Rossella Farra,a Fioretta Asaro,b Romano Lapasin,a Gabriele Grassic and Mario Grassi*a
Here we focus the attention on the physical characteristics of a highly biocompatible hydrogel made up of
crosslinked alginate and Pluronic F127 (PF127). This is a composite polymeric blend we propose for artery
endoluminal delivery of an emerging class of molecules named nucleic acid based drugs (NABDs). The
physical characterization of our composite gel, i.e. mesh size distribution and PF127–alginate mutual
organization after crosslinking, can significantly determine the NABDs release kinetics. Thus, to explore
these aspects, different technical approaches, i.e. rheology, low/high field NMR and TEM, were used.
While rheology provided information at the macroscopic and nano-level, the other three approaches
gave details at the nano-level. We observe that Pluronic micelles, organizing in cubic ordered domains,
generate, upon alginate crosslinking, the formation of meshes (z150 nm) larger than those occurring in
a Pluronic-free alginate network (z25 nm). Nevertheless, smaller alginate meshes are still on and can
just host un-structured Pluronic micelles and water. Accordingly, the gel structure is quite
inhomogeneous, where big meshes (filled by crystalline Pluronic) co-exist with smaller meshes (hosting
water and un-structured PF127 micelles). While big meshes offer a considerable hindering action on a
diffusing solute, smaller ones represent a sort of free space where solute diffusion is faster. The presence
of big and small meshes indicates that drug release may follow a double kinetics characterized by a fast
and slow release. Notably, this behavior is considered appropriate for endoluminal drug release to the
arterial wall.
Introduction
Coronary stenosis, a common atherosclerosis consequence, hasbeen treated in the past by percutaneous transluminal coronaryangioplasty (PTCA), a procedure able to enlarge the stenoticportion of the coronary by means of an expanding balloon.Nevertheless, the high incidence of re-stenosis (30–40%)following PTCA,1 presses to consider alternative approaches.The use of stents, rigid scaffolds positioned in correspondenceof the coronary stenotic portion, considerably reduced the re-stenosis occurrence compared to PTCA alone.2,3 However, stentsdid not completely solve the re-stenosis problem. Whereas theycould prevent the coronary wall early elastic recoil and late re-modeling, two known events leading to re-stenosis, theyinduced neointima iperplasia (In Stent Restenosis – ISR). Thislast event is mainly due to the iper-proliferation of vascularsmooth muscle cells (VSMCs)4,5 present in the artery wall. The
introduction of drug eluting stents (DES), devices able to releasedrugs with potent anti-proliferative properties, substantiallyreduced VSMCs iper-proliferation and thus re-stenosis rate.6,7
Unfortunately, also DES, so far the best approach for ISRprevention, do not represent the ideal solution. Their use isassociated to some problematic occurrencies such as late stentthrombosis and delayed restenosis.8 Themain reasons for thesedrawbacks are considered to be both patient/lesion-related andantiproliferative agent/stent platform-related.9 With regard tothe antiproliferative agent, a protable alternative to commonlyused drug (i.e. sirolimus) is represented by nucleic acid baseddrugs (NABDs), that proved to be reliable in suppressing theVSMCs exuberant proliferation.10,11 NABDs are short DNA orRNA molecules able to recognize, in a sequence-specicfashion, a target which, depending on the different NABD, canbe represented by a nucleic acid or a protein. Upon binding,NABDs are either able to induce the destruction or biologicalimpairment of the target. Thus, they can be used to counteractthe effects of gene inducing diseases.12
Due to the fragile nature, NABD incorporation onto DES isproblematic (both in the naked or carrier-complexed form). Apossible alternative for their in situ delivery is represented bythe endoluminal gel paving technique (EGP),13 coupled withimplantation of a bare metal stent. EGP consists of the use of a
Soft Matter, 2014, 10, 729–737 | 729
Soft Matter Paper
gel covering the entire balloon injury site immediately aer theballoon ination. The polymeric solution is delivered by meansof a catheter to the endoluminal surface, and crosslinked in situto yield a thin layer of degradable polymer. This layer, adheringto the vessel wall and embedding the stent, acts both as a drug(NABD) reservoir and creates a physical barrier between thedamaged coronary wall and the overowing thrombogenic andinammatory elements present in the blood stream.14
In order to meet the requirements of the EGP–stentapproach, we need a polymer system that: (1) behaves as asolution when it ows inside the catheter and that (2) becomesgel soon aer deposition on the vessel wall, (3) the gel issufficiently strong to resist to the blood ow erosion. Thepolymeric blend composed by Pluronic F127 (PF127) andalginate can guarantee this behavior. PF127 is a syntheticpoly(oxyethylene)-poly(oxypropylene) block copolymer withreverse thermo-responsive properties in aqueous solutions. Atroom temperature it behaves as a low viscosity solution while,at higher temperature, it forms cubic liquid crystalline struc-tures with gel-like mechanical behaviour. Due to this property,PF127 can be combined with the NABD complexes attemperature around 4–8 �C.15,16 Alginates, the second compo-nent of our mixture, are constituted by linear polymers con-taining b-D-mannuronic (M) and a-L-guluronic (G) acid. By addingan aqueous solution containing divalent cations, they formtemperature-insensitive strong physical gels. Taking advantage ofthe properties of the two gel components, our aim is to apply theaqueous mixture PF127–alginate–NABD to the endoluminalsurface of the artery vessel; due to the body temperature PF127undergoing gelation soon aer in situ release. The inner surfaceof the gel-like matrix is then briey exposed to a solution rich indivalent cations inducing the gelation of the alginate component.While the strong alginate part, in contact with blood ow,prevents from premature gel erosion and NABD migrationtowards the blood ow, the so PF127 layer, facing the coronarywall, favours NABD delivery to the coronary wall.
As all the abovementioned gel properties strongly depend onthe polymeric blend nano-structure, this paper is aimed atcharacterization, by means of rheology, low/high eld NMR andTEM, of the PF127–alginate gel nano-structure.
ExperimentalMaterials
Alginates are linear polymers extracted from brown algae livingin the northern seas. They are constituted by b-D-mannuronic(M) and a-L-guluronic (G) acid linked 1/ 4.17 In the presence ofan aqueous solution containing divalent cations, they formtemperature-insensitive, strong and erosion resistant physicalgels.18 The alginate used in this paper (molecular weight z 106
Da), kind gi from FMC Biopolymer Ltd, UK, was characterizedby a high G content (z70% G and 30% M).
Pluronic F127, a synthetic poly(oxyethylene-oxypropylene-oxyethylene) tri-block copolymer, added to water forms amicellar liquid solution below room temperature while, atphysiological temperature, micelles rapidly assemble into acubic liquid crystalline structure generating a so gel.19
730 | Soft Matter, 2014, 10, 729–737
Pluronic F127 was purchased from Sigma-Aldrich ChemieGmbH, Germany.
Two model drugs were considered in this study to getinformation about the gel structure. The rst one was theoph-ylline (TPH), a small organic molecule (molecular weight 198Da), purchased from Sigma-Aldrich Chemie GmbH, Germany.The second one, an oligonucleotide 51 nt long (DNA GT15H),was purchased by Eurons MWG Operon, Ebersberg, Germany.
All other chemicals were of analytical grade.
Methods
Gel preparation. Three kinds of gels were prepared: the rstone contained only alginate (2% w/w; A2) the second one con-tained only Pluronic F127 (18% w/w; P18) and the third con-tained alginate (2%w/w) and PF127 (18% w/w) (A2P18). ThePF127/A2 ratio was suggested by previous studies.15 The poly-meric blend A2P18 was prepared using the so-called “coldmethod” proposed by Schmolka.20 In brief, a proper amount ofalginate powder was slowly added to stirred distilled watercontained in a beaker maintained at 7 �C. Subsequently, thedesired amount of Pluronic akes were slowly added to thealginate solution. The system was stirred until complete poly-mer dissolution and then kept at 4 �C for 12 h before use. Aproper amount of polymeric solution was then poured into abottom at beaker to get a lm of thickness approximatelyequal to 1 mm (7 �C). The solution was heated to 37 �C to reachthe thermal gelation of Pluronic (A2PF18 thermal gelation startsat 20 �C and is complete at 24 �C).16 Subsequently, a CaCl2 watersolution (Ca2+ concentration equal to 5 g l�1) was rapidlysprayed on the gel surface to promote alginate crosslinking. Inorder to prevent possible lacks of Ca2+, the volume of thesprayed crosslinking solution was approximately equal to gelvolume. Aer a 5 min contact, the crosslinking solution wasremoved and the crosslinked lm was immediately and gentlycleaned with laboratory paper. The P18 gel was preparedfollowing the same procedure, except for the addition of algi-nate and the crosslinking solution (P18 thermal gelation startsat 22 �C and is complete at 26 �C).16 The A2 gel was obtainedusing the same method, except for the addition of PluronicF127. Considering that high eld NMR analysis requiresdeuterated solvents, high eld NMR samples have beenprepared by dissolving polymers in a D2O solution, containingthe model drug (10 mg cm�3 for theophylline and 1mg cm�3 foroligonucleotide). Indeed, D2O presence was necessary to silencethe water protons that otherwise would have hiddenmodel drugsignals.
Polarized light microscopy. P18 and A2P18 samples, bothbefore and aer cross-linking, were checked using polarizedlight microscopy (Leitz Pol-Orthoplan microscope).
Rheological investigations. Rheological characterization wascarried out at 37 �C using a Haake RS-150 controlled stressrheometer equipped with a thermostat Haake F6/8 andmounting a parallel plate device with serrated surfaces (PP35Ti:diameter ¼ 35 mm). Due to the rigid nature of A2 and A2P18gels, the gap setting was optimized according to a proceduredescribed elsewhere.21 Conversely, the gap was xed equal to 1
mm in the case of the so P18 gel. The measuring device waskept inside a glass bell at constant humidity conditions to avoidevaporation effects. The rheological tests were performed underoscillatory shear conditions. In particular, the linear viscoelasticregions were assessed, at 1 Hz, through stress sweep experi-ments. Frequency sweep tests were carried out in the frequency(f) range 0.01–10 Hz at constant stress s ¼ 5 Pa (well within thelinear viscoelastic range for all studied gels). Each test wascarried out in triplicate.
The generalized Maxwell model22 was used for theoreticaldependence of the elastic (G0) and viscous (G0 0) moduli onpulsation u ¼ 2pf, f being the frequency:
G0 ¼ Ge þXn
i¼1
Gi
ðliuÞ21þ ðliuÞ2
; Gi ¼ hi=li (1)
G00 ¼Xn
i¼1
Gi
uli
1þ ðliuÞ2; (2)
where n is the number of the Maxwell elements considered, Gi,hi and li represent, respectively, the spring constant, thedashpot viscosity and the relaxation time of the ith Maxwellelement while Ge is the spring constant of the last Maxwellelement which is supposed to be purely elastic.22 The simulta-neous tting of eqn (1) and (2) to experimental G0 and G0 0 datawas performed assuming that relaxation times (li) were scaledby a factor of 10.22 Hence, the parameters of the model were 2 +n (i.e. l1, Ge plus Gi). Based on a statistical procedure,23 n wasselected in order to minimize the product c2 � (2 + n), where c2
is the sum of the squared errors. According to the generalizedMaxwell model, the elastic modulus varies between two limitingvalues: Gmin (¼ Ge) and Gmax (¼ Ge + SGi), sum of all the elasticcontributions.24
Starting from Flory's theory25 the polymeric network cross-link density rx (dened as moles of junctions between differentpolymeric chains per gel unit volume) can be determined fromthe elastic shear modulus G through:
rx ¼ G/lRT (3)
where R is the universal gas constant, T is the temperature andl is a correcting parameter, called front factor, accounting forthe dissimilarity between the real gel structure and the idealmodel postulated by the Flory theory.25 In the case of alginategels, such difference is related to the non-punctual nature ofthe junction zones formed among the G-blocks belonging todifferent alginate chains.26 Accordingly, l coincides with thenumber of egg-box structures within the junction in the algi-nate hydrogel. While in the ideal Flory network we have l ¼ 1,in our case l is around 14.26 Finally, the equivalent networktheory27 allows evaluating the average network mesh size x
according to:
x ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi6=prxNA
3p
(4)
where NA is the Avogadro number and (1/(NArx)) is the sphericalvolume competing to each cross-link.
Low eld NMR. Low Field NMR (LFNMR) characterizationwas performed, at 37 �C, using a Bruker Minispec mq20 (0.47 T,20 MHz). Transverse relaxation time (T2) measurements werecarried out according to the (Carr-Purcell-Meiboom-Gill; CPMG)sequence (number of scans ¼ 4; delay ¼ 5 s) adopting 90–180�
pulse separation times s of 0.25ms. In order to determine the T2discrete distribution, the signal intensity I(t), related to thedecay of the transverse component of the magnetization vector(Mxy), was tted by the following sum of exponential functions:
IðtÞ ¼Xmi¼1
Aie�t=T2i h1=T2i ¼
Xmi¼1
Ai=T2i
�Xmi¼1
Ai (5)
where t is time, Ai are the pre-exponential factors (dimension-less) proportional to the number of protons relaxing with therelaxation time T2i and h1/T2i is the average value of the inverserelaxation time of protons. Again, m was determined by mini-mizing the product c2 � 2m, where c2 is the sum of the squarederrors and 2m represents the number of tting parameters ofeqn (5).23 Measurements were led in triplicate.
The relaxation (T2) experiments were used for estimation ofthe mesh size of the alginate gel. Indeed, owing to the interac-tions between water molecules and polymeric chains, waterprotons near the surface of the polymeric chains relax fasterthan those in the bulk.28,29On the basis of the Scherer theory,30 itcan be demonstrated31,32 that, for diluted gel systems (polymervolume fraction 4 # 0.1), the average polymeric network meshsize (x) can be expressed as:
x ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3p
ð1� 4Þ4
sRf (6)
where Rf is the radius of the polymeric chain. In addition, the“ber-cell” theory28 ensures that the following relation holds:
�1
T2
�¼ 1
T2H2O
þ 2
a
hM ix
a ¼ffiffiffiffiffiffiffiffiffiffiffiffi1� 4
43p
s(7)
where h1/T2i is the average of the inverse relaxation time of thewater protons trapped within the polymeric gel network, T2H2O
is the relaxation time of the protons of the bulk water (i.e.protons of the free water, whose relaxation is not affected by thepresence of the polymeric chains) and hM i (length/time) is anempirical parameter (relaxation sink strength) accounting forthe effect of polymer chains surface on proton relaxations.While eqn (7) holds on average for all the polymeric networkmeshes, similar expressions can be written for polymericnetwork meshes of different size (xi):
1
T2i
¼ 1
T2H2O
þ 2
a
hM ixi
(8)
where T2i is the relaxation time of the water molecules protonsentrapped in polymeric meshes of size xi. Eqn (7) and (8) hold infast-diffusion regime, i.e. when the mobility of the watermolecules, expressed by their self-diffusion coefficient D, ishigh compared with the rate of magnetization loss, identiablewith hM i � Rc (i.e. hM iRc/D � 1). Rc indicates the distance fromthe polymer chain axis where the effect of polymeric chains on
Soft Matter, 2014, 10, 729–737 | 731
Fig. 1 Mechanical spectra referring to alginate (A2), Pluronic (P18) andalginate–Pluronic (A2P18) gels at 37 �C. Close and open symbolsrepresent, respectively, storage (G0) and loss (G0 0) modulus while solidlines represent the best fitting of the generalized Maxwell modelcomposed by 5 elements (eqn (1) and (2)). Vertical bars indicatestandard error.
Soft Matter Paper
water protons relaxation becomes negligible and it can beexpressed by:28
Rc ¼ Rfffiffiffi4
p (9)
As h1/T2i (see eqn (7)), T2H2O and x (see eqn (6)) areknown, eqn (7) allows the determination of hM i. Further-more, by knowing hM i and T2i (see eqn (5)), eqn (8) makespossible the evaluation of xi for each class of polymericnetwork meshes.
In order to study water mobility inside the gel network,pulsed gradient spin echo (PGSE) measurements were per-formed at 37 �C. The applied sequence consisted in the classicalecho sequence with two equal gradient pulses (of length d ¼ 1ms) occurring at x1 ¼ 1 ms and x2 ¼ 1 ms aer the 90 and 180�
pulses, respectively. The time separation, indicated by D (z s �x1 � d + x2), is related to the water molecule diffusion time tdaccording to td ¼ (D � d/3). The determination of the movingspecies self-diffusion coefficient was led tting the followingequation to experimental data:33
At ¼Xp
i¼1
A0ie�q2tdDwi q ¼ ggd A0 ¼
Xp
i¼1
A0i (10)
where At and A0 are, respectively, the measured amplitude ofthe signal at the echo with and without gradient applied, g isthe proton gyromagnetic ratio, g is the known magnetic eldgradient while A0i are the fractions of protons characterizedby a self-diffusion coefficient Dwi. In the case of a homoge-neous system, of course, the summation limits to the rstterm (p ¼ 1) as all the protons are characterized by the sameself-diffusion coefficient. Also in this case, p was determinedminimizing the product c2 � 2p where c2 is the sum of thesquared errors and 2p represents the number of ttingparameters (A0i, Dwi) of eqn (10).23 Measurements were carriedout in triplicate.
High eld NMR. Self-diffusion NMR measurements (PGSTE)were carried out at 37 �C on a Varian 500 MHz NMR spec-trometer (11.74 T) operating at 500 MHz for 1H, equipped with amodel L650 Highland Technology pulsed eld gradient (PFA)amplier (10 A) and a standard 5 mm indirect detection, PFGprobe. The lock was made on D2O and solvent suppression wasaccomplished by pre-saturation. A one-shot sequence has beenemployed for theophylline gels diffusion measurements,34,35
with 20 different z-gradient strengths, Gz, between 0.02 and 0.54T m�1, a pulsed gradient duration, d, of 2 ms, and at differentdiffusion interval (D). At each gradient strength, 64 transientshave been accumulated employing a spectral width of 5.5 kHz(11 ppm) over 16k data points.
Oligonucleotide samples have been analyzed thanks to anexcitation sculpting PGSTE experiment.36 PGSTE NMR spectrawere processed using MestRenova and self-diffusion coeffi-cients were determined by means of eqn (10).
TEM. A2 and A2P18 gels were dehydrated and embedded inEpoxy resin. Then, ultrathin sections were contrasted usingPb3(C6H5O7)2. Images were recorded on a Philips EM 208 (100KV) Transmission Electron Microscope.
732 | Soft Matter, 2014, 10, 729–737
Results and discussion
Stress sweep tests revealed (data not shown) that, for all thestudied gels (A2, P18 and A2P18), the linear viscoelastic rangeholds for stresses above and beyond the value adopted forfrequency sweep tests (s ¼ 5 Pa).
Fig. 1 reports the mechanical spectra of the A2, A2P18 andP18 systems. It can be seen that for all three systems, the storagemodulus G0 is quite independent of pulsation u (¼ 2pf) andneatly prevalent on G0 0. This behavior resembles that displayedby other physical gels and other structured polymeric anddisperse systems.22 Both G0 and G0 0 decrease in the order A2,A2P18, P18, thus indicating that Pluronic addition hinders thealginate crosslinking process. Conversely, as previously found,16
the presence of un-crosslinked alginate enhances micellesorganization determining an anticipation of the F127 sol–geltemperature transition without altering the nal rheologicalproperties of the alginate–Pluronic gel.
The statistical procedure presented in the “Rheologicalinvestigation” paragraph revealed that a parallel combination offour Maxwell elements and one purely elastic is necessary forobtaining a statistically reliable tting of the mechanicalspectra reported in Fig. 1 (see solid lines). Best tting parame-ters values are reported in Table 1.
Table 1 (see F-test) and Fig. 1 reveal that the three tting arestatistically good and that the mesh size of the polymericnetwork should be comprised between 12 and 19 nm for the A2gel and between 14 and 22 nm for the A2P18 gel. In the A2P18case, the evaluation of the mesh size appears questionable asPluronic does not give origin to a network topology comparableto that assumed by Flory25 and Scherer.30 Nevertheless, the x
increase, in comparison with the A2 case, is another way todetermine the hindering behavior of Pluronic on the alginatecrosslinking process.
Low eld NMR tests revealed that, for the P18 gel, threerelaxation times are necessary to describe the relaxation of the
Table 1 Parameters relative to eqn (1) and (2) best fitting to the experimental data (systems A2, A2P18, P18) shown in Fig. 1.Ge,G1,G2,G3 andG4,are the spring constants of the generalized Maxwell model, l1 is the relaxation time of the first viscoelastic Maxwell element, Gmin (¼ Ge)
and Gmaxð¼ Ge þX4i¼1
GiÞ indicate, respectively, the minimum and maximum value of the shear modulus, rx is the crosslink density, x is the mesh
size of the polymeric network while F(5, 30) indicate the F-test results about the statistical acceptability of data fitting
transverse component (Mxy) of the magnetization vector (M).Conversely, in the A2 and A2P18 cases, four relaxation times areneeded as reported in Table 2.
Although it is never simple associating a relaxation time to aparticular protons status, in the case of P18 we can argue thatthe highest relaxation time (T21) essentially corresponds to“free” water protons, i.e. it corresponds to the water moleculesthat are weakly inuenced by P18 micelles and that permeateinto the micelles three-dimensional network. This hypothesis issupported by their abundance (A1% z 84%, see Table 2) and bythe relatively high value of the relaxation time (T21 ¼ 2280 ms,see Table 2) in comparison to that of the bulk water that, at 37�C, is approximately 3700 ms.32 The last two relaxation times(T22, T23) could be attributed to Pluronic micelles and boundwater to them. This hypothesis is supported by the fact the sumof T22 and T23 abundance (16.3%) is close to that theoreticallycompeting to Pluronic protons (15.9%). In addition, the averageT22 and T23 value (weighted by their relative abundance) (280ms) is close to the relaxation time (224 ms) we measured forPluronic protons in the P18-D2O gel (37 �C).
In the A2 case, the fastest relaxation times (T22, T23, T24)should correspond to the protons of the water trapped insidethe polymeric meshes as their values are very low in comparison
Table 2 Relaxation times (T2i) and relative weights (Ai%) referring to the th
as Ai% ¼ 100Ai
�Xmi¼1
Ai (see also eqn (5)). Finally, the average inverse rela
to free water relaxation at 37 �C (3700 ms (ref. 32)) and theyrepresent about 80% of the relaxing protons. Oppositely, therst relaxation time (T21) is too high to be associated to watertrapped in the polymeric meshes and could be related to thewater present on the lm surface due to the unavoidable algi-nate shrinkage upon crosslinking as also found elsewhere.26
Due to the low alginate concentration, the contribution ofalginate protons is not detectable (z0.64%). The A2P18 gelshows a relaxation behavior similar to the A2 one (see Table 2),with a main difference occurring for the slowest relaxation time(T21). Indeed, the Pluronic presence in the alginate networkclearly decreases T21 value and weight (A1%) that becomesapproximately one/third. This mentioned evidence furthersupports the hypothesis that T21 is associated to surface waterdue to network shrinkage. Indeed, in the presence of Pluronic,network shrinking is considerable reduced (data not shown)and it is also reasonable that the surface water is more “bound”due to the presence of Pluronic. Due to the existence of twostructures (one of alginate and one of Pluronic) the physicalinterpretation of T22, T23 and T24 in the A2P18 case is not easy.Anyway, their values are statistically equal to those competing tothe A2 gel (see Table 2) and differences arise for what concernstheir relative abundance (see Table 2). Thus, we could conclude
ree gels considered (A2P18, A2, P18). The relative weights are evaluated
Fig. 3 Logarithmic decrease, ln(At/A0), of the ratio between themeasured amplitude of the signal at the echo with (At) and without (A0)gradient applied, versus q2 (q ¼ g � g � d, see eqn (10)) for the threegels studied (A2, P18, and A2P18) assuming a diffusion time td ¼ 29.66ms (D ¼ 30 ms) (37 �C). Symbols indicate the experimental data whilesolid line indicate eqn (10) best fitting. Vertical bars indicate standarderror.
Soft Matter Paper
that Pluronic presence does not qualitatively change the struc-tures present in the A2 gel, but induces a variation of theirrelative abundance. Interestingly, A2P18 is the gel characterizedby the lowest average relaxation time among the three consid-ered gels.
In the A2 case, the theory shown in the Low eld NMR sectioncan be considered for the determination of the polymericnetwork mesh size distribution. Bearing in mind that polymervolume fraction (4) is 0.0114 and alginate ber radius (Rf) isequal to 0.8 nm,37 eqn (6) returns x ¼ 22.9 nm. x knowledge andeqn (7) allow to conclude that the relaxivity hM i is equal to 3.24� 10�7 m s�1. This estimation was carried out calculating theaverage inverse relaxation time (h1/T2i ¼ 9.6 � 10�3 ms�1)based on the three fastest relaxation times (T22, T23 and T24 inTable 2), as the rst one was not attributed to water blocked inthe polymeric network. Considering that the water self-diffu-sion coefficient at 37 �C in the A2 gel (see Fig. 4) is around 3 �10�9 m2 s�1 (i.e. close to that of free water38) and that Rc ¼ 69.4nm (see eqn (9)), it turns out that hM i Rc/D ¼ 8 � 10�7, i.e., �1.Accordingly, fast conditions are met and eqn (8) can be adoptedto estimate the mesh size (xi) corresponding to relaxation timesT22, T23 and T24: x2 ¼ 114 nm (11%), x3 ¼ 28 nm (59%) and x4 ¼13.6 nm (30%). It can be noticed that the x range, as perrheology (12–19 nm; see Table 2), is not so far from the x3–x4range that represents, according to the low eld NMR analysis,the 89% of the network meshes.
Both estimations are supported by the TEM image of A2 gel(Fig. 2) showing the presence of few big meshes (z100 nm), aswell as much smaller and frequent meshes.
In order to get more insights on A2P18 gel structure, thedetermination of the water self-diffusion coefficient (D), thanksto PGSE experiments can be useful. As an example, Fig. 3 showsthe trend of ln(At/A0) versus q
2 (see eqn (10)) for the three gelsstudied (A2, P18, and A2P18) and considering D ¼ 30 ms (i.e. td¼ 29.66 ms). It is easy to observe that in the A2 case only one ofthe p exponentials appearing in eqn (10) is necessary for reliabledata tting.
Fig. 2 TEM image referring to the alginate (A2) gel.
734 | Soft Matter, 2014, 10, 729–737
On the contrary, for P18 and A2P18 gels, two exponentials areneeded. This means that in the A2 gel all the water moleculesdiffuse in the same way (obviously, the crosslinked alginatechains cannot diffuse), while in the other two systems twodistinct diffusion modes are detectable. Fig. 4 extends thisnding for all the D, or td, considered. For the A2 gel, the waterself-diffusion coefficient (DwA2, see crosses) is independent of td,or its square root, thus the A2 polymeric network is substan-tially homogeneous (interconnected mesh) as its hinderingaction on water molecules diffusion is the same whatever the tdconsidered.39 Moreover, its hindering action is very limited asDwA2 is very close to the bulk water self-diffusion coefficient(37 �C; 3.04 � 10�9 m2 s�1 (ref. 38)). In the P18 case, on the
Fig. 4 Diffusion coefficients dependence on diffusion time (td). Whilein the A2 gel only one diffusion component can be seen (water, DwA2),two diffusion modes are detectable for the P18 and A2P18 gels. Dw1
P18 and Dw1 A2P18, indicate the free water self-diffusion coefficient inthe P18 and the A2P18 gel, respectively. D2 P18 andD2 A2P18, indicate,respectively, the self-diffusion coefficient of the pluronic micelles plusbound water in the P18 and the A2P18 gel, respectively. Vertical barsindicate standard error.
contrary, two diffusing species can be found. The rst one, Dw1
P18, can be attributed to free water (water molecules that areweakly inuenced by P18) while the second one, D2 P18, shouldcorrespond to Pluronic micelles and the water strictly bound tothem. Indeed, not only Dw1 P18 is very close to bulk water self-diffusion coefficient at 37 �C, but also eqn (10) data ttingevidences that, despite the diffusion time td, about 80% of theprotons diffuse with self-diffusion coefficient equal to Dw1 P18while the remaining 20% diffuses as D2 P18. This outcomedenitely agrees with what found in relaxation experiments (seeTable 2) and with the gel theoretical composition (18% Plur-onic, 82% water). Furthermore, PGSE experiments carried outon the P18–D2O gel, reveal that only one diffusing species canbe found and its self-diffusion coefficient is, regardless of td,around 2.7 � 10�11 m2 s�1, i.e. close to D2 P18. In the A2P18 gel,again, two diffusion components can be detected: Dw1 A2P18and D2 A2P18.
Their relative abundance (80%, 20%) is equal to that of theP18 case and it agrees with the gel theoretical composition forwhat concerns mobile species (18% Pluronic, 80% water). Thus,the slow diffusion could be attributed to Pluronic micelles andbound water while the fast diffusion component could beattributed to free (unbound) water. The presence of alginatemakes D2 A2P18 values slightly smaller than those competing toD2 P18 (see Fig. 4). In conclusion, also PGSE experiments revealdifferent characteristics for the A2 and A2P18 gel. This conclu-sion is also supported by the high eld NMR analysis employedto study the self-diffusion coefficient of theophylline (DTH) inour gel systems.
Fig. 5 shows the DTH dependence on the square root of thediffusion time, td, in a D2O–theophylline solution (10 mg cm�3,37 �C) and in the three gels prepared with the same D2O–theophylline solution. It can be noticed that, in the D2O solution,DTH is independent of td and its value is a little bit smaller thanthat in H2O at the same temperature40 (8.2� 10�10 m2 s�1). Thissmall difference can be explained by the higher viscosity of D2Oas compared with H2O. In the A2 gel, again, DTH is independent
Fig. 5 Diffusion time (td) dependence of the theophylline self-diffu-sion coefficient (DTH) in D2O (open circles), alginate (A2, open squares),Pluronic (P18, open triangles) and alginate–Pluronic (A2P18, blackdiamonds with dotted line) gels (37 �C). Vertical bars indicate standarderror.
of td and this is a clear indication of a homogeneous network thatoffers the same hindering action on theophylline diffusion,whatever the distance explored in the diffusion time. In addition,DTH is slightly decreased by the presence of the polymericnetwork, as its value is not so far from that in D2O. This meansthat theophylline diameter (0.78 nm)40 has to be small incomparison to network meshes, as per rheology (12–19 nm) andlow eld NMR (114–13 nm) previous estimations. In the P18 gel,DTH is still constant, but its reduction with reference to that inD2O is now notable. Thus, the mentioned gel is homogeneousbut its architecture is completely different from that of A2. As theA2P18 gel is characterized by a td dependent DTH, it is nothomogeneous. This means that, for small td, theophyllinemobility is almost unaffected by the gel structure (see Fig. 5),while its mobility is considerably reduced when it has the chanceto explore wider space portions (big td) where the probability ofmatching an obstacle is considerably increased. Interestingly, theDTH value corresponding to the highest td explored (see Fig. 4) islower than that competing to the PF18 system, due to thesimultaneous presence of alginate and Pluronic in the A2P18 gel.A nal conrmation of the non-homogeneous nature of theA2P18 gel is given in Fig. 6 that reports the comparison betweenthe TEM picture of the A2 gel (that of Fig. 2) and the A2P18 gel.
It is clear that the alginate chains (black traces; Pluronicmicelles are not visible) form wider meshes when Pluronic ispresent (A2P18 image in Fig. 6). In addition, the coexistence ofsmall and big meshes in the A2P18 gel (Fig. 6) can be explainedby the non-uniform spatial distribution of Pluronic micelles inthe polymeric network as found by Frisman and co-workers.41
Indeed, dealing with a similar system (photo-crosslinkedPEGylated brinogen–Pluronic F127 gels), these authors foundthat Pluronic micelles were still present aer PEGylated brin-ogen crosslinking and that their ordering depended on Pluronicconcentration. When Pluronic F127 concentration was <7% w/w, a homogeneous spatial distribution of micelles occurred,while for higher Pluronic F127 concentration, micelles orga-nized into dense aggregates whose packing increased withPluronic concentration. Moreover, they observed the coexis-tence of areas of randomly distributed micelles and areas ofordered micelles. Such a non-uniform state of micelle aggre-gation reasonably occurs also in our case, thus giving origin tothe coexistence of bigger and smaller meshes. Oppositely, astructure characterized by smaller meshes is formed in theabsence of Pluronic (A2 image in Fig. 6). Rheology, low and high
Fig. 6 Comparison between the TEM images referring to the algi-nate–Pluronic (A2P12) and the alginate (A2) gels.
Soft Matter, 2014, 10, 729–737 | 735
Fig. 7 Schematic representation of the A2P18 gel nanostructure, asper our characterization. Pluronic micelles, organizing in cubicdomains, induce the distortion of the alginate network. Thus, bigmeshes, filled by ordered domains, coexist with smaller meshes thatcan host only single micelles. Water permeates the whole structure.
Soft Matter Paper
eld NMR results lead us to conclude that the structure of theA2P18 gel can be schematized as reported in Fig. 7. Fig. 7 wasbuilt catching the alginate chains architecture from Fig. 6(A2P18) and then inserting the cubic structures formed byPluronic micelles (stars of diameter z 20 nm;41,42 stars are inscale with the mesh size). Depending on size, it is possible todistinguish meshes which can host the Pluronic liquid crystal-line arrangement (stars lattice) and other meshes that cannotdo it, because of their small dimensions. Small meshes can hosteither unstructured micelles, or just water. Interestingly, Fig. 7is reassuringly close to that of Frisman and co-workers41 whoworked on a similar gel made up by photo-crosslinked PEGy-lated brinogen and Pluronic F127.
Hence, our model drug (theophylline) can freely diffuse insmaller meshes (those without Pluronic micelles ordereddomains) while its diffusion is obstructed in large mesheswhere the Pluronic micelles ordered domains can form. Theabsence of birefringence both in P18 and A2P18 gels proved asimilar organization of Pluronic micelles in these two systemsand gels isotropy, as the cubic crystalline phase is generated byP18 above about 27–28 �C.43
Finally, high eld NMR allowed also to estimate the self-diffusion coefficient (DO) of our second model drug (oligonu-cleotide, used to mimic NABD) in the A2P18 gel. It turned outthat in the explored diffusion time range (300 ms < td < 1800ms), DO decreases from 2 � 10�11 to 3 � 10�12 m2 s�1 (37 �C).Thus, for small td, its diffusivity is about 1/3 of that measured inpure D2O (6.6 � 10�11 m2 s�1; 37 �C) while this ratio falls toabout 1/20 for longer td. Again, this behavior should be due tothe inhomogeneous structure of the A2P18 gel.
Conclusions
The joint use of rheology, TEM, low and high eld NMR allowedto understand the structure of a gel made up of alginate andPluronic and conceived for artery drug delivery according to the
736 | Soft Matter, 2014, 10, 729–737
endoluminal gel paving approach. This study revealed thatPluronic micelles, organizing in cubic domains, generate, uponalginate crosslinking, the formation of meshes bigger than thatoccurring in the Pluronic free alginate network. Nevertheless,smaller alginate meshes are still on and can just host unstruc-tured Pluronic micelles and water. As a results, Pluronic pres-ence gives origin to an inhomogeneous structure formed by bigmeshes (lled by ordered Pluronic micelles), where the diffu-sion of a solute is considerably hindered and smaller mesheswhere solute diffusion is faster. This aspect can be very inter-esting from a delivery point of view as it is supposed to roughlyinvolve a two-stage release kinetics: an initial fast stage followedby a slow one. The combination of the rst stage, due to drugpresence in the meshes lled by packed PF127 micelles (bigones), and the second, due to drug presence in the meshes freefrom packed PF127 Pluronic micelles (small ones), shouldensure an optimal balance between slow and fast release.Indeed, previous simulations44 indicated the synergy of fast anda slow release stages as the desired release kinetics to obtain analmost constant drug concentration in the artery wall.
Acknowledgements
This work was supported by the Italian Ministry of Education(PRIN 2010-11 (20109PLMH2)) and by the University of Trieste“FRA 2009” fund. Fondazione CRT-Trieste is acknowledged forthe purchase of Varian 500 NMR spectrometer. The authorswould like to thank Mr Gamboz for realization of TEM images,Dr Daniela Giacomazza and Dr Maria Grazia Ortore for helpfuldiscussions.
References
1 R. M. Califf, Am. Heart J., 1995, 130, 680–684.2 P. W. Serruys, P. de Jaegere, F. Kiemeneij, C. Macaya,W. Rutsch, G. Heyndrickx, H. Emanuelsson, J. Marco,V. Legrand and P. Materne, N. Engl. J. Med., 1994, 331,489–495.
3 D. L. Fischman, M. B. Leon, D. S. Baim, R. A. Schatz,M. P. Savage, I. Penn, K. Detre, L. Veltri, D. Ricci andM. Nobuyoshi, N. Engl. J. Med., 1994, 331, 496–501.
4 P. N. Ruygrok, M. W. Webster, J. J. Ardill, C. C. Chan,K. H. Mak, I. T. Meredith, J. T. Stewart, J. A. Ormiston andS. Price, Cathet. Cardiovasc. Interv., 2003, 59, 165–171.
5 R. Moreno, C. Fernandez, F. Alfonso, R. Hernandez,M. J. Perez-Vizcayno, J. Escaned, M. Sabate, C. Banuelos,D. J. Angiolillo, L. Azcona and C. Macaya, J. Am. Coll.Cardiol., 2004, 43, 1964–1972.
6 J. W. Moses, M. B. Leon, J. J. Popma, P. J. Fitzgerald,D. R. Holmes, C. O'Shaughnessy, R. P. Caputo,D. J. Kereiakes, D. O. Williams, P. S. Teirstein, J. L. Jaegerand R. E. Kuntz, N. Engl. J. Med., 2003, 349, 1315–1323.
7 G. W. Stone, S. G. Ellis, D. A. Cox, J. Hermiller,C. O'Shaughnessy, J. T. Mann, M. Turco, R. Caputo,P. Bergin, J. Greenberg, J. J. Popma and M. E. Russell, N.Engl. J. Med., 2004, 350, 221–231.
8 J. W. Jukema, J. J. W. Verschuren, T. A. N. Ahmed andP. A. Quax, Nat. Rev. Cardiol., 2012, 9, 53–62.
9 J. W. Jukema, J. J. W. Verschuren, T. A. N. Ahmed andP. A. Quax, Nat. Rev. Cardiol., 2012, 9, 79–90.
10 G. Grassi, P. Dawson, G. Guarnieri, R. Kandolf andM. Grassi, Curr. Pharm. Biotechnol., 2004, 5(4), 369–386.
11 B. Dapas, R. Farra, M. Grassi, C. Giansante, N. Fiotti, L. Uxa,G. Rainaldi, A. Mercatanti, A. Colombatti, P. Spessotto,V. Lacovich, G. Guarnieri and G. Grassi, Mol. Med, 2009,15, 297–306.
12 G. Grassi, B. Scaggiante, B. Dapas, R. Farra, F. Tonon,G. Lamberti, A. Barba, S. Fiorentino, N. Fiotti,F. Zanconati, M. Abrami and M. Grassi, Curr. Med. Chem.,2013, 20, 3515–3538.
13 M. J. Slepian and J. A. Hubbell, Adv. Drug Delivery Rev., 1997,24, 11–30.
14 J. L. West and J. A. Hubbell, Proc. Natl. Acad. Sci. U. S. A.,1996, 93, 13188–13193.
15 G. Grassi, A. Crevatin, R. Farra, G. Guarnieri, A. Pascotto,B. Rehimers, R. Lapasin and M. Grassi, J. Colloid InterfaceSci., 2006, 301, 282–290.
16 G. Grassi, R. Farra, E. Noro, D. Voinovich, R. Lapasin,B. Dapas, O. Alpar, C. Zennaro, M. Carraro, C. Giansante,G. Guarnieri, A. Pascotto, B. Rehimers and M. Grassi, J.Drug Delivery Sci. Technol., 2007, 17, 325–331.
17 K. I. Draget, G. Skjak-Bræk, B. E. Christensen, O. Gaserødand O. Smidsrød, Carbohydr. Polym., 1996, 29, 209–215.
18 X. Liu, L. Qian, T. Shu and Z. Tong, Polymer, 2003, 44, 407–412.
19 R. K. Prud'homme, G. Wu and D. K. Schneider, Langmuir,1996, 12, 4651–4659.
20 I. R. Schmolka, J. Am. Oil Chem. Soc., 1977, 54, 110–116.21 A. J. Kuijpers, G. H. M. Engbers, J. Feijen, S. C. De Smedt,
T. K. L. Meyvis, J. Demeester, J. Krijgsveld, S. A. J. Zaat andJ. Dankert, Macromolecules, 1999, 32, 3325–3334.
22 R. Lapasin and S. Pricl, Rheology of industrial polysaccharides:theory and applications, Blackie Academic & Professional,London, 1995.
23 N. R. Draper and H. Smith, Applied Regression Analysis, JohnWiley & Sons, Inc., New York, 1966.
24 E. Pasut, R. Toffanin, D. Voinovich, C. Pedersini, E. Muranoand M. Grassi, J. Biomed. Mater. Res., Part A, 2008, 87, 819–824.
25 P. J. Flory, Principles of polymer chemistry, Cornell UniversityPress, Ithaca, USA, 1953.
26 G. Turco, I. Donati, M. Grassi, G. Marchioli, R. Lapasin andS. Paoletti, Biomacromolecules, 2011, 12, 1272–1282.
27 J. Schurz, Prog. Polym. Sci., 1991, 16, 1–53.28 M. M. Chui, R. J. Phillips and M. J. McCarthy, J. Colloid
Interface Sci., 1995, 174, 336–344.29 K. R. Brownstein and C. E. Tarr, Phys. Rev. A, 1979, 19, 2446–
2453.30 G. W. Scherer, J. Sol-Gel Sci. Technol., 1994, 1, 285–291.31 M. Grassi, S. Fiorentino, R. Farra, B. Dapas and G. Grassi, in
Polysaccharide hydrogels: characterization and biomedicalapplications, Pan Stanford publishing, Singapore,2013, inpress.
32 T. Coviello, P. Matricardi, F. Alhaique, R. Farra, G. Tesei,S. Fiorentino, F. Asaro, G. Milcovich and M. Grassi,eXPRESS Polym. Lett., 2013, 7, 733–746.
33 V. D. Skirda, I. Y. Aslanyan, O. E. Philippova, N. S. Karybiantsand A. R. Khokhlov,Macromol. Chem. Phys., 1999, 200, 2152–2159.
34 M. D. Pelta, G. A. Morris, M. J. Stchendroff andS. J. Hammond, Magn. Reson. Chem., 2002, 40, S147–S152.
35 C. S. Johnson Jr, Prog. Nucl. Magn. Reson. Spectrosc., 1999, 34,203–256.
36 T. L. Hwang and A. J. Shaka, J. Magn. Reson., Ser. A, 1995, 112,275–279.
37 B. G. Amsden, Macromolecules, 1998, 31, 8382–8395.38 M. Holz, S. R. Heil and A. Sacco, Phys. Chem. Chem. Phys.,
2000, 2, 4740–4742.39 T. Stait-Gardner, S. A. Willis, N. N. Yadav, G. Zheng and
W. S. Price, Diffusion Fundamentals, 2009, 11, 1–22.40 M. Grassi, G. Grassi, R. Lapasin and I. Colombo,
Understanding drug release and absorption mechanisms: aphysical and mathematical approach, CRC Press, BocaRaton, USA, 2007, pp. 1–627.
41 I. Frisman, D. Seliktar and H. Bianco-Peled, Acta Biomater.,2012, 8, 51–60.
42 P. Alexandridis and T. A. Hatton, Colloids Surf., A, 1995, 96,1–46.
43 L. Gentile, G. De Luca, F. E. Antunes, C. O. Rossi andG. A. Ranieri, Appl. Rheol., 2010, 20(52081), 1–9.
44 L. Davia, G. Grassi, G. Pontrelli, R. Lapasin, D. Perin andM. Grassi, Comput. Biol. Chem., 2009, 33, 33–40.
