ISSN 0959-9428

0959-9428(2010)20:33;1-L

Volume 20 | N

umber 33 | 2010

Journal of Materials C

hemistry

Pages 6817–7044

www.rsc.org/materials Volume 20 | Number 33 | 7 September 2010 | Pages 6817–7044

PAPERS. Sánchez-Salcedo et al.Biopolymer-coated hydroxyapatite foams: a new antidote for heavy metal intoxication

PAPEREva Enz and Jan LagerwallElectrospun microfibres with temperature senstitive iridescence from encapsulated cholesteric liquid crystal

PAPER www.rsc.org/materials | Journal of Materials Chemistry

Biopolymer-coated hydroxyapatite foams: a new antidote for heavy metalintoxication

S. S�anchez-Salcedo,ab M. Vila,ab I. Izquierdo-Barba,ab M. Cicu�endezab and Mar�ıa Vallet-Reg�ı*ab

Received 29th April 2010, Accepted 17th May 2010

DOI: 10.1039/c0jm01260b

Novel 3D-macroporous biopolymer-coated hydroxyapatite foams are potential devices for the

treatment of heavy-metal intoxication by ingestion. These foams are designed to exhibit a fast and

efficient metal ion immobilization into the HA structure in acidic media. The capture process of metal

ions is stable, not releasing any metal ion when the foams are soaked in clean basic media afterwards.

These two steps mimic a digestion process.

1. Introduction

Nowadays, heavy-metal intoxication via ingestion has become

increasingly prominent due to industrial activities which

endanger population health and result in adverse social impacts.

In addition to this, the high amount of accidental or voluntary

uptake of pesticides has led to the requirement of a fast way of

administrating effective systems able to prevent their absorption

by the gastrointestinal tract.

Heavy metal poisoning can provoke serious effects in health

and, currently, there is no treatment via oral administration that

can be used before intestinal absorption or without requiring

hospital admission. The treatment for most heavy metal

poisoning is chelation therapy1–3 and this process may be lengthy

and with a number of side effects. Other available options

(gastric lavage or activated charcoal uptake) can be painful or

unpleasant and normally are administrated combined with

laxatives.

Here we present novel 3D-macroporous biopolymer-coated

hydroxyapatite (HA) foams as new and potential devices for the

treatment of heavy-metal intoxication by ingestion.

As is well known, HA is biocompatible and its crystal structure

is tolerant to many ionic substitutions and complete replacement

of Ca2+ by Ba2+, Sr2+, Cd2+, Pb2+, Zn2+ and Cu2+ is possible.4–7

Although HA foams are well known in tissue engineering and the

use of polymers is very effective to increase their mechanical

properties,8–10 there are only examples of HA applied in heavy

metal intoxication in the form of powders, although not offering

an excellent alternative for such application.11

Herein, ceramic HA has been designed to be macroporous

foams as those systems should improve and increase the ionic

exchange and heavy metal immobilization due to their higher

diffusion and transport-mass.7 Later, they were biopolymer-

coated for decreasing their solubility (normally very high in

acidic media such as the one in the stomach pH ¼ 1.2) to avoid

toxic large amounts of calcium in the digestive tract, disaggre-

gation of HA pieces and to improve their handling.

aDept. Qu�ımica Inorg�anica y Bioinorg�anica, Facultad de Farmacia,Universidad Complutense de Madrid, Plaza de Ram�on y Cajal s/n, 28040Madrid, Spain. E-mail: vallet@farm.ucm.esbCentro de Investigaci�on Biom�edica en Red, Bioingenier�ıa, Biomateriales yNanomedicina, CIBER-BBN, Spain

6956 | J. Mater. Chem., 2010, 20, 6956–6961

The strategy was to produce the foams and to analyze their

stability in acidic/basic conditions simulating both gastric and

intestinal fluids as well as the capability to immobilize different

metal ions when such devices are soaked in polluted fluids,

mimicking a serious intoxication case.

2. Experimental

2.1 Synthesis of macroporous hydroxyapatite foams

These biopolymer-coated 3D-macroporous HA foams have been

synthesized by the sol–gel technique12 including a non-ionic

surfactant, Pluronic F127 (EO106PO70EO106), as macropore

inducer in the accelerated evaporation induced self assembly

(EISA) method.

Aqueous sols were prepared hydrolyzing triethylphosphite

P(OCH2CH3)3 (TIP) and adding it onto an ethanol solution of

the non-ionic surfactant. Four different molar ratios x of

F127:TIP have been tested in order to obtain different macro-

porosity. In all cases, the concentration of surfactant in ethanol

has been always kept constant. After 30 min continuous stirring,

an aqueous 4 M calcium nitrate solution Ca(NO3)2$4H2O was

added. In all cases, the Ca/P ratio is 1.67, which corresponds to

HA phase.13 The mixed sol was stirred for 15 min and subse-

quently was aged at 60 �C for 6 h. After that, all sols were diluted

with ethanol to a molar ratio of 30 to improve the homogeneity

of resulting material and then was maintained at 60 �C during

72 h.

The resulting mixture was transferred into an open Petri dish

and placed in an oven for 1 h at 100 �C to evaporate the solvent

and to obtain the 3D-macroporous foams. Finally, in order to

remove the surfactant and to obtain HA phase the samples were

calcined at 550 �C for 6 h in air atmosphere.

Four different molar ratios x of F127:TIP have been tested in

order to obtain different macroporosity as it was observed by

tailoring the F127/TIP ratio the macroporosity of the resulting

HA foams could be controlled.

2.2 Coating of macroporous hydroxyapatite foams with

biopolymers

Foams were coated with different biocompatible polymers

approved by the US Food and Drug Administration (FDA). In

one case, two different gelatine concentrations in water (1.2 and

This journal is ª The Royal Society of Chemistry 2010

2.4% (w/v)) were prepared and mixed with a solution 0.5% (v/v)

of glutaraldehyde in stirring conditions during 1 h at 20 �C.

Gelatine was, previously, crosslinked with glutaraldehyde to

reduce its solubility in water.14,15 The crosslinking rate was

measured by following an indirect method by complex formation

with 2,4,6-trinitrobenzenesulfonic acid which is described else-

where.16

After that, HA foams were soaked in such biopolymer solu-

tions, extracted and dried at room temperature. In the second

case, the coating was made of 3-polycaprolactone (PCL), the

pieces were soaked in a solution of 5% (w/v) of PCL in

dichloromethane.

2.3 In vitro assays

In vitro tests were carried out in two different media with

different pH: simulated gastric fluid (SGF) and phosphate buffer

solution (PBS: (0.15 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1

mM Na2HPO4, pH 7.4)). SGF was prepared by dissolving 6 g of

NaCl with 2 mL of 12 N HCl until 1 L with pH adjusted to 1.2.17

As a first step, swelling assays and degradation were performed

in both media during 24 h and 7 days, respectively at 37 �C with

vigorous orbital stirring.

The HA stability was determined by the study of calcium

concentration variation in both solutions. Calcium concentra-

tion leached to saline solutions versus time was monitored by

colorimetric analysis based on EASY-Ca-01 method at a wave-

length of 620 nm. For these measurements a SmartChem140

automatic discrete analyzer (Alliance Instruments, AMS France)

was employed. Pb2+, Cd2+ and Cu2+ ions were measured by

inductively coupled plasma/optical emission spectrometry (ICP/

OES) in a Perkin Elmer OPTIMA 3300 DV device.

Swelling behaviour of the HA foams after being biopolymer-

coated was determined by gravimetric (%W) analysis as

follows:18,19

%W(SWELLING) ¼ 100 � (Wt � Wd)/Wd (1)

Where Wd is the weight of dried foam and Wt is the weight of

hydrated foams at time t (0–24 h). For this swelling test the

samples were soaked in SGF and PBS solutions, respectively and

subsequently extracted and gently wiped with absorbent paper

and weighed. The weights of the hydrated samples were

measured along time until the foams reached the swelling ratio

equilibrium.

In vitro Pb2+, Cd2+ and Cu2+ immobilization assays were per-

formed by soaking 10 and 20 mg of the coated-HA pieces in 5 mL

of SGF containing 500 ppm of PbCO3, Cd(NO3)2, Cu(NO3)2

salts, respectively, during 2 h at 37 �C in vigorous orbital stirring

(100 rpm), mimicking the gastric step. The variations of Pb2+,

Cd2+, Cu2+and Ca2+ concentration were evaluated at different

times (10 min, 30 min, 1 h and 2 h). After this treatment, each

sample was soaked in clean PBS solution, which does not contain

any Pb2+, Cd2+ or Cu2+ ions, respectively, during 22 h in vigorous

stirring. This last step pretends to mimic the intestinal step giving

information if the captured metal ions in the gastric step are

lixiviated in pH value of 7.4. Metal ions were measured by

inductively coupled plasma/optical emission spectrometry (ICP/

OES) in a Perkin Elmer OPTIMA 3300 DV device.

This journal is ª The Royal Society of Chemistry 2010

2.4 Characterization

The resulting macroporous HA and coated-HA foams were

characterized by X-ray diffraction (XRD) in a Philips X’Pert

diffractometer using Cu-Ka radiation. To determine the quan-

titative phase composition of the samples, after the digestion

process, the XRD patterns were refined by the Rietveld method

using FullProf software.20 Fourier Transform Infrared spec-

trometry was performed in attenuated total reflectance geometry

(ATR-FTIR; Nicolet Nexus) using a Nicolet GoldenGate device

operating with a single germanium crystal.

Scanning electron microscopy (SEM) was performed in

a JEOL JSM 6335F field emission scanning microscope. Trans-

mission electron microscopy (TEM) studies have been performed

and were carried out with a JEOL 3000 FEG electron microscope

operating at 300 kV (Cs 0.6 mm, resolution 1.7 �A) fitted with

a double tilting goniometer stage (�45�) and with an Oxford

LINK EDS analyzer. TEM images were recorded using a CCD

camera (MultiScan model 794, Gatan, 1024 � 1024 pixels, size

24 mm). Elemental analysis was performed in a Perkin Elmer

2400 CHN and Analyzer Thermogravimetric (TG) analyses were

carried out with a Perkin-Elmer Pyris Diamond TG/DTA

instrument, between 30 and 900 �C in air at a flow rate of

100 ml min�1 and a heating rate of 10 �C min�1. Nitrogen

adsorption studies were performed with a Micromeritics

ASAP2010C system. Hg porosimetry measurements were carried

out in an AutoPore III porosimeter (Micromeritics Instrument

Corporation, Norcross, GA, USA).

3. Results and discussion

As four different molar ratios x of F127:TIP were tested in order

to obtain different macroporosity, it was observed that by

tailoring the F127/TIP ratio the macroporosity of the resulting

HA foams could be controlled. The most homogeneous HA

foam, with the highest pore volume and total porosity of 90%

was obtained for x ¼ 11, being x ¼ 0 and 2.5 the other ratios

tested, giving rise to dense or semi-dense samples, respectively.

The optimized foam exhibits a high volume and size of inter-

connected macroporosity in the range 1–400 mm as can be

observed by Digital Imaging and SEM (Fig. 1a and Fig. 1b

respectively). Its XRD pattern corresponds to pure HA phase

and a porous network with a pore size of 10–15 nm is observed by

TEM (Fig. 1c). This fact could be due to the elimination of the

surfactant during the calcination process.

The higher magnification TEM image and FT diffractogram

shown in Fig. 1c and d evidence the presence of pure HA in both

samples, showing the 002, 112 and 211 reflections of an apatite-

like phase. Selected foams were coated with two different gela-

tine/glutaraldehyde concentrations in water (1.2 and 2.4% (w/v))

(samples named HA1.2G/Glu and HA2.4G/Glu), and with 3-poly-

caprolactone (PCL) giving rise to the HAPCL sample. The

obtained crosslinking rates (gelatine/glutaraldehyde) were 20 and

15% for the samples HA1.2G/Glu and HA2.4G/Glu, respectively.

As shown in Fig. 2, the Hg intrusion porosity analysis, before

and after coating the pieces, shows a slight decrease of total

pore volume in the range of 100–300 mm. The amount of

biopolymer corresponding to coated-HA foams was determined

by thermogravimetric analyses showing a 40% polymer in

J. Mater. Chem., 2010, 20, 6956–6961 | 6957

Fig. 1 (a) Digital photograph. (b and inset) SEM micrograph at

different magnifications. (c and d) TEM studies at different magnifica-

tions. (Insets) XRD patterns and FT diffractogram. All corresponding to

a representative HA foam.

HAPCL and HA2.4G/Glu samples and 20% for the HA1.2G/Glu

sample.

HA foams have similar morphology before and after the

coating process (inset of Fig. 2) and it does not affect the struc-

tural characteristics of the formed HA phase.

To evaluate the performance and chemical stability of these

foams, the in vitro degradation and swelling assays tests were

carried out in two different media with different pH: simulated

gastric fluid (SGF: pH ¼ 1.2) and phosphate buffer solution

(PBS: pH ¼7.4).

The upper figure in Fig. 3 shows the calcium concentration in

the acidic media (SGF) as a function of time directly related to

degradation of the foam. The uncoated HA foam exhibits the

highest HA solubility rate media (dissolving the 100% of the

initial calcium in the foam) compared to biopolymer-coated.

These results are in very good agreement with the solubility rate

Fig. 2 Pore size distribution by Hg intrusion corresponding to HA,

HAPCL, HA1.2G/Glu and HA2.4G/Glu samples. (Inset) SEM micrograph

corresponding to a representative coated-foam.

6958 | J. Mater. Chem., 2010, 20, 6956–6961

in acid medium for the HA material.21 A considerable decrease

in the HA solubility rate is observed in SGF assays as

follows HA1.2G/Glu (58%) ¼ HA2.4G/Glu (63%) > HAPCL(29%),

(percentages compared to the initial calcium present in the foam)

showing for the latter a lower calcium concentration in the

medium. This fact could be a function of the hydrophilicity rate

of each polymer which is higher for gelatin crosslinking

compared to 3-polycaprolactone. Therefore, hydrophilic poly-

mers (such as gelatine) allow a higher accessibility of water

molecules increasing the solubility of the foams.

The same stability studies but in PBS (Fig. 3 lower figure)

showed that HAPCL, HA1.2G/Glu and HA2.4G/Glu samples main-

tain their integrity, even after 7 days of test. Although their

solubility in PBS in lower than in the SGF medium, it also

decreases with the biopolymer coating, but there is no significant

differences between the polymers. From Fig. 3 we can also

calculate the percentages of dissolved calcium in the PBS medium

related to the total calcium of the foam being 0.4% HAPCL and

HA2.4G/Glu and 0.03% for HA1.2G/Glu.

Swelling ratio (%W) studies (see Fig. 4) of HAPCL, HA1.2G/Glu

and HA2.4G/Glu in SGF and PBS, show that HA1.2G/Glu foam

absorbs 200%W of solution more than HA2.4G/Glu which

could be explained in base by gelatin content and the degree of

crosslinking reduction that provoke water uptake into the

sample. On the contrary, HAPCL foam exhibits a contraction in

Fig. 3 Upper figure: Variation of calcium concentration as function of

the time in SGF, and in PBS (lower figure), corresponding to HA foam

samples before and after being coated with the different biopolymers.

This journal is ª The Royal Society of Chemistry 2010

both media. This effect is due to a different hydrophobicity

of PCL polymer compared to gelatin and also could explain

the lower calcium solubility rate in SGF exhibited for this

sample.

After the foams showed ability to resist such acid/basic media,

in vitro metal ion immobilization assays were performed to check

their efficiency as ion capturers. Biopolymer-coated HA foams

were immersed in SGF containing a very high concentration of

lead, cadmium and copper, respectively (representative usual

contaminants) as a simulation case of serious intoxication.

In the case of lead immobilization, as can be seen in Fig. 5, for

the 20 mg samples (125 mm3), results show a very efficient and

fast immobilization (done in 2 h) for all samples. The fact that the

capture is performed also demonstrates the permeability of the

polymers to the ions. Is worth to notice that for sample

HA1.2G/Glu, almost the complete Pb2+ capture is made in the first

10 min. Moreover, although the 10 mg (62.5 mm3) samples

capture efficiently the Pb2+, they do it at a slower rate, so we can

conclude that the immobilization of Pb2+ can be faster with the

weight of the foam as it implies more surface available for the

Fig. 4 Swelling ratio (%W) studies of HAPCL, HA1.2G/Glu and HA2.4G/

Glu in SGF and PBS.

Fig. 5 Upper figure: Variation of Pb2+ and Ca2+ (inset) concentration in

SGF as function of time corresponding to the HAPCL, HA1.2G/Glu and

HA2.4G/Glu 10 mg samples after soaking them in Pb2+ containing SGF

(388 ppm). Lower figure: same results for the 20 mg sample.

This journal is ª The Royal Society of Chemistry 2010

uptaking process. In summary, only 20 mg of HA foam equiv-

alent to a very small prism is enough to immobilize in 10 min

(sample HA1.2G/Glu) the total amount of lead in the polluted SGF

in every case.

From the data, it can be seen that the immobilization rate is

faster for the case of HA1.2G/Glu and HA2.4G/Glu than for the

HAPCL. The different immobilization rate between the samples

coated with different polymers could be explained in base by the

mechanism of lead uptake by HA material.22 In this case, the

Pb2+ immobilization implies a partial-dissolution of HA and re-

precipitation mechanism of a pyromorphite phase,

Pb10(PO4)6(Cl)2, which is much more stable than the HA phase.23

This is in agreement with the stability studies above described for

the cases of HA1.2G/Glu and HA2.4G/Glu foams. Those samples

exhibit higher solubility (related to higher hydrophilicity of the

polymer) and therefore higher immobilization rate of lead. To

confirm such mechanism Ca2+ release has been monitored (see

Fig. 5 (inset)). As expected, it is observed a progressive calcium

release versus incubation time being more acute for HA1.2G/Glu

and HA2.4G/Glu than for HAPCL samples.

Comparing calcium and lead releasing and uptaking values

respectively, in the SGF medium, it is observed that there is

J. Mater. Chem., 2010, 20, 6956–6961 | 6959

a higher rate of calcium dissolution than lead immobilization as

calcium release is not only related to lead capture but to foam

dissolution processes (Fig. 3). After the total lead uptaking of the

solution, this calcium dissolution still continues up to a 2% of the

total initial calcium (Fig. 5).

To confirm the phase changes directly related to lead uptake,

XRD studies of these samples after 2 h in polluted-SGF are

displayed in Fig. 6 (upper Fig.). Diffractograms show the

formation of a new phase coexisting with initial HA phase which

has been identified as pyromorphite,24 which confirms such

immobilization mechanism.

After the polluted-SGF treatment during 2 h such samples

were soaked in clean PBS (pH¼ 7.4) during 22 h (intestinal step).

From colorimetric analysis and ICP measurements respectively,

no calcium or lead release has been observed confirming the lead

immobilization even after 22 h of incubation time at pH equal to

7.4. As can be seen in Fig. 6 (lower Fig.), XRD patterns after PBS

exhibit similar profiles to those after polluted SGF treatment

confirming the permanence of both phases (pyromorphite

and HA).

Performing Rietveld refinement on the XRD patterns of the

foams after the complete process, we obtain a representative

34%wt. of pyromorphite phase and a 66%wt. of HA phase for all

the samples. We have to take into account that from the initial

100% of HA phase there is also an amount of HA dissolved to the

medium without posterior reprecipitation as has been com-

mented before.

Fig. 6 XRD patterns corresponding to the HAPCL, HA1.2G/Glu and

HA2.4G/Glu foams after 2 h in SGF containing Pb2+ (upper) and after

treatment in metal ion free PBS (lower). The identified phases correspond

to HA and (*) pyromorphite.

6960 | J. Mater. Chem., 2010, 20, 6956–6961

These results confirm that in the intestine-like pH, biopolymer-

coated HA foams do not release lead, and thus, should be

evacuated by defecation eliminating the total of the polluting

agent captured.

Moreover, as shown by Hg porosimetry in Fig. 7, after the 2 h

study in Pb2+ contaminated SGF (upper fig.), all coated samples

maintain their initial porous structure and the integrity of the

samples. There are no significant changes in the pore volume and

pore diameter centered at 100 mm. However, a slight increase in

the percentage of porosity at values of lower than 1 mm is

observed which could be attributed to partial-dissolution of the

foam-like structure. In addition, after PBS treatment during 22 h

there is a slight decrease of total pore volume and pore diameter.

This fact is very pronounced for the case of HAPCL sample and it

could be related to the contraction behaviour of this sample in

PBS (Fig. 4). It is important to point out that in all the cases a c.a.

70% of the macroporous structure of biopolymer coated HA

foams is maintained along the in vitro digestive process as can be

seen in the inset of Fig. 7. Similar values were obtained for

HA-biopolymer foams in Cd2+ and Cu2+ immobilization assays.

In the case of cadmium and copper a very fast and effective

immobilization of these ions takes place, capturing c.a. 57–60%

of Cd2+ and 62–65% of Cu2+ from polluted SGF after only

10 min. There is not significant variation in this value even when

the incubation time is increased (see Table 1). The different

removal efficiency of Pb2+, Cd2+ and Cu2+ responds basically to

Fig. 7 Upper figure: Pore size distribution by Hg intrusion corre-

sponding to HAPCL, HA1.2G/Glu and HA2.4G/Glu samples after 2 h in

Pb2+containing SGF, and after posterior treatment 22 h in PBS (lower

figure). Inset: representative foam after the simulated digestive process.

This journal is ª The Royal Society of Chemistry 2010

Table 1 Immobilization capability of Cd2+ and Cu2+ metal ions corre-sponding to biopolymer coated HA foam after 10 min and 2 h in SGFcontaining high concentration of metal ions, respectivelya

Samples % Cd2+ % Cu2+

HAPCL (10 min) 60.0 � 0.3 65.6 � 0.2HA1.2G/Glu (10 min) 60.0 � 0.3 63.0 � 0.2HA2.4G/Glu (10 min) 57.0 � 0.3 62.3 � 0.2HAPCL (2 h) 63.0 � 0.3 62.6 � 0.2HA1.2G/Glu (2 h) 67.0 � 0.3 66.4 � 0.2HA2.4G/Glu (2 h) 62.0 � 0.3 61.8 � 0.2

a In all cases 20 mg of each piece have been used for the immobilizationtest.

the pH of the medium, in acid conditions the Pb2+ immobiliza-

tion is facilitated more than that of Cd2+ or Cu2+ ions.25 In these

particular cases, the capture mechanism by HA is different

compared with lead capture, producing in these cases an ion-

exchange mechanism.26,27 As well as in lead immobilization

assays there is an increase in the calcium release after polluted

SGF incubation and no release of metal ions during the PBS

soaking step, confirming again for these ions the immobilization

process.

4. Conclusions

In summary, biopolymer-coated HA foams have shown a fast

and high efficiency for capturing Pb2+, Cd2+ and Cu2+ ions in

acidic media not releasing any metal ions when the samples are

soaked in clean basic media. Both steps mimic the digestion

process. All the processes are carried out without any disinte-

gration of the foam. These systems are here proposed as very

efficient antidotes against metal ion intoxication before intestinal

absorption and hospital treatment.

Acknowledgements

This work has been financially supported by the Spanish CICYT

through project MAT-2008-00736, Spanish National CAM

project S2009/MAT-172 and the Marie Curie FP7-PEOPLE-

2007-2-2-ERG.

This journal is ª The Royal Society of Chemistry 2010

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