydrogels due to


DOXO-hydrogel electrostatic interactions and this efficiency canbe improved as the pKa values of the –COOH group increase[8,44]. The DOXO-loading process takes place much faster forhydrogels with high charge density and high pKa. At the same time,due to thermodynamic equilibrium conditions, the release of theloaded drug proceeds more slowly and in a sustained manner.The rate and the amount of released DOXO can be further controlledby external magnetic stimulation. Presence of a magnetic fieldinduces enhancement in the diffusion-controlled release kinetics.

Importantly, complexation of DOXO into the hydrogels pro-vided resistance of the drug against hydrolytic degradation [53]and in vitro sustained release for months. This could reduce thenumber of repeated DOXO administrations to cancer patients. Pre-liminary in vitro experiments on cervix adenocarcinoma cells in thepresence of DOXO-loaded hydrogels have demonstrated thathydrogel-released DOXO induces cell death in the same way asthe native drug. Moreover, the rate of cell death is proportionalto the amount of applied hydrogel. The results discussed hereincould constitute an initial basis for the improvement of thecontrolled release of DOXO-containing administration systems.Such encouraging perspectives are underway in our laboratories.

Acknowledgements

We thank Prof. Pantelis N. Trikalitis (Department of Chemistry,University of Crete, Heraklion, Greece) for TEM measurements. Firb(RBAP11ZJFA_003) and Prin (2010M2JARJ_004) projects providedpartial funding for this research.




Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ejpb.2014.06.005.

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Long-term doxorubicin release from multiple stimuli-responsive hydrogels based on α-amino-acid residues