Institute of Technical Sciences of SASA, Knez Mihailova 35/4, 11000 Belgrade, SerbiaDepartment of Advanced Materials, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, SloveniaDepartment of Radiolabels and Radiopharmaceuticals, Institute of Nuclear Sciences “Vinca”, Mike Petrovica-Alasa 12-14, 11000 Belgrade, SerbiaDivision of Biomaterials and Bioengineering, University of California, San Francisco, CA 94143, USA
r t i c l e i n f o
rticle history:eceived 15 June 2010eceived in revised form5 September 2010ccepted 15 September 2010vailable online 24 September 2010
a b s t r a c t
Biodegradable poly(d,l-lactide-co-glycolide) (PLGA) and bioactive hydroxyapatite (HAp) are selected forthe formation of a multifunctional system with the specific core-shell structure to be applied as a carrierof a drug. As a result, both components of PLGA/HAp core-shells are able to capture one part of the drug.Polymeric shells consisting of small nanospheres up to 20 nm in size act as a matrix in which one partof the drug is dispersed. In the same time, ceramic cores are formed of rod-like hydroxyapatite particlesat the surface of which another part of the drug is adsorbed onto the interface between the polymer
and the ceramics. The content of the loaded drug, as well as the selected solvent/non-solvent system,have a crucial influence on the resulting PLGA/HAp morphology and, finally, unimodal distribution ofcore-shells is obtained. The redistribution of the drug between the organic and inorganic parts of thematerial is expected to provide an interesting contribution to the kinetics of the drug release resulting
The formation of multi-component systems for biomedicalpplication enables more efficient tailoring of the functionality ofhese materials. Basic bifunctional drug delivery devices are usuallyormed with the purpose of ensuring two functions: slow releasef the drug and elimination of the matrix from the organism afterhe delivery of the drug is completed [1]. The formation of multi-unctional devices by combining different functions within a singlearrier (degradation, slow release of the drug, elimination of therug carrier and regeneration of the place at which the implantas placed, for example) enable the creation of a new type of drug
elivery systems whose local application can be highly effective invercoming problems of some medicaments related to low avail-bility and unwanted side effects [2].
There are two critical features in the formation of these multi-functional implants: a proper choice of the implant materials, i.e.,the drug carriers, and a proper choice of the structure of particlesthat comprise the material.
As far as the structure is concerned, it has been shown thatsimultaneous control of the shape, size and surface structureenables possibility to create multifunctional particles for a varietyof biological applications, such as theranostics, diagnostic assaysand drug delivery [3]. In recent years, there has been an increas-ing interest in the formation and practical application of core-shellstructures, which are applied for different purposes: luminescencelabelling, manipulation by magnetic field [4], as a nano-reactor fortargeted drug delivery of specific enzymes, antigens, cells, genes,peptides and pharmaceutical agents [2,5–7]. The main reason fortheir application in medication is the possibility to ensure thoroughcontrol over the healing process. Regular morphology is especiallyimportant for drug delivery systems: a slower rate of drug releaseand the reduction of the initial burst effect were detected after theapplication of particulate drug carriers with narrower particle size
distributions [8]. Nanocarrier-based drug delivery strategies havebeen intensively studied and it have been shown that they havehigh potential for site-specific medication [9].
In terms of the material type, various materials including syn-thetic or natural polymers were applied for the formation of the
hell of core-shell structures, with an empty core providing thepace for the incorporation of a drug (in the case of hollow cap-ules) [2] or a core filled with some organic or inorganic drug carrierr drug itself. In general, the formation of an efficient material foriological application requires features like degradability, low toxi-ity and cell specificity [7] and many different materials: polymers,ioceramics and composites thereof have been applied as poten-ial drug delivery carriers [10]. The main advantage of polymersn comparison to other materials rests in the possibility to obtain a
ide range of topological, chemical and architectural modificationsy the application of different processing techniques or throughombination with other materials, which is a crucial advantagen meeting ever increasing demands concerning the new designf drug delivery devices [7,11]. The benefits of the application ofiodegradable polymers in comparison to non-biodegradable ones7,11] related to the encapsulation efficiency, foreign body reactionnd controllable kinetics of drug release are already well-known.oly(d,l-lactide-co-glycolide) (PLGA) is a biodegradable polymerith non-toxic degradation products that are easily removed from
he organism by natural metabolite pathways; it has a controllableegradation rate approved by the US Food and Drug Administrationor Commercial Application and is already applied for the encapsu-ation of numerous biologically active substances [12–17]. HAp haslso been extensively investigated as a possible drug delivery vehi-le [18–21]. Its compatibility with the composition of natural bone,apacity to promote and stimulate the regeneration of bone tissueue to bioactivity and the ability to be resorbed at the implanta-ion site during hard tissue repair, make it a natural candidate forcarrier of drugs against bone infections [19,22].
The formation of the desired structure is strongly dependentn the selection of the processing technique [23]. Double emulsionethods are usually applied for the encapsulation of water-soluble
ubstances [24,25]. It has been shown that ultrasonication dur-ng encapsulation improves both the loading efficiency and theesulting morphology [26–28]. The amplitude, intensity, frequencynd power of ultrasound can be modified so as to control thegitation-induced homogenization of the composite phases at thene scale and can take part in the formation of unusual structures29,30].
The morphology and topology of organic–inorganic core-shellaterials should be precisely controlled in order to promote desir-
ble applications [31]. According to our best knowledge there is noeported data in which the concept of a PLGA/HAp multifunctionalaterial in the form of core-shell structures is considered as a drug
arrier. In this work, we applied PLGA and HAp in order to formmultifunctional drug delivery carrier in the form of a core-shell
tructure with loaded clindamycin within both the polymeric shellnd the ceramic core. The morphological regularity is especiallymphasized because it is expected to have an important influencen the kinetics of the drug release and interaction between theurface of the material and cells.
. Materials and methods
.1. Materials
Calcium nitrate pentahydrate (Ca(NO3)2·5H2O (Sigma, Aldrich,ermany)), ammonium-dihydrogen phosphate (NH4H2PO4 (Sigmaldrich, Germany)), and urea ((NH2)2CO (Alfa, Aesar, Germany))ere used for the synthesis of HAp. PLGA (mol. wt 45,000–75,000)
ith co-monomer ratio 50:50 (Sigma Aldrich, Germany) andoly(vinyl-pyrrolidone) (PVP) (Sigma Aldrich, Germany) were used
n the preparation procedure. Clindamycin (CHEPHASSAR GmbH,t. Ingbert, Germany) was applied in the loading process. Theolvents/non-solvents applied were ethanol, isopropanol and ace-
s B: Biointerfaces 82 (2011) 404–413 405
tone (Sigma Aldrich, Germany). All chemicals and reagents were ofanalytical grade.
2.2. Formation of PLGA/HAp with loaded clindamycin
Clindamycin-containing PLGA/HAp core-shell nanostructureswere prepared by an optimization of our previously reportedmethod for the production of PLGA/HAp particles using ultra-sonic processing [29]. In the first step, HAp was synthesized bya previously reported homogeneous sonochemical precipitationmethod [32]. Briefly, precipitation was performed from dilute solu-tions of Ca and P precursors which were mixed and heated to88 ◦C. After heating, an aqueous solution of urea was added, whichwas followed by sonication under the following conditions: thetemperature of the medium Tmax = 90 ◦C, ultrasonic field powerP = 600 W, frequency of the ultrasonic field f = 20 kHz and effectivetime of the pulsed sonication t = 3 h with on:off = 02:01 s periodsof pulsation and relaxation. The obtained precipitate was aged for12 h and separated from the supernatant by centrifugation during1 h at 4000 rpm. Sonochemically obtained HAp was additionallyprocessed in a high intensity ultrasonic field using the same exper-imental parameters as in the preceding HAp synthesis (P = 600 W,f = 20 kHz and on:off = 02:01) but with a smaller volume of theliquid medium (10 ml of ethanol). The ultrasonication time was10 min after which the pallet was separated by centrifugation at4000 rpm for 1 h. In the third step, PLGA was dissolved in acetoneor acetonitrile, which were applied as polymeric solvents. Eachsolution was cooled to the temperature of 8 ◦C, after which thewater solution of clindamycin (in two forms: active clindamycin-base and proactive clindamycin-2-phosphate) was added to thePLGA solution dropwise with a constant mixing at 1200 rpm. Inthe case of PLGA solution in acetonitrile, three different concen-trations of clindamycin in water were applied (c = 38.4, 19.2 and9.6 mg ml−1). In all other cases, the highest concentration of clin-damycin in water (c = 38.4 mg ml−1) was used. In the fourth step,the PLGA/HAp composite material, with 90:10% (w/w) polymer-to-ceramic part ratio, was processed according to the modifiedprocedure described in Jevtic et al. [29] with the application ofdifferent polymeric solvents (acetone/acetonitrile) and polymericnon-solvents (ethanol/isopropanol/water applied), and an addi-tional step which allowed for the loading of clindamycin. SolidHAp was added to the mixture of PLGA and clindamycin solutionsand re-dispersed using ultrasonic field with the following parame-ters: ultrasonic field power P = 142.5 W, frequency of the ultrasonicfield f = 20 kHz and duration of the ultrasonic pulsing t = 2 min withon:off = 01:04 s of the pulsing and relaxation periods. After dis-persing HAp within the PLGA/clindamycin mixture, a polymericnon-solvent (organic or water) was added to initiate the precipita-tion of the polymer and formation of the PLGA/HAp composite withloaded clindamycin. The milky PLGA/HAp/clindamycin dispersionwas added to a large volume of PVP solution (200 ml, c = 20 mg ml−1
in water) to stabilize the surface of the formed drops before theirsolidification into particles, centrifuged at 4000 rpm during 2 h tospin down the precipitate and air dried. The same experimentalprocedure was additionally applied in order to load clindamycinwithin PLGA or HAp separately. All experiments were performedby an Ultrasonic Processor for High Volume Applications VCX 750,Newtown, Connnecticut, USA.
2.3. Characterization methods
2.3.1. High performance liquid chromatography (HPLC)HPLC analyses were performed on supernatants obtained after
the precipitation processes using a Hewlett Packard 1050 Liq-uid Chromatograph (consisting of a quaternary Pump System-HP 1050, HP 1050 Autosampler, Agilient ChemStation and HP
4 urface
VHrOot�aflt
2
Zcwtmlwwfst(
2
sAepsvfiMur
3
3o
ioppfiopdscobsmrItoc
06 M. Vukomanovic et al. / Colloids and S
ectra XA system controller). The system was equipped withP1050 UV-Vis detectors. The separation was carried out on a
eversed phase column C18 (250 mm × 4.6 mm × 5 mm), NUCLE-SIL 100-5-C18 (Macherey–Nagel). The elution solvent consistedf acetonitrile and water (60:40 v/v, flow rate was 0.7 ml min−1 andhe injection volume was 20 �l). The monitor wavelength was set atmax(ε) = 210 nm and the chromatographic analysis was operatedt 25 ◦C. The data were acquired and processed using HP softwareor HP 1050 HPLC Modules. All measurements were repeated ateast three times and presented data are the mean values of at leasthree repetition integrations.
.3.2. Field emission scanning electron microscopy (FESEM)Morphological analysis was performed using a SUPRA 35 VP Carl
eiss field emission scanning electron microscope. Sputtering witharbon was applied to prevent charging. A stereological analysisas carried out to determine the morphological parameters of par-
icles on the basis of the 2D projection images obtained from FESEMicrographs. The area analysis was performed using an image ana-
yzer (Image Tool Win 3.0 Software). Morphological parametersere determined on 550–700 particles. The following parametersere determined using particle projections from FESEM images:
eret diameter, area section, roundness and elongation. A furthertructural analysis based on exploring the individual nanostruc-ures was performed by transmission electron microscopy (TEM)JEM-2010F).
.3.3. X-ray photoelectron spectroscopy (XPS)Surface and profile analyses were carried out on the PHI-TFA XPS
pectrometer exciting a sample’s surface by X-ray radiation from anl-monochromatic source. The survey and narrow scan spectra ofmitted photoelectrons were taken with 187 eV and 29 eV. Depthrofile analyses were performed by sequential sputtering of theample and removing the layers with an argon ion beam (sputteringelocity ∼1.7 nm min−1). XPS analyses were performed after everyve sputtering cycles (5-min duration). The data were treated byultipak program, version 8.1. The sensitivity of applied method is
p to 0.05 at.%. All data are obtained by averaging of at least threeepeated integrations of the peaks.
. Results
.1. Ultrasonic processing of PLGA/HAp/clindamycin structuresbtained by organic non-solvent of polymer
In the first stage of the evaluation of the process-ng and loading of clindamycin within PLGA/HAp, anrganic/water/solid/organic/water dispersion method accom-anied with ultrasonic processing was applied (Fig. 1). In ourrevious study, we had applied ultrasonic processing for theormation of PLGA/HAp (90:10%, w/w) particles, showed thenfluence of different processing parameters on the morphologyf the particles and came up with the optimal conditions for thereparation of well-shaped, spherical particles with the meaniameter of circa 300 nm [29]. These conditions were primarilyelected for the preparation of PLGA/HAp particles loaded withlindamycin. However, the morphology of the PLGA/HAp particlesbtained using this procedure drastically changed after they hadeen loaded with clindamycin. The obtained particles were stillpherical, though irregular in shape, and had a few dozen oficrometers in size (Fig. 1a). On the surface of these spheres,
od-like particles of clindamycin were observed (insert in Fig. 1a).n the described procedure, acetone and ethanol were selected ashe solvent/non-solvent system. Having in mind the low solubilityf clindamycin in acetone and its insolubility in ethanol [33,34], itan be expected that when ethanol was added to precipitate the
s B: Biointerfaces 82 (2011) 404–413
polymer, it also brought about the precipitation of clindamycin. Asa result, an amount of clindamycin became attached to the surfaceof PLGA/Hap, while the rest remained as a separate phase.
In order to improve the resulting morphology, the influ-ence of different solvent/non-solvent systems on the mor-phological properties of the PLGA/HAp/clindamycin particlesobtained by organic/water/solid/organic/water dispersion methodwas further analysed by the application of different organicsolvent/non-solvent systems, including acetone/isopropanol, ace-tone/(ethanol:water = 50:50%, v/v), acetonitrile/isopropanol andacetonitrile/(ethanol:water = 50:50%, v/v).
It can be noticed that in all cases when acetone (as agood solvent of PLGA and poor solvent of clindamycin) wasapplied as a polymeric solvent, the obtained morphology of thePLGA/HAp/clindamycin structures was similar: irregular spheres,few dozen of micrometers in size (Fig. 1b and c). The particle sizeof clindamycin attached to the polymer surface was reduced byincreasing its solubility in the polymeric non-solvent. For example,the micrometer-sized rod-like particles observed on the surface ofPLGA/HAp spheres obtained using ethanol (insert in Fig. 1a) werereduced to only a few hundreds of nanometers in size when iso-propanol was used (insert in Fig. 1b). Eventually these particleswere not detected at all on the surface of the structures obtainedusing dilute ethanol (insert in Fig. 1c) as an organic polymeric non-solvent. As for ethanol, it has been shown that the solubility ofclindamycin increases in binary water–ethanol solvent mixtureswith the increase of the water content [34]. These large sphereswere formed of smaller submicron spheres, which can be clearlyobserved when dilute ethanol was applied as a polymeric non-solvent (insert in Fig. 1c). When the solubility of clindamycin in theorganic polymeric non-solvent decreased, the morphology of thesesubmicron spheres was affected and they tended to agglomerate,undergo ripening and form larger spheres.
When acetone was replaced with acetonitrile as the polymericsolvent (implying an increase in the solubility of both the poly-mer and clindamycin), the formation of the large spheres wasnot observed (Fig. 1d and e). When acetonitrile, as a good sol-vent for PLGA and clindamycin, was applied with isopropanol asan organic polymeric non-solvent, only small spherical particleswere observed, but their morphology was affected due to the lowsolubility of clindamycin in this solvent. In addition, the particleswere partially agglomerated and ripened (insert in Fig. 1d). Finally,when acetonitrile, as a good solvent for PLGA and clindamycin, anddilute ethanol, as a good solvent for clindamycin, were applied, avery regular morphology of PLGA/HAp/clindamycin spherical parti-cles, without any sign of agglomeration and ripening was observed(Fig. 1e). However, the resulting particle size distribution was notnarrow, and among small spherical particles with about 200 nmmean diameter (insert in Fig. 1e), larger ones with mean diameterof about 1 �m were observed too (Fig. 1e).
3.2. Ultrasonic processing of PLGA/HAp/clindamycin structuresobtained using water as a polymeric non-solvent
In a further stage of research, the organic/water/solid/water/water ultrasonic method was applied and the organic poly-meric non-solvent was replaced with water (a very good solventfor clindamycin). When the only organic solvent was acetone, agood solvent for PLGA and poor for clindamycin, the morphologywas similar to that obtained in the previously analysed method,that is, large sphere-like agglomerates made of smaller partially
ripened spheres (Fig. 1f). When a binary mixture of acetone andacetonitrile (50:50%, v/v) was applied as the polymeric solvent thesolubility of clindamycin increased and the obtained morphologywas similar to that obtained in the previously described case whenacetonitrile/dilute ethanol was applied as solvent/non-solvent sys-
M. Vukomanovic et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 404–413 407
F ol (a)( etonen nanos
tF
ttit(t
ig. 1. PLGA/HAp/clindamycin obtained by ultrasonic method using: acetone/ethand) and acetonitrile/(ethanol:water) (e) as solvent/organic non-solvent systems; acon-solvent systems with a planar spatial organization of sphere-like particles and
em. Along with small and uniform spherical particles (insert inig. 1g), larger ones were formed, too (Fig. 1g).
Finally, when the solubility of both clindamycin and PLGA inhe polymeric solvent, as well as the solubility of clindamycin in
he polymeric non-solvent increased, small and uniform spher-cal structures were observed (Fig. 1h and i). It was noticedhat smaller particles formed in acetonitrile/(ethanol:water),acetonitrile:acetone)/water and all the particles formed in acetoni-rile/water tended to form planar spatial organization (Fig. 1h and
, acetone/isopropanol (b) and acetone/(ethanol:water) (c); acetonitrile/isopropanol/water (f), (acetone:acetonitlile)/water (g) and acetonitrile/water as solvent/watertructured morphology (h–j).
i). These plate-like structures displayed a very regular arrangementof sphere-like particles on their surfaces, while in their interiorparticles were randomly organized and partially agglomerated.
It should be mentioned that these small, submicrometre-sized
sphere-like structures did not have smooth surfaces, they werecomposed of sphere-like particles with less than 20 nm in size(Fig. 1j), and their structure was comparable to the structure oflarger micrometer spheres which were mentioned earlier with asingle crucial difference related to drastically reduced sizes. The for-
408 M. Vukomanovic et al. / Colloids and Surface
Table 1Loading data obtained according to HPLC results.
a m0 – quantity of initially added clindamycin before loading.b m – quantity of loaded clindamycin.c ı – loading efficiency.
ation of such sphere-like structures consisting of spheres of verymall dimensions can be brought into relationship with a much bet-er solubility of PLGA in acetonitrile in comparison to its solubilityn acetone, which were applied as polymeric solvents.
.3. Analysis of the loading data in the system with the mostegular morphology
Having in mind that the optimal morphology, in terms of smallnd uniform sphere-like structures, was obtained with acetonitriles the polymeric solvent and water as the polymeric non-solventnd the initiator of the precipitation, this system was further thor-ughly analysed. The chromatograms of clindamycin in water, theupernatant obtained after the processing of PLGA/HAp withoutlindamycin, and the supernatants obtained after loading clin-amycin within PLGA, HAp and the PLGA/HAp (90:10%, w/w)omposite allowed for an insight into the efficiency of the load-ng process. The loading efficiencies were 30%, 35% and 15% for theLGA/HAp particles comprising c = 10, 5 and 1 wt% of clindamycin,espectively (Table 1). After the concentration of clindamycin inhe supernatants obtained after the processing of PLGA and HApith clindamycin separately had been determined, it was obvious
rom the results that each of the components of PLGA/HAp had theapacity to retain an amount of the drug. It was calculated that
pproximately c = 41 wt% of the initially introduced clindamycinas retained within PLGA, while about c = 28 wt% was kept byAp (Table 1). These data show that HAp as the solid phase inpplied dispersion process has the capacity to adsorb one part oflindamycin from the organic/water liquid medium.
Fig. 2. Morphology of HAp/clindamycin (a, b)
s B: Biointerfaces 82 (2011) 404–413
3.4. Morphology of PLGA/clindamycin and HAp/clindamycinparticles
The morphology of PLGA and HAp particles processed withclindamycin separately was also analysed. FESEM micrographsin Fig. 2 represent the morphology of the HAp (Fig. 2a and b)and PLGA (Fig. 2c and d) particles processed with clindamycin inthe acetonitrile/water system. The morphology of HAp particleswith clindamycin was rod-like and the rods formed thin plate-like structures similar to those previously observed [32]. However,in contrast to the micrometre length of the plate-like structuresobtained after the homogeneous precipitation method, the addi-tional ultrasonic treatment in a small volume of an inert mediumand processing with clindamycin resulted in significant reductionof the size of these plate-like structures, which is in agreement withthe well-known relationship between the morphological influenceof ultrasonic field and geometry, medium and processing parame-ters applied during the process of de-agglomeration and breakingdown of larger particles into smaller ones [35–37]. On the otherside, the morphology of the PLGA particles processed with clin-damycin was spherical with the particle size in the range between100 and 300 nm. In that sense, they exhibited a similar morphologyto that of the PLGA/HAp particles with c = 10 wt% of clindamycin.
3.5. Morphology of PLGA/HAp/clindamycin particles withdifferent drug contents
The morphology of PLGA/HAp structures obtained using ace-tonitrile as the polymeric solvent and water as the polymericnon-solvent after different concentrations of clindamycin had beenloaded was also analysed. It was noticed that clindamycin hadan additional effect on the morphology of PLGA/HAp. An obviousevidence for this comes from the comparison of the parametersobtained after the stereological analysis applied with the maingoal of quantifying the extent of the morphological change. Fig. 3shows the distributions of feret diameters and roundness stere-
ological factors of composite particles with different content ofclindamycin. The mean values of these parameters as well asthe mean values of the area section and elongation, along withtheir standard deviations, are summarized in Table 2. It can benoticed that the increase of the content of clindamycin within
and PLGA/clindamycin particles (c, d).
M. Vukomanovic et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 404–413 409
F rticles(
ttttmtao(3hmHPtctt
TMd
ig. 3. Morphology, distributions of feret diameter and roundness of PLGA/HAp pa�) relative and (�) cumulative distributions.
he PLGA/HAp composite affects the distribution of feret diame-ers of the obtained sphere-like particles in such a manner thathey become narrower when there is more clindamycin withinhe composite. Accordingly, clindamycin may play the role of a
orphological stabilization agent which affects the surface interac-ions and prevents the agglomeration of small sphere-like particlesnd their ingrowths into the larger spheres. This is most obvi-usly pronounced in the case of the smallest content of clindamycinc = 1 wt%) when the mean value of the obtained particles was about00 nm and the feret diameter distribution was very broad. Forigher contents of clindamycin (c = 5 and 10 wt%), the obtainedean values of the feret diameter were similar (160 and 190 nm).owever, the distribution of the feret diameters belonging toLGA/HAp with c = 10 wt% of clindamycin was unimodal, whereas
he same distribution of the PLGA/HAp particles with c = 5 wt% oflindamycin was bimodal with one fraction of particles shifted tohe higher values of feret diameters. The mean values for area sec-ion indicate a trend similar to that of feret diameters. While the
able 2ean values of stereological parameters of PLGA/HAp/clindamycin particles with
ifferent contents of antibiotic.
Stereological parameters 10 wt% a 5 wt% a 1 wt% a
Mean value of feret diameter [nm] 160 ± 50 190 ± 50 300 ± 20Mean value of area [�m2] 0.02 ± 0.01 0.03 ± 0.02 1.14 ± 0.15Mean value of roundness 0.89 ± 0.05 0.89 ± 0.03 0.89 ± 0.21Mean value of elongation 1.10 ± 0.10 1.09 ± 0.07 0.99 ± 0.65
a Concentrations of clindamycin within PLGA/HAp.
with: 1% (a)–(c), 5% (d)–(f) and 10% (g)–(i) of clindamycin, respectively; markers:
mean values of the area section of particles with c = 5 and 10 wt% ofclindamycin were similar, they were markedly increased for parti-cles with c = 1 wt% of clindamycin. On the other hand, the roundnessand the elongation factors of particles were the same or highlysimilar for all analysed samples. The distributions of the round-ness were slightly different, indicating the narrowing of the particleroundness with the increase of the clindamycin content.
The morphology of PLGA/HAp with 10 wt% of clindamycin wasfurther investigated by a TEM analysis (Fig. 4). Fig. 4a presents rod-like HAp particles within a PLGA matrix. Additionally, crystallinedomains of HAp within amorphous PLGA were detected close to theedge of the spheres (Fig. 4b) and in their bulk (Fig. 4c). Finally, amor-phous PLGA layers were detected on the surface of these structureswith the presence of crystalline entities inside them, indicating theformation of core-shell structures. The identification of these struc-tures could not show the exact place at which clindamycin waspresent: within the polymeric matrix or on the interface betweenthe polymer and the ceramics. For that reason, a further, morespecific, analysis was carried out.
3.6. Surface and profile chemistry
In the surface spectra of HAp with and without clindamycin
(Fig. 5a), the signals of calcium, phosphorus and oxygen weredetected together with the signal of carbon that corresponds tothe carbonates present in sonochemically obtained HAp [32] withurea as the homogeneous precipitation agent. On the basis of thepresence of the nitrogen (400 eV) and chlorine signals (272.5 eV
410 M. Vukomanovic et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 404–413
F e HApt
aaatHoCu
F(P
ig. 4. TEM micrographs of PLGA/HAp core-shells with 10% of clindamycin: rod-likhe amorphous PLGA shell.
nd 200.7 eV) in the spectrum of the surface of HAp/clindamycin,s well as to the different Ca/C ratio on the surface of HAp withnd without the drug, the presence of clindamycin adsorbed onto
he surface of HAp was confirmed. The profile spectra of HAp andAp/clindamycin showed the same elemental compositions with-ut nitrogen and chlorine signals (Fig. 5b and c). Different Ca/P anda/O ratios of the surface of these materials and their similar val-es along their profiles were in accordance with the last statement.
ig. 5. XPS spectra of the surface of HAp and HAp/clindamicin (a); depth profiles of: HAp (a); O 1s (b) and C1s (c) high-resolution spectra of PLGA/HAp/clindamycin; C1s higLGA/HAp/clindamycin (f).
core (a) and its crystalline domains near the surface (b) and inside the bulk (c) of
A similar analysis was applied for the detection of the absorptionof biphosphonate pamidronate onto the surface of plasma sprayedhydroxyapatite coatings [38].
The surface XPS spectrum of PLGA/HAp/clindamycin showedsignals of oxygen (39 at.%) and carbon (61 at.%) (Fig. 5d) that corre-spond to the polymeric phase of the composite. The signals in theO1s spectrum of PLGA/HAp/clindamycin (Fig. 5e) showed oxygenwithin C–O–C and O C groups, respectively, while signals in the C1s
b) and HAp/clindamycin (c); XPS spectrum of the surface of PLGA/HAp/clindamycinh-resolution spectrum of PLGA/HAp (d); depth profiles of PLGA/HAp (e) and
urface
ss[iwtaowePblm
4
cmdcsionw
omibPnsmoaacoowctiisiptsfrwsmttcosm
s
M. Vukomanovic et al. / Colloids and S
pectra of PLGA/HAp with (Fig. 5f) and without (Fig. 5g) the drughowed carbon within C–C/C–H, C*–O–C O and O–C O groups39]. The envelope contents show that the carboxylic carbon wasncreased, the content of the neighbouring carbon was decreased,
hereas the content of aliphatic carbon remained unchanged inhe composite with clindamycin. The spectra of PLGA/HAp withnd without clindamycin (Fig. 5h and j), show the increasing trendf C/O and Ca/P ratios along the profiles of these materials, whichas more pronounced in the samples loaded with drug. The pres-
nce of nitrogen was observed in all spectra along the profile ofLGA/HAp/clindamycin, indicating the presence of the antibioticoth near the surface and within the bulk of the material. Simi-
ar observations based on the presence of the nitrogen signal wereade when paclitaxel was loaded within PLGA particles [40].
. Discussion
Ultrasonic processing method was evaluated for the loading oflindamycin within the PLGA/HAp (90:10%, w/w) multifunctionalaterial in the form of core-shell nanospheres and the influence of
ifferent polymeric solvents and non-solvents on the morphologi-al properties of the obtained structures was analysed. It has beenhown that the solubility of each component of this material dur-ng the processing stage has a marked influence on the propertiesf the final product. The selection of the non-solvent in the combi-ation with the proper solvent influenced the loading process, asell as the size and shape uniformity of the obtained structures.
In terms of the size of the resulting structures, the increasef the solubility of both PLGA and clindamycin within the poly-eric solvent and the increase of the solubility of clindamycin
n the polymeric non-solvent (replacement of organic solventy water) resulted in the reduction of the diameter of theLGA/HAp/clindamycin particles from few dozen micrometers toanostructured submicron spheres. When the polymer was highlyoluble in a selected solvent, it was difficult to initiate the replace-ent of the molecules of the polymeric solvent with the molecules
f water, as a polymeric non-solvent, around the precursor particlesnd solutes even when the non-solvent was added in a twice largermount. The simplest indication of this was a significantly longerentrifugation time required to completely precipitate PLGA andbtain a clear supernatant above the precipitate after the additionf a non-solvent to an acetonitrile solution of PLGA in comparisonith the solution in acetone. For that reason, in this case, the pre-
ipitation of the polymer is a slow process and the primary particleshus obtained are rather small with submicron diameters. Havingn mind the dynamic environment in which the ultrasonic process-ng is carried out, i.e. intensive mixing and homogenization, thelow formation of these particles decreases the probability for theirntensive collisions during the ultrasonic dispersion. These primaryarticles tend to form submicron spheres owing to their tendencyo lower the surface energy through aggregation; albeit that, theytill exist as individual entities. The obtained morphological uni-ormity may be brought into relationship with the homogeneityesulting from this rather slow precipitation process. In contrast,hen the solubility of the polymer in a selected solvent and non-
olvent (water or organic) is decreased, its precipitation proceedsuch faster, the primary particles are larger and their concentra-
ion is significantly increased, indicating that the probability ofheir collision is high. For that reason, these particles are in constantollision during ultrasonic mixing, which results in the formation
f large and irregular, secondary spherical aggregates. Due to inten-ive collisions, these particles undergo ripening and lead to larger,icrometre-sized secondary particles.The solubility of these components in the selected solvent/non-
olvent (organic/organic or organic/water) had an additional effect
s B: Biointerfaces 82 (2011) 404–413 411
on the loading efficiency of the antibiotic within the PLGA/HApstructures. The increase in the solubility of clindamycin resultedin the size reduction of the particles attached to the surface ofthe PLGA/HAp spheres. A further increase in the solubility of clin-damycin allowed loading these particles within PLGA/HAp withouttheir attaching to PLGA/HAp surface. When the solubility of clin-damycin in a polymeric solvent was poor, the additional decreasingof its solubility through the selection of an adequate organic non-solvent and the combination with a rapid increase of the viscosity ofthe system obtained by rapid precipitation of the polymer resultedin a simultaneous precipitation of clindamycin and the polymer.For that reason, an amount of clindamycin remained outside thecomposite spheres, attached to their surface. An opposite situa-tion was noticed when the solubility of the polymer was high andits precipitation slow (organic/water system). Having in mind thatthe solubility of clindamycin in these solvents was good and thechange in of the viscosity initiated by the precipitation of the poly-mer was slower, clindamycin was retained in the solution withoutprecipitating and it became loaded within the core-shell particlesonly by physical capturing from the solution in the moment of theirformation. In that case, the attachment of clindamycin to the sur-face of PLGA/HAp spheres was not observed and clindamycin wascompletely loaded into the PLGA/HAp core-shells.
The summary of the discussed results with respect to the influ-ence of solubility on the morphological and loading properties ofPLGA/HAp/clindamycin is represented in Fig. 6. The outlined factsmay be important in the application of this method for the encap-sulation or immobilization of this or any other substance within aPLGA/HAp multifunctional material applied as a drug carrier.
The most regular morphological properties of PLGA/HApcore-shells loaded with the antibiotic and the most significantreduction of the particle size were obtained using acetoni-trile/water as solvent/non-solvent (organic/water) system. Fig. 7represents a hypothesized mechanism of the formation of core-shell nanospheres of PLGA/HAp loaded with clindamycin in casewhen the antibiotic is highly soluble in a selected solvent/non-solvent system.
During the loading process, clindamycin had an additional effecton the morphology: it played the role of a morphological stabiliza-tion agent and had a special role in the regulation of the distributionof size and shape of the resulting core-shell structures. Similar influ-ence of the concentration of the loaded drug was observed in caseof the formation of poly(ethylene glycole)-block-poly(l-lactide)(PEG-b-PLLA) and poly(ethylene glycole)-block-poly(d-lactide)(PEG-b-PDLA) core-shells loaded with paclitaxel. Such an influ-ence was explained by the change of the resulting PEG-b-PLLAor PEG-b-PDLA/paclitaxel supermolecular structures [41]. Higherconcentrations of clindamycin resulted in more regular structureswith the narrowest distribution of the roundness factor and a nar-row unimodal distribution of diameters with the mean value of160 nm. These spheres were nanostructured and were composedof smaller spheres up to 20 nm in diameter. Possible electrostaticinteractions due to the presence of polar groups on the one hand,and steric interactions due to the branched structure of clindamycinmolecule on the other, can explain why clindamycin behaves likea highly efficient surface active substance and morphological sta-bilizer. Clindamycin was not detected on the surface of thesesphere-like particles and it was localized within the inner side ofthe polymeric layer – within the polymeric matrix and on the sur-face side of the ceramic layer – in the interface between PLGA andHAp. Nanotechnology allowed for tremendous progress in the field
of drug delivery because of the possibility to ensure control over therate and periods of delivery of drugs to a specific target [42]. It hasbeen shown that nanostructured polymeric materials can enablea unique application since their size scale is comparable to that ofbiological components, viruses and proteins, and for that reason
412 M. Vukomanovic et al. / Colloids and Surfaces B: Biointerfaces 82 (2011) 404–413
Fig. 6. Illustration on the effects of solubility of solvent/non-solvent pairs (organic/(organic or water)) on the morphological properties of PLGA/HAp particles comprisingclindamycin.
l PLG
m[swpf
sTcsdtdtHmatag[
Fig. 7. Proposed mechanism for the formation of spherica
any different techniques have been applied for their formation43]. The above described ultrasonic loading method can ensure apecific morphology of PLGA/HAp core-shell nanoparticles loadedith clindamycin and it can be expected that the obtained mor-hology can be highly useful in the application of this material inuture.
It has been noticed that both components of PLGA/HAp core-hells, PLGA and HAp, are able to capture one part of clindamycin.he precipitation of the polymer during the formation of PLGA/HApore-shells allows to load the antibiotic within the polymer. Aimilar situation occurred during the processing of PLGA with clin-amycin without HAp. On the other hand, it has been noticedhat HAp can capture one part of clindamycin, too. When clin-amycin was processed with HAp, hydroxyapatite was applied ashe solid phase. Having in mind the ordered crystal structure ofAp obtained using the homogeneous sonochemical precipitationethod [32], clindamycin could not be loaded within this structure:
possible route to attach clindamycin on HAp is through adsorp-
ion. The capacity of HAp to adsorb different drugs was shown laternd explained by electrostatic interactions between Ca2+ or PO4
3−
roups of HAp with oppositely charged groups of drug molecule44]. It can also be noticed that the percent of the antibiotic retained
A/HAp core-shell nanoparticles loaded with clindamycin.
by the PLGA/HAp composite was approximately the average of thepercent values of the antibiotic retained separately by PLGA andHAp. Different capacity of PLGA and HAp to capture clindamycinwhen they are separated and when they are in the composite mightbe related to a change in the surface charge density, which becomespartially compensated between PLGA and HAp within the compos-ite. Having in mind that both PLGA and HAp are negatively chargedunder most pH conditions [45], as well as that the magnitude ofzeta-potential of PLGA is larger compared to that of HAp [46], itis possible that clindamycin may be attached to the PLGA and HApthrough its more hydrophobic part where the pyrrole ring occupiesthe central place (Fig. 7) rather than through its highly hydroxylatedmethylsylfanyl side.
It has been shown that the formation of biohybrids exhibitsimproved structural and functional properties of the resultingmaterial [47]. From that point of view, it can be expected thatthe degradation of the polymeric part of the PLGA/HAp compos-
ite will be modified in contrast to the degradation of pure PLGAdue to the presence of the ceramic phase. A similar effect wasnoticed in the case of the degradation of the polymeric part ofthe PLGA/�-tricalcium phosphate (PLGA/TCP) composite. In thiscase, the dissolution of TCP and the consequent release of hydroxyl
urface
itpbo
awpiwict
5
icfcocdbdsmtdtoto
A
StbRB
R
[[[
[
[[
[[
[
[
[[
[[
[[
[
[
[[
[[[
[[[[[[
[[[
[[[
[[46] S. Fischer, C. Foerg, S. Ellenberger, H.P. Merkle, B. Gander, J. Control. Release
M. Vukomanovic et al. / Colloids and S
ons in the solution allowed for its neutralization by counteringhe effect of acidification following the degradation of the polymerhase, which would otherwise further accelerate the process of itsreaking down in an autocatalytic manner. As a result, the presencef TCP prolonged the degradation time of PLGA [48].
It was observed that the first part of the incorporated drugdsorbed onto the surface of HAp is mainly the phosphate formhich is a prodrug form of clindamycin. On the other hand, theolymeric matrix has been mainly the base form of this drug, which
s an active form of this antibiotic. The presence of clindamycinithin the both components of PLGA/HAp particles may likewise
nfluence the kinetics of the release of clindamycin from PLGA/HApore-shells and will present the subject of a further study (Part 2 ofhis manuscript) [49].
. Conclusions
Having in mind the obtained results, the ultrasonic process-ng method applied to load a drug within core-shell drug carrieran be favoured as a simple, low-cost and highly efficient methodor the processing of materials for controlled drug delivery. Itan also be expected that the new type of multifunctional antibi-tic carrier, formed from a biodegradable polymer and a bioactiveeramic phase in the form of polymeric shell and ceramic core withrug dispersed inside the polymeric matrix and on the interfaceetween the polymer and the ceramics, will become an efficientrug delivery material for advanced biomedical applications. Itspecial benefits are expected to show in the local application ofultifunctional implants based on this material for the medica-
ion of infectious bone tissue diseases, having in mind that slowegradation of PLGA can ensure the control of the drug release, thathe remaining drug on the surface of HAp can be additionally des-rbed (prolonging the period of drug release) and, finally, that afterhe medication treatment is finished, bioactive HAp can promotesteointegration in the place where the implant was inserted.
cknowledgements
Authors are grateful to Dr. J. Kovac and Dr. T. Filipic from Jozeftefan Institute, Department of Surface Engineering and Optoelec-ronics, for assistance in XPS analyses. This research was supportedy The Ministry of Science and Technological Development of theepublic of Serbia, under Grant No. 142006 and Serbian-Slovenianilateral Scientific Collaboration.
eferences
[1] C. Wischke, A. Lendlein, Pharm. Res. 27 (2010) 527–529.[2] G.B. Sukhorukov, A.L. Rogach, B. Zebli, T. Liedl, A.G. Skirtach, K. Kohler, A.A.
Antipov, N. Gaponik, A.S. Susha, M. Winterhalter, W.J. Parak, Small 1 (2005)194.
[3] S. Bhaskar, K.M. Pollock, M. Yoshida, J. Lahann, Small 6 (2010) 404.
[[[
s B: Biointerfaces 82 (2011) 404–413 413
[4] S.H. Hu, S.Y. Chen, D.M. Liu, C.S. Hsiao, Adv. Mater. 20 (2008) 2690.[5] N. Sounderya, Y. Zhang, Recent Pat. Biomed. Eng. 1 (2008) 34.[6] X. Gong, S. Peng, W. Wen, P. Sheng, W. Li, Adv. Funct. Mater. 19 (2009) 292.[7] G. Rethore, A. Pandit, Small 22 (2010) 488.[8] Q. Xu, M. Hashimoto, T.T. Dang, T. Hoare, D.S. Kohane, G.M. Whitesides, R.
Langer, D.G. Anderson, Small 5 (2009) 1575.[9] W. Ulbrich, A. Lamprecht, J. R. Soc. Interface 7 (2010) S55.10] V. Mourino, A.R. Boccaccini, J. R. Soc. Interface 7 (2010) 209.11] L.Y. Qiu, Y.H. Bae, Pharm. Res. 23 (2006) 1.12] C.G. Ambrose, G.R. Gogola, T.A. Clyburn, A.K. Raymond, A.S. Peng, A.G. Mikos,
Clin. Orthop. Relat. Res. 415 (2003) 279.13] M.R. Virto, B. Elorza, S.M.D. Torrado, L.A. Elorza, G. Frutos, Biomaterials 28
(2007) 877.14] M. Stevanovic, J. Savic, B. Jordovic, D. Uskokovic, Colloids Surf. B 59 (2007) 215.15] M. Stevanovic, A. Radulovic, B. Jordovic, D. Uskokovic, J. Biomed. Nanotechnol.
4 (2008) 1.16] M. Stevanovic, D.P. Uskokovic, Curr. Nanosci. 5 (2009) 1.17] N.L. Ignjatovic, P. Ninkov, R. Sabetrasekh, D.P. Uskokovic, J. Mater. Sci.: Mater.
Med. 21 (2010) 231.18] V.C. Amaro Martins, G. Goissis, A.C. Ribeiro, E. Marcantonio Jr, M.R. Bet, Artif.
Organs 22 (3) (1998) 215.19] M.A. Rauschmann, T.A. Wichelhaus, V. Stirnal, E. Dingeldein, L. Zichner, R.
Schnettler, V. Alt, Biomaterials 26 (2005) 2677.20] S.P. Victor, T.S.S. Kumar, J. Biomed. Nanotechnol. 4 (2) (2008) 203.21] S. Lepretre, F. Chai, J.C. Hornez, G. Vermet, C. Neut, M. Descamps, H.F. Hilde-
brand, B. Martel, Biomaterials 30 (2009) 6086.22] T.A. Ostomel, Q. Shi, C.K. Tsung, H. Liang, G.D. Stucky, Small 2 (2006) 1261.23] M. Enayati, Z. Ahmad, E. Strideand, M. Edirisinghe, J. R. Soc. Interface 7 (2010)
667.24] V. Uskokovic, M. Drofenik, Adv. Colloid Interface Sci. 133 (2007) 23–34.25] E. Cohen-Sela, M. Chorny, N. Koroukhov, H.D. Danenberg, G. Golomb, J. Control.
Release 133 (2009) 90.26] K. Makino, T. Mizorogi, S. Ando, T. Tsukamoto, H. Ohshima, Colloids Surf. B 22
(2001) 251.27] S. Avivi (Levi), Y. Nitzan, R. Dror, A. Gedanken, J. Am. Chem. Soc. 125 (51) (2003)
15712.28] D.-M. Qi, Y.-Z. Bao, Z.-X. Weng, Z.-M. Huang, Polymer 47 (2006) 4622.29] M. Jevtic, A. Radulovic, N. Ignjatovic, M. Mitric, D. Uskokovic, Acta Biomater. 5
(2009) 208.30] J. Wu, Y.-J. Zhu, S.-W. Cao, F. Chen, Adv. Mater. 22 (2010) 749.31] J. Liu, Q. Yang, L. Zhang, H. Yang, J. Gao, C. Li, Chem. Mater. 20 (2008) 4268.32] M. Jevtic, M. Mitric, S. Skapin, B. Jancar, N. Ignjatovic, D. Uskokovic, Cryst.
Growth Des. 8 (7) (2008) 2217.33] Y. Chen, J.K. Wang, J. Chem. Eng. Data 52 (2007) 1908.34] L. Zhu, Y. Chen, J.-K. Wang, J. Chem. Eng. Data 53 (2008) 1984.35] H. Li, H. Li, Z. Guo, Y. Liu, Ultrason. Sonochem. 13 (2006) 359.36] M.D. Luque de Castro, F. Priego-Capote, Ultrason. Sonochem. 14 (6) (2007) 717.37] Y.Y. Huang, T.P.J. Knowles, E.M. Terentjev, Adv. Mater. 21 (2009) 3945.38] K. McLeod, S.R.St. Kumar, C. Smart, N. Dutta, N.H. Voelcker, G.I. Anderson, R.
Sekel, Appl. Surf. Sci. 253 (2006) 2644.39] N.T. Paragkumar, D. Edith, J.L. Six, 253, Appl. Surf. Sci. (2006) 2758.40] L. Mu, S.S. Feng, Pharm. Res. 20 (2003) 1864.41] J.P.K. Tan, S.H. Kim, F. Nedeberg, E.A. Appel, R.M. Waymouth, Y. Zhang, J.L.
Hedrick, Y.Y. Yang, Small 5 (2009) 1504.42] N. Doshi, S. Mitragotri, Adv. Funct. Mater. 19 (2009) 3843.43] D.A. Bernards, T.A. Desai, Soft Matter 6 (2010) 1621.44] B. Palazzo, M. Iafisco, M. Laforgia, N. Margiotta, G. Natile, C.L. Bianchi, D. Walsh,
S. Mann, N. Roveri, Adv. Funct. Mater. 17 (2007) 2180.45] Y. Zhang, Y. Yokogawa, J. Mater. Sci.: Mater. Med. 19 (2) (2008) 623.
111 (2006) 135.47] E. Ruiz-Hitzky, M. Darder, P. Aranda, K. Ariga, Adv. Mater. 22 (2010) 323.48] Z. Yang, S.M. Best, R.E. Cameron, Adv. Mater. 21 (2009) 3900.49] M. Vukomanovic, S.D. Skapin, I. Poljansek, E. Zagar, B. Kralj, N. Ignjatovic, D.
