Colloidsand Surfaces A:Physicochem.Eng.Aspects 472 (2015)78-84Contents lists available at ScienceDirectCORRANColloidsand SurfacesA:PhysicochemicalandEngineeringAspectsELSEVIERjournalhomepage:www.eisevier.com/locate/coisurfaModulationofreleasebehaviorsofmethylenebluefromdegradableCrossMarksilica-methyleneblue@octacalciumphosphatepowderswithdifferent shellstructuresChengfeng Lia*,Xiaolu Geb,Yadong Lia,Jiahai Baia,Guochang Lia,Changhong SudZanzhongYangaaSchoolofMaterialsScienceandEngineering.ShandongUniversityofTechnology,255049Shandong.PRChinabSchoolof Science,Shandong Universityof Technology,255049Shandong,PR ChinaHIGHLIGHTSABSTRACTGRAPHICAL.The coating ofOCP on silica-MBwasachieved byafacile sol-gel method.(c)80·The release behavior of MB fromsamples was sensitive with the pHvalue8n?The shell structure modulated these(d)release behavior of MB from silica-MB@OCP.e40? The structure-destruction of silica-MB was promoted after the deposi-tionofoCP20(b)800(a)102030506070After72hof incubationat37CTime (h)INFOARTICLEABSTRACTArticle history:Methylene blue-loaded silica (silica-MB) was coated by a shell of polyethylene glycol (PEG), citrate ionsReceived 14 January 2015and octacalcium phosphate (OCP) through a facile sol-gel method.The influences of molecular weightReceived in revised form 23 February 2015ofPEG,additionamountofPEGand citricacidonthephase,morphologyand chemical compositionofAccepted26February2015samples werecharacterizedbyX-raydiffraction,scanningelectronmicroscopyandFouriertransformAvailable online6March2015infrared spectroscopy,respectively.Compared with that of silica-MB,the sustained-release behavior ofMBfrom silica-MB@OCPwassensitivetotheshell structureandthepHvalueof culturesolution.AtKeywords:eachtimeinterval,theratioofabsorbanceofMBmonomersoverthatofMBdimersreleasedfromsilica-Drug carrierMB@OCp was higherthan that from silica-MB,indicating that the significant influence of interactionSilicadensity among the network of PEG, citrate ions and OCP on the release behavior of MB. Silica-MB andMethylene bluesilica-MB@OCP were degraded when MB molecules diffused to the culture solution from solid silicaOctacalcium phosphatematrix, which would shed light on the self-destruction investigation of drug/drug carrier systems in theDegradationbiological system.2015 Elsevier B.V.All rights reserved.*Correspondingauthor.Tel.:+865332782232:fax:+865332781665E-mail address: cfli@sdut.edu.cn (C. Li)http://dx.doi.org/10.1016/j.colsurfa.2015.02.0410927-7757/@ 2015 Elsevier B.V.All rights reserved
Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 Contents lists available at ScienceDirect Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa Modulation of release behaviors of methylene blue from degradable silica-methylene blue@octacalcium phosphate powders with different shell structures Chengfeng Li a,∗, Xiaolu Ge b, Yadong Li a, Jiahai Bai a, Guochang Li a, Changhong Sua, Zanzhong Yanga a School of Materials Science and Engineering, Shandong University of Technology, 255049 Shandong, PR China b School of Science, Shandong University of Technology, 255049 Shandong, PR China h i g h l i g h t s • The coating of OCP on silica-MB was achieved by a facile sol–gel method. • The release behavior of MB from samples was sensitive with the pH value. • The shell structure modulated the release behavior of MB from silicaMB@OCP. • The structure-destruction of silicaMB was promoted after the deposition of OCP. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 14 January 2015 Received in revised form 23 February 2015 Accepted 26 February 2015 Available online 6 March 2015 Keywords: Drug carrier Silica Methylene blue Octacalcium phosphate Degradation a b s t r a c t Methylene blue-loaded silica (silica-MB) was coated by a shell of polyethylene glycol (PEG), citrate ions and octacalcium phosphate (OCP) through a facile sol–gel method. The influences of molecular weight of PEG, addition amount of PEG and citric acid on the phase, morphology and chemical composition of samples were characterized by X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy, respectively. Compared with that of silica-MB, the sustained-release behavior of MB from silica-MB@OCP was sensitive to the shell structure and the pH value of culture solution. At each time interval, the ratio of absorbance of MB monomers over that of MB dimers released from silicaMB@OCP was higher than that from silica-MB, indicating that the significant influence of interaction density among the network of PEG, citrate ions and OCP on the release behavior of MB. Silica-MB and silica-MB@OCP were degraded when MB molecules diffused to the culture solution from solid silica matrix, which would shed light on the self-destruction investigation of drug/drug carrier systems in the biological system. © 2015 Elsevier B.V. All rights reserved. ∗ Corresponding author. Tel.: +86 533 2782232; fax: +86 533 2781665. E-mail address: cfli@sdut.edu.cn (C. Li). http://dx.doi.org/10.1016/j.colsurfa.2015.02.041 0927-7757/© 2015 Elsevier B.V. All rights reserved
79C.LietaL./Colloidsand SurfacesA:Physicochem.Eng.Aspects472(2015)78-841. Introductionpore walland lowcross-linking degreein silica matrix.Forexample,the degradation of silica nanospheres with mesoporous structureThe essential features of drug/drug carrier systems includedwascompletein24hforsampleswithhighspecificsurfaceareaeffective cellular uptake, controllable release of drug and self-of632m-/gandporesizeof10nm25].Throughtheexposuretodestruction of drug carrier for safe excretion from the biologicalthe iron chelates, approximately 84% of the iron(Il) doped into thesystem [1]. The surfaces of inorganic powders, like silica and cal-silicananoshell was removedafter3daysinan80C waterbath,cium phosphate, are hydrophilic and favorable for cellular uptake.and silica samples were degraded completely after at least 17 daysSilica isgenerallyaccepted asa promisingcarrierduetoitsnon-of cultureinphysiological conditions[26l.Solid silicawithMB-toxicityandversatilefunctionaftersurfacemodificationJ1-4l.Asrich nuclei degraded in two weeks after the release of encapsulatedthemaininorganiccomponentofbiologicalhardtissues,calciumMBmolecules[1].Herein,MBmoleculeswerefirstlyencapsulatedphosphate is currently used for gene delivery (transfection), lumi-in silica particles (silica-MB), followed by a deposition of octacal-nescent labels, or luminescent drug carriers [5-9]. However, drugcium phosphate(OCP) shell on silica-MB (silica-MB@OCP)with theloadingandcontrolledreleasearealsolimitedduetotheweakfavorofpolyethyleneglycol(PEG)moleculesandcitrateions.Theinteractionsbetweeninorganicpowdersanddrugmoleculesabsorbanceofthesupernatantinphosphatebufferedsaline(PBS)Asa drug,methyleneblue(MB)playsa crucial roletotreatandlysosome-likebufferwasmeasured and usedto investigatethe release behaviors of MB from hybrid powders. The occurrencemethemoglobinemia, urinary tract infections and malaria infec-tion [10,11]. In recent studies, MB is used as an antagonist againstof degradation in a fast rate was expected through the modula-tion of diffusion behavior of MB from the core-shell structuredheat-shockresponse gene expression in cancer cells[12] or asa photosensitizer in photochemotherapy for the treatment forpowders. The degradation of drug carriers would shed light on thetumortissues[13-17l.MonomersanddimersofMBhavedistinctself-destruction investigation of drug/drug carrier systems for theirphotochemical reactions and cancer-killing efficacies [18]. Afterpotentialdiagnosticortherapeuticfunctionstotreattumorissues.optical pumping.the monomer in a singlet state generally under-goes intersystem crossing (isC)to a metastable triplet state with a2.Experimentalhigh quantum yield. Then, exchanged energy is transferred fromthe triplet MB molecule to oxygen molecule and results in the2.1. Synthesisgeneration of singlet oxygen molecule ('O2)to exert an antipro-liferative effect. MB monomer is easily reduced and oxidized in2.1.1. Silica-MBthe biological media, which makes it cross membranes to treatSilica-MB was synthesized by a modified Stober method [27,28]methemoglobinemia [19-21]. Although larger affinity to nega-Typically, 0.10g of methylene blue (MB) was dissolved in 92 ml oftivelychargedinterfacesandtomelanin[22,23].MBmonomerwasethanol, 13.8mlofdeionized waterand2.38mlofaqueousammo-reduced easily and excreted rapidly from biological system, whichnia (NH3.H20) under stirring. After 15 min, 3.44 ml of tetraethylresultedinitsineffectivenesstostainmostofthetumortissuesandorthosilicate (TEOS)was added and then the stirring ofthereactionthe hindrance of the widespread clinical application of MB [24].mixture was continued for another 4h. Precipitations of silica-MBRaising the dosage of administrated MB would cause the forma-werecollectedbycentrifugation,washedwithethanoltwiceandtion of hardly-reduced MB dimers and the serious influence on theused to prepare a dispersion of silica-MB in 10 ml of ethanol (withhealthofnormaltissues.Asustained-releaseofMBmonomersfroma concentration of 53mg/ml).drug carriers could be expected to maintain its circulated concen-trationinblood,whichwould effectivelyimproveitsaccumulation2.1.2.Silica-MB@OCPintumortissuesThe coating of ocP on silica-MB was carried out by a modifiedThe elimination ofdrug carrierfrom the biologic system is ratherPechini sol-gel process [27,29], which was illustrated in Scheme 1.difficultaftertheaccomplishmentofthediagnosticortherapeuticInatypicalreaction,4.50gofcitricacidmonohydrate(CiA),2.48gfunctions, which remains as one of the major obstacles impedingof tetrahydrate calcium nitride and 0.85g of diammonium hydro-potentialclinicaltranslationofdrug/drugcarriersystems[1,25,26].gen phosphate were dissolved in 46.1ml of ethanol and 138.3mlRecently.thedegradationof silicaarousedgreatresearch interest-of deionized water. The pH value of the solution was adjusteding and was achieved through introducing large pores with thinto 9 by the addition of NH-H20, and then 9.22g of PEG with a(PEG)(CiA)(MB monomer)HPO,2HPO,2O'Ca*POCa2*ihiPO,Caz+IPO,2PO,I:MBmonomer,I:MB dimersCore: silica-MB; Shell: OCP(MB dimers)Scheme 1. The schematic illustration of the preparation of silica-MB@OCP
C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 79 1. Introduction The essential features of drug/drug carrier systems included effective cellular uptake, controllable release of drug and selfdestruction of drug carrier for safe excretion from the biological system [1]. The surfaces of inorganic powders, like silica and calcium phosphate, are hydrophilic and favorable for cellular uptake. Silica is generally accepted as a promising carrier due to its nontoxicity and versatile function after surface modification [1–4]. As the main inorganic component of biological hard tissues, calcium phosphate is currently used for gene delivery (transfection), luminescent labels, or luminescent drug carriers [5–9]. However, drug loading and controlled release are also limited due to the weak interactions between inorganic powders and drug molecules. As a drug, methylene blue (MB) plays a crucial role to treat methemoglobinemia, urinary tract infections and malaria infection [10,11]. In recent studies, MB is used as an antagonist against heat-shock response gene expression in cancer cells [12] or as a photosensitizer in photochemotherapy for the treatment for tumor tissues [13–17]. Monomers and dimers of MB have distinct photochemical reactions and cancer-killing efficacies [18]. After optical pumping, the monomer in a singlet state generally undergoes intersystem crossing (ISC) to a metastable triplet state with a high quantum yield. Then, exchanged energy is transferred from the triplet MB molecule to oxygen molecule and results in the generation of singlet oxygen molecule (1O2) to exert an antiproliferative effect. MB monomer is easily reduced and oxidized in the biological media, which makes it cross membranes to treat methemoglobinemia [19–21]. Although larger affinity to negatively charged interfaces and to melanin [22,23], MB monomer was reduced easily and excreted rapidly from biological system, which resulted in its ineffectiveness to stain most ofthe tumor tissues and the hindrance of the widespread clinical application of MB [24]. Raising the dosage of administrated MB would cause the formation of hardly-reduced MB dimers and the serious influence on the health of normaltissues.Asustained-release of MB monomers from drug carriers could be expected to maintain its circulated concentration in blood, which would effectively improve its accumulation in tumor tissues. The elimination of drug carrier from the biologic system is rather difficult after the accomplishment of the diagnostic or therapeutic functions, which remains as one of the major obstacles impeding potential clinicaltranslation of drug/drug carrier systems [1,25,26]. Recently, the degradation of silica aroused great research interesting and was achieved through introducing large pores with thin pore wall and low cross-linking degree insilicamatrix. For example, the degradation of silica nanospheres with mesoporous structure was complete in 24 h for samples with high specific surface area of 632 m2/g and pore size of 10 nm [25]. Through the exposure to the iron chelates, approximately 84% of the iron(III) doped into the silica nanoshell was removed after 3 days in an 80 ◦C water bath, and silica samples were degraded completely after at least 17 days of culture in physiological conditions [26]. Solid silica with MBrich nuclei degraded in two weeks after the release of encapsulated MB molecules [1]. Herein, MB molecules were firstly encapsulated in silica particles (silica-MB), followed by a deposition of octacalcium phosphate (OCP) shell on silica-MB (silica-MB@OCP) with the favor of polyethylene glycol (PEG) molecules and citrate ions. The absorbance of the supernatant in phosphate buffered saline (PBS) and lysosome-like buffer was measured and used to investigate the release behaviors of MB from hybrid powders. The occurrence of degradation in a fast rate was expected through the modulation of diffusion behavior of MB from the core–shell structured powders. The degradation of drug carriers would shed light on the self-destruction investigation of drug/drug carrier systems for their potential diagnostic or therapeutic functions to treat tumor issues. 2. Experimental 2.1. Synthesis 2.1.1. Silica-MB Silica-MB was synthesized by a modified Stöber method [27,28]. Typically, 0.10 g of methylene blue (MB) was dissolved in 92 ml of ethanol, 13.8 ml of deionized water and 2.38 ml of aqueous ammonia (NH3•H2O) under stirring. After 15 min, 3.44 ml of tetraethyl orthosilicate (TEOS) was added and then the stirring of the reaction mixture was continued for another 4 h. Precipitations of silica-MB were collected by centrifugation, washed with ethanol twice and used to prepare a dispersion of silica-MB in 10 ml of ethanol (with a concentration of 53 mg/ml). 2.1.2. Silica-MB@OCP The coating of OCP on silica-MB was carried out by a modified Pechini sol–gel process [27,29], which was illustrated in Scheme 1. In a typical reaction, 4.50 g of citric acid monohydrate (CiA), 2.48 g of tetrahydrate calcium nitride and 0.85 g of diammonium hydrogen phosphate were dissolved in 46.1 ml of ethanol and 138.3 ml of deionized water. The pH value of the solution was adjusted to 9 by the addition of NH3•H2O, and then 9.22 g of PEG with a Scheme 1. The schematic illustration of the preparation of silica-MB@OCP
80C Li et al. /Colloids and SurfacesA: Physicochem.Eng.Aspects 472 (2015)78-84molecular weight of 2000 g/mol (PEG2000) was added under stir-(010)ring. After 1 h, the dispersion of silica-MB in 10 ml of ethanol wasadded and the reaction mixture was then stirred for another 3h.Finally,silica-MB@OCP was collected by centrifugation, washed(h)美with ethanoltwiceand driedat60Cfor 12h.Differentsamples(O)10兰of silica-MB@OCP were synthesized through the above-mentioned(ce)aprocess with thevariation ofmolecularweight of PEG,amount ofPEG or CiA addition as shown in Table 1.ke(d)2.2.Characterization(c)(b)Phase identification by X-ray diffraction (XRD) was carried outon a D8 advanced X-ray polycrystalline diffractometer with Cu Koxradiation (wavelength: ^=0.15406 nm). The scanning range (20)(a)umhlywas from 5 to 70° at a scan speed of 0.1°min-1. The crystallite size102060304050of sample was estimated from the Scherrer's equation:2 theta (degree)kad=(1)Fig. 1. XRD patterns of standard OCP: JCPDS file No. 26-1065 (a). silica-MB (b).(FWHM.cos 9)silica-MB@OCP400 (c), silica-MB@OCP2000 (d), silica-MB@OCP6000 (e). silica-MB@0CP20001 (). silica-MB@0CP20003 (g), silica-MB@0CP2000-CiA1 (h), andwhere k is the Scherrer constant (0.89), the value of and the fullsilica-MB@OCP2000-CiA3 (i)width at half maximum (FWHM) are herein determined from thebroadening of (010) diffraction peak of OCP. The crystal morphol-solutions at pH=9. When increasing molecular weight of PEG andogy was characterized by scanning electron microscopy (SEM, FEIdecreasing addition of CiA, the (010) diffraction peak of silica-SIRION200).Fourier transform infrared spectra(FTIR,Nicolet5700,MB@OCPbecamebroad (Fig.1)and the crystallite size decreasedThermo, USA) were obtained with the wavenumbers recorded from(see Table 1), indicating that PEG and CiA affected the crystalliza-400 to 4000cm-1 at a 1cm-1 resolution.tion ofOCP.For silica-MB@OCP6000and silica-MB@OCP2000-CiA1,In order to investigate the release behavior of MB, 0.02g ofthe diffraction peaks of ocp became broad and weak as a resultsilica-MB or silica-MB@OCP powders were incubated in 25 ml ofof poor crystallization. Amount of PEG addition had slightlyPBS (pH=7.2-7.4) or lysosome-like buffer (pH=4.7)at 37°C underrotary shaking (120 rpm). Ultraviolet-visible (UV-Vis) absorptioninfluenced on thecrystallite size of OCPfor silica-MB@0CP20001,silica-MB@0CP2000 and silica-MB@0CP20003 (Table 1)spectrum of the supernatant was recorded using a UV-Vis spec-trophotometer(TU1901)atcertaintimeinterval.Theconcentration3.2.SEMcharacterizationof released MB was determined by the corresponding calibrationcurve. The degradation percent was calculated according to theThrough 4h of sol-gel reaction, spherical silica-MB particlesresidual weight ratio after incubating 0.10g of silica-MB and silica-were synthesized with size of about 600 nm as shown in Fig.2(a).MB@OCP400 in 125ml of PBS or lysosome-like buffer for 72 h. Inaddition, PBS was composed of 137 mM sodium chloride (NaCl).With absence of MB in the reaction solution, silica particles were2.7 mM potassium chloride (Kcl), 4.3 mM of disodium hydrogenhardly collected after reaction for 4h due to the slow precipitationrate of silica [27]. As a quaternary ammonium salt, the basic dyephosphate (Na2HPO4) and 1.4mM of potassium dihydrogen phos-of MB dissociates to a positive ammonium cation.The electrostaticphate (KH2PO4). Lysosome-like buffer was prepared with 4.4mMof citric acid and 5.6mM of sodium citrate.interaction occurred between thecationicgroup ofthe dye and thenegatively-charged functional group on the surface of silica nuclei(Scheme 1),which would result in the promotion of nucleation and3.Results and discussiongrowth of silica with loading of MB molecules [1,30-32].As shown in Fig.2,powders of silica-MB@OCP400,silica-3.1. XRD characterizationMB@0CP2000 and silica-MB@0CP6000 werelarger than silica-MB,indicating the deposition of OCP shell on core silica-MB. DuringPhases of silica-MB and silica-MB@OCP were identified bysynthesis of silica-MB@OCP (Scheme 1), calcium ions were firstlyXRD as shown in Fig. 1. For silica-MB, the broad peak centeredchelated by citrate ions and then reacted with HPO4and PO43-at 22° was a typical diffraction peak of amorphous silica [27]. Inions to precipitate OCP when the pH value of reaction solution wassolutions containing water, ethanol, MB and NHg·H2O, TEOS wasraised to 9. The formation of a network via the interaction amongfirstly hydrolyzed to reactive silanol group, and then condensed tocitrate ions, OCP, OH groups of silica-MB and PEG as a cross-linkingthe Si-O-Si network, finally precipitated as amorphous silica. Foragentresulted inthecoating ofOCPon silica-MBthrougha modifiedsilica-MB@OCP,phases of OCP and amorphous silica was observedPechini sol-gel process [27,29]in Fig. 1(c)-(i), indicating the precipitation of OCP in reactionTable1Reaction conditions (molecular weight of PEG (MW), PEG addition and CiA addition), crystallite size and loaded MB of silica-MB@OCP with different shell structures.MW (g/mol)PEG (g)CiA (g)SampleCrystallite size (nm)Loaded MB (mg/g)76.3Silica-MB9.224004.5030.918.0Silica-MB@0CP40020009.224.5026.542.3Silica-MB@0CP2000Silica-MB@0CP600060009.224.5017.531.94.50Silica-MB@0CP2000120004.6140.535.3913.834.5034.725.92Silica-MB@0CP2000320002.2520009.223.154.04Silica-MB@0CP2000-cia120009.226.7543.532.21Silica-MB@0CP2000-cia3
80 C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 molecular weight of 2000 g/mol (PEG2000) was added under stirring. After 1 h, the dispersion of silica-MB in 10 ml of ethanol was added and the reaction mixture was then stirred for another 3 h. Finally, silica-MB@OCP was collected by centrifugation, washed with ethanol twice and dried at 60 ◦C for 12 h. Different samples of silica-MB@OCP were synthesized through the above-mentioned process with the variation of molecular weight of PEG, amount of PEG or CiA addition as shown in Table 1. 2.2. Characterization Phase identification by X-ray diffraction (XRD) was carried out on a D8 advanced X-ray polycrystalline diffractometer with Cu K radiation (wavelength: = 0.15406 nm). The scanning range (2) was from 5 to 70◦ at a scan speed of 0.1◦ min−1. The crystallite size of sample was estimated from the Scherrer’s equation: d = k (FWHM • cos ) (1) where k is the Scherrer constant (0.89), the value of and the full width at half maximum (FWHM) are herein determined from the broadening of (0 1 0) diffraction peak of OCP. The crystal morphology was characterized by scanning electron microscopy (SEM, FEI SIRION 200). Fourier transform infrared spectra (FTIR, Nicolet 5700, Thermo, USA) were obtained with the wavenumbers recorded from 400 to 4000 cm−1 at a 1 cm−1 resolution. In order to investigate the release behavior of MB, 0.02 g of silica-MB or silica-MB@OCP powders were incubated in 25 ml of PBS (pH = 7.2–7.4) or lysosome-like buffer (pH = 4.7) at 37 ◦C under rotary shaking (120 rpm). Ultraviolet–visible (UV–Vis) absorption spectrum of the supernatant was recorded using a UV–Vis spectrophotometer (TU1901) at certain time interval. The concentration of released MB was determined by the corresponding calibration curve. The degradation percent was calculated according to the residual weight ratio after incubating 0.10 g of silica-MB and silicaMB@OCP400 in 125 ml of PBS or lysosome-like buffer for 72 h. In addition, PBS was composed of 137 mM sodium chloride (NaCl), 2.7 mM potassium chloride (KCl), 4.3 mM of disodium hydrogen phosphate (Na2HPO4) and 1.4 mM of potassium dihydrogen phosphate (KH2PO4). Lysosome-like buffer was prepared with 4.4 mM of citric acid and 5.6 mM of sodium citrate. 3. Results and discussion 3.1. XRD characterization Phases of silica-MB and silica-MB@OCP were identified by XRD as shown in Fig. 1. For silica-MB, the broad peak centered at 22◦ was a typical diffraction peak of amorphous silica [27]. In solutions containing water, ethanol, MB and NH3•H2O, TEOS was firstly hydrolyzed to reactive silanol group, and then condensed to the Si–O–Si network, finally precipitated as amorphous silica. For silica-MB@OCP, phases of OCP and amorphous silica was observed in Fig. 1(c)–(i), indicating the precipitation of OCP in reaction Fig. 1. XRD patterns of standard OCP: JCPDS file No. 26-1065 (a), silica-MB (b), silica-MB@OCP400 (c), silica-MB@OCP2000 (d), silica-MB@OCP6000 (e), silicaMB@OCP20001 (f), silica-MB@OCP20003 (g), silica-MB@OCP2000-CiA1 (h), and silica-MB@OCP2000-CiA3 (i). solutions at pH = 9. When increasing molecular weight of PEG and decreasing addition of CiA, the (0 1 0) diffraction peak of silicaMB@OCP became broad (Fig. 1) and the crystallite size decreased (see Table 1), indicating that PEG and CiA affected the crystallization of OCP. For silica-MB@OCP6000 and silica-MB@OCP2000-CiA1, the diffraction peaks of OCP became broad and weak as a result of poor crystallization. Amount of PEG addition had slightly influenced on the crystallite size of OCP for silica-MB@OCP20001, silica-MB@OCP2000 and silica-MB@OCP20003 (Table 1). 3.2. SEM characterization Through 4 h of sol–gel reaction, spherical silica-MB particles were synthesized with size of about 600 nm as shown in Fig. 2(a). With absence of MB in the reaction solution, silica particles were hardly collected after reaction for 4 h due to the slow precipitation rate of silica [27]. As a quaternary ammonium salt, the basic dye of MB dissociates to a positive ammonium cation. The electrostatic interaction occurred between the cationic group of the dye and the negatively-charged functional group on the surface of silica nuclei (Scheme 1), which would result in the promotion of nucleation and growth of silica with loading of MB molecules [1,30–32]. As shown in Fig. 2, powders of silica-MB@OCP400, silicaMB@OCP2000 and silica-MB@OCP6000 were larger than silica-MB, indicating the deposition of OCP shell on core silica-MB. During synthesis of silica-MB@OCP (Scheme 1), calcium ions were firstly chelated by citrate ions and then reacted with HPO4 − and PO4 3− ions to precipitate OCP when the pH value of reaction solution was raised to 9. The formation of a network via the interaction among citrate ions, OCP, OH groups of silica-MB and PEG as a cross-linking agent resultedinthe coating of OCP onsilica-MBthroughamodified Pechini sol–gel process [27,29]. Table 1 Reaction conditions (molecular weight of PEG (MW), PEG addition and CiA addition), crystallite size and loaded MB of silica-MB@OCP with different shell structures. Sample MW (g/mol) PEG (g) CiA (g) Crystallite size (nm) Loaded MB (mg/g) Silica-MB – – – – 76.3 Silica-MB@OCP400 400 9.22 4.50 30.9 18.0 Silica-MB@OCP2000 2000 9.22 4.50 26.5 42.3 Silica-MB@OCP6000 6000 9.22 4.50 17.5 31.9 Silica-MB@OCP20001 2000 4.61 4.50 40.5 35.39 Silica-MB@OCP20003 2000 13.83 4.50 34.7 25.92 Silica-MB@OCP2000-cia1 2000 9.22 2.25 3.1 54.04 Silica-MB@OCP2000-cia3 2000 9.22 6.75 43.5 32.21
81C.LietaL/Colloids andSurfacesA:Physicochem.Eng.Aspects 472(2015)78-84b2um(d)(c)1umTFig. 2. SEM characterization of silica-MB (a), silica-MB@OCP400 (b), silica-MB@OCP2000 (c), and silica-MB@OCP6000 (d)3.3.FTIR spectrasmaller than that of free carboxylate ions (ca. 188 cm-1), indi-cating the citrate ions are bonded to OCP nanoparticles throughFTIR spectra of silica-MB and silica-MB@OCP were shown ina bidentate-type bonding [35]. While, only a single absorbanceFig. 3. Absorbance bands of silica for silica-MB in Fig.3(a) appearedband of carboxylate ion was observed for silica-MB@0CP20001at 1099.0, 949.7. 802.3 and 471.0cm-1 [33]. A broad band atand silica-MB@OCP2000CiA1.As shown in Table 2,these bands3410.4cm-1 represented the asymmetric stretchingvibration (u3)werehardlyobservedforsilica-MB@OCP6000duetofewcitrateof OH groups of H2O.Absorbance bands of MB included theions present in the shell, which was similar with its XRD patternsring stretching vibration at 1609.2cm-1, the symmetric stretch-(Fig. 1(e). The red shift of the symmetric stretching vibration banding vibration of C-N at 1394.7 cm-1, and symmetric deformation(U)ofOHgroupsinH2Oforsilica-MB@OCPwascausedbytheof-CH3 at 1339.6 cm-1 [34]strong hydrogen bond between H2O and powders [36]. The inter-Besides those of silica and MB,absorbance bands assigned toactionbetweenMBandpowderswasaccordancewiththeshiftofPO,3-and citrate ionsfor silica-MB@OCP wereobserved inFig.3.theringstretchingbandofMBforsilica-MB@OcP,incomparisonTypically,for silica-MB@OCP400 (Fig.3(b), absorbance bands ofwith that for silica-MB as shown in Table 2.PO43- appeared at 547.2 and 603.6cm-1. The absorbance bandsat 1573.4 and 1541.0cm-1 were respectively assigned to asym-3.4.Releasebehavior of MBmetric (vas(COO-)) and symmetric stretching (vs(COO-)) modelsfor the carboxylate group of citrate ion, respectively. The differ-3.4.1.Release behavior of MB from silica-MBence between vas(CO0-) and vs(CO0-) is about 32.4cm-1 andAfter silica-MB was etched by hydrofluoric acid, its loadingcapacity of MB was determined as 76.3 mg/g according to theabsorbance of MB in the solution [37].The release behavior of MB(h)fromsilica-MBexhibitedaslowrateinbothPBSandlysosome-likebufferasshowninFig.4.After72h,only8.4%and11.8%ofloaded(g)MB were released to PBS and lysosome-like buffer, respectively.(f)The diffusion of H+ ions into silica matrix would result in the pro-tonation of orthosilicic acid (pKm = 9.8[38).and thus weaken the(e)electrostatic interaction between MB molecules and silica.Hence,(d)(c)Table21541.0FTIR analysis results of silica-MB and silica-MB@OCP (ring stretching vibration(b)mode:RSV,stretchingvibrationmode: SV).1573.4603.0SampleRSV (MB)SV (COO-)SV (U,(OH)(a)101t609Silica-MB1609.2471.0Silica-MB@OCP4001613.71541,15733237.45001000150020002500300035004000Silica-MB@0CP20001611.21540, 15743220.61607.4Weak3236.0Silica-MB@OCP6000Wavenumber (cm")Silica-MB@0CP200011616.815483220.63238.2Silica-MB@0CP200031615.41541,1576Fig. 3. FTIR spectra of silica-MB (a), silica-MB@OCP400 (b), silica-MB@OCP2000Silica-MB@0CP2000-cia11609.415473236.0(c).and silica-MB@0CP6000(d).silica-MB@0CP20001 (e).silica-MB@0CP20003 (n).1611.51540, 15743216.6Silica-MB@OCP2000-cia3silica-MB@0CP2000-CiA1 (g), and silica-MB@OCP2000-CiA3 (h)
C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 81 Fig. 2. SEM characterization of silica-MB (a), silica-MB@OCP400 (b), silica-MB@OCP2000 (c), and silica-MB@OCP6000 (d). 3.3. FTIR spectra FTIR spectra of silica-MB and silica-MB@OCP were shown in Fig. 3. Absorbance bands of silica for silica-MB in Fig. 3(a) appeared at 1099.0, 949.7, 802.3 and 471.0 cm−1 [33]. A broad band at 3410.4 cm−1 represented the asymmetric stretching vibration (3) of OH groups of H2O. Absorbance bands of MB included the ring stretching vibration at 1609.2 cm−1, the symmetric stretching vibration of C–N at 1394.7 cm−1, and symmetric deformation of –CH3 at 1339.6 cm−1 [34]. Besides those of silica and MB, absorbance bands assigned to PO4 3− and citrate ions for silica-MB@OCP were observed in Fig. 3. Typically, for silica-MB@OCP400 (Fig. 3(b)), absorbance bands of PO4 3− appeared at 547.2 and 603.6 cm−1. The absorbance bands at 1573.4 and 1541.0 cm−1 were respectively assigned to asymmetric (as(COO−)) and symmetric stretching (s(COO−)) models for the carboxylate group of citrate ion, respectively. The difference between as(COO−) and s(COO−) is about 32.4 cm−1 and Fig. 3. FTIR spectra of silica-MB (a), silica-MB@OCP400 (b), silica-MB@OCP2000 (c), and silica-MB@OCP6000 (d), silica-MB@OCP20001 (e), silica-MB@OCP20003 (f), silica-MB@OCP2000-CiA1 (g), and silica-MB@OCP2000-CiA3 (h). smaller than that of free carboxylate ions (ca. 188 cm−1), indicating the citrate ions are bonded to OCP nanoparticles through a bidentate-type bonding [35]. While, only a single absorbance band of carboxylate ion was observed for silica-MB@OCP20001 and silica-MB@OCP2000CiA1. As shown in Table 2, these bands were hardly observed for silica-MB@OCP6000 due to few citrate ions present in the shell, which was similar with its XRD patterns (Fig. 1(e)). The red shift of the symmetric stretching vibration band (1) of OH groups in H2O for silica-MB@OCP was caused by the strong hydrogen bond between H2O and powders [36]. The interaction between MB and powders was accordance with the shift of the ring stretching band of MB for silica-MB@OCP, in comparison with that for silica-MB as shown in Table 2. 3.4. Release behavior of MB 3.4.1. Release behavior of MB from silica-MB After silica-MB was etched by hydrofluoric acid, its loading capacity of MB was determined as 76.3 mg/g according to the absorbance of MB in the solution [37]. The release behavior of MB from silica-MB exhibited a slow rate in both PBS and lysosome-like buffer as shown in Fig. 4. After 72 h, only 8.4% and 11.8% of loaded MB were released to PBS and lysosome-like buffer, respectively. The diffusion of H+ ions into silica matrix would result in the protonation of orthosilicic acid (pKm S1 = 9.8 [38]), and thus weaken the electrostatic interaction between MB molecules and silica. Hence, Table 2 FTIR analysis results of silica-MB and silica-MB@OCP (ring stretching vibration mode: RSV, stretching vibration mode: SV). Sample RSV (MB) SV (COO−) SV (1(OH)) Silica-MB 1609.2 – – Silica-MB@OCP400 1613.7 1541, 1573 3237.4 Silica-MB@OCP2000 1611.2 1540, 1574 3220.6 Silica-MB@OCP6000 1607.4 Weak 3236.0 Silica-MB@OCP20001 1616.8 1548 3220.6 Silica-MB@OCP20003 1615.4 1541, 1576 3238.2 Silica-MB@OCP2000-cia1 1609.4 1547 3236.0 Silica-MB@OCP2000-cia3 1611.5 1540, 1574 3216.6
82C Li et aL /Colloids and Surfaces A: Physicochem. Eng, Aspects 472 (2015) 7884 silica-MB (in PBS)- silia-MB (in PBS)(a)(b)-o-silica-MB-o- silica-MB[(in lsosome-like buffer)(in lysosome-like buffer)0.8610aaae2030405060Time (h)Time (h)Fig. 4. Release behaviors (a) and ratio of Am/Ap (b) of MB molecules from silica-MB in PBS and lysosome-like bufferthe release rate of MBfrom silica-MB in lysosome-like buffer wasthesupernatant.ReleasingofMBmonomerdominatedatthebegin-higherthan that in PBS.ning for silica-MB,and the releasing ofMB dimer started to taketheInaqueoussolutions,monomersanddimersofMBhaveamax-leadasaresultofradialMBconcentrationgradientinsilicamatriximum absorbance (A)ata wavelength of664nm (Am)and610nm[1].InreactionsolutioncontainingMBwithhighconcentration,dye(Ap), respectively. As shown in Fig. 4, the ratio of Am/Ap was andimmers were formed and encapsulated in the silica nuclei duringindicativeoftheconcentration ofmonomerversus thatofdimerinthe sol-gel process (Scheme 1). With prolong of reaction time, the(a(b)1.4.arlica-MB@OCP400 silica-MB@OCP400A-silica-MB@OCP2000-silica-MB@OCP2000ilica-MB@OCP600 silica-MB@OCP60001.2"10Ca0.650405RTime (h)Time (h)(d)(c)-silica-MB@OCP20001o—silica-MB@OCP20001-silica-MB@OCP200034-silica-MB@OCP20003o4010.150enTime(h)Time (h)(f)o- silica-MB@OCP2000-CiA1o-silica-MB@OCP2000-CiA!- silica-MB@OCP2000-CiA3A-silica-MB@OCP200-CiA3230-210Time (h)Time (h)Fig. 5. Release behavior (a, c and e) of MB molecules released from silica-MB@OCP and ratio of Am/Ap (b, d and f) in PBS (solid symbols) and lysosome-like buffer (hollowsymbols)
82 C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 (a) (b) Fig. 4. Release behaviors (a) and ratio of AM/AD (b) of MB molecules from silica-MB in PBS and lysosome-like buffer. the release rate of MB from silica-MB in lysosome-like buffer was higher than that in PBS. In aqueous solutions, monomers and dimers of MB have a maximum absorbance (A) at a wavelength of 664 nm (AM) and 610 nm (AD), respectively. As shown in Fig. 4, the ratio of AM/AD was an indicative of the concentration of monomer versus that of dimer in the supernatant.Releasing ofMBmonomerdominatedatthe beginning for silica-MB, and the releasing of MB dimer started to take the lead as a result of radial MB concentration gradient in silica matrix [1]. In reaction solution containing MB with high concentration, dye dimmers were formed and encapsulated in the silica nuclei during the sol–gel process (Scheme 1). With prolong of reaction time, the (a) (b) (c) (d) (e) (f) Fig. 5. Release behavior (a, c and e) of MB molecules released from silica-MB@OCP and ratio of AM/AD (b, d and f) in PBS (solid symbols) and lysosome-like buffer (hollow symbols)
83C.LietaL./ColloidsandSurfacesA:Physicochem.Eng.Aspects472(2015)78-8490interval.For silica-MB@OCP synthesized with different addition ofPBSPEG or citric acid, the release rate of MB is relatively high from80lysosome-likebuffersilica-MB@0CP20003or silica-MB@0CP2000-CiA3,respectively.70 The improved sustained-releaseprofileof MBfrom silica-MB@OCPwas due to the presence of the network crossed by PEG, citrate(oeer60and oCP particles. The interaction density among them, that is, thesteric hindrance for outward diffusion of MB was varied with the50PEGchainlength,additionof PEG and CiA,which wassimilarwith40 the observations of red shift ofthe symmetric stretching band (u)ofOHbandsinH,Oandblueshiftofringstretchingbandof MBin5Fig.3.20At each duration, the ratio of Am/Ap of MB released from silica-MB@OCP in Fig. 5 was larger than that from silica-MB (see Fig. 4).10as a result of the steric hindrance of the network of OCP,PEG andcitrateionsontheoutwarddiffusionofMBdimersandmonomerssilica-MBsilica-MBThe ratioofAm/Ap decreasedfor MB released from silica-MB@OCPsilica-MBsilica-MB@OCP6000synthesized with increasing addition of PEG (Fig.5(d)) and CiA@OCP400@OCP2000(Fig. 5(f)). For silica-MB@0CP6000 and silica-MB@OCP2000-CiA3,Fig.6.Degradation percent of silica-MBand silica-MB@OCPafter72h of incubationthe value of Am/Ap of MB released at every time duration ininPBSandlysosome-likebufferlysosome-like buffer was higher than that released in PBS.dimers decreased after the consumption of MB and monomers of3.5.DegradationofsamplesinPBSand lysosome-likebufferMB were then loaded in the outside silica.In culture solution, the degradation of silica and ocP occurred3.4.2.ReleasebehaviorofMBfromsilica-MB@OCPaftertheoutwarddiffusionofMBasshowninFig.6.Theaver-ThereleasebehaviorsofMBfromsilica-MB@OCPwereinterestage degradation percent of silica-MB after immersion in PBS andingly dependent on both the pH value of culture solution and thelysosome-likebufferfor72hisabout28.8%and33.7%,respectivelyshell structure as shown in Fig. 5. Typically, about 81.7% and 81.6%Compared with that of silica-MBbefore degradation in Fig.2(a),theofMBwerereleasedfromsilica-MB@OCP-4OOinPBSandlysosome-particle size was hardly changed (Fig. 7(a)) after 72 h of culture inlike buffer,respectively. An initial burst release of 23.2% in 6 h wasPBS,indicating that thedegradation occurred from insideto out-followedbyasustainedreleasetillto72hofincubationinPBS.Inside.Inlysosome-likebuffertheparticlebecameswelled(Fig.7(b))contrast,after an initial burst release of 20.6% in 6h, thereleaseandtheoutwarddiffusionofMBwasenhancedduetotheinfluencerateremainedconstantandthenbecamefastagainataround24hofprotonationofsilica(-Si-OH)ontheinteractionbetweensilicain lysosome-likebuffer,where theprotonation ofcitrate ions (pKa:and MB.Thedegradation increased once silica network was broken,3.13,4.76and6.40)intheshell (Scheme1)mighthindertheoutwhich was consistent with the previous report [1].ward diffusion of MB molecules with positive charges.Only afterAs shown in Fig. 6, the average degradation percent of silica-theself-destructionofshellstructures,thediffusionofMBfromtheMB@0CP400isdeterminedas60.3%and70.2%after72hofculturehybrid powders was enhanced with a high release rate. Relative toinPBSandlysosome-likebuffer,respectively.TheOCPshellremainsthat from silica-MB@OCP40o, the release rates of MB from silica-almost intact after 72 h of incubation in PBS after the destructionMB@0CP6000 and silica-MB@0CP2000 decreased at every timeof core silica and the outward diffusion of loaded MB as shown(b)μumμm(d)μmFig.7. SEM characterization of silica-MB (a and b)and silica-MB@OCP-PEG40o (c and d)after culture in PBS (a and c)and lysosome-like buffer (b and d) for 72 h
C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 83 Fig. 6. Degradation percent of silica-MB and silica-MB@OCP after 72 h of incubation in PBS and lysosome-like buffer. dimers decreased after the consumption of MB and monomers of MB were then loaded in the outside silica. 3.4.2. Release behavior of MB from silica-MB@OCP The release behaviors of MB from silica-MB@OCP were interestingly dependent on both the pH value of culture solution and the shell structure as shown in Fig. 5. Typically, about 81.7% and 81.6% ofMB were released fromsilica-MB@OCP-400 inPBS and lysosomelike buffer, respectively. An initial burst release of 23.2% in 6 h was followed by a sustained release till to 72 h of incubation in PBS. In contrast, after an initial burst release of 20.6% in 6 h, the release rate remained constant and then became fast again at around 24 h in lysosome-like buffer, where the protonation of citrate ions (pKa: 3.13, 4.76 and 6.40) in the shell (Scheme 1) might hinder the outward diffusion of MB molecules with positive charges. Only after the self-destruction of shell structures,the diffusion of MB from the hybrid powders was enhanced with a high release rate. Relative to that from silica-MB@OCP400, the release rates of MB from silicaMB@OCP6000 and silica-MB@OCP2000 decreased at every time interval. For silica-MB@OCP synthesized with different addition of PEG or citric acid, the release rate of MB is relatively high from silica-MB@OCP20003 or silica-MB@OCP2000-CiA3, respectively. The improved sustained-release profile of MB from silica-MB@OCP was due to the presence of the network crossed by PEG, citrate and OCP particles. The interaction density among them, that is, the steric hindrance for outward diffusion of MB was varied with the PEG chain length, addition of PEG and CiA, which was similar with the observations of red shift of the symmetric stretching band (1) of OH bands in H2O and blue shift of ring stretching band of MB in Fig. 3. At each duration, the ratio of AM/AD of MB released from silicaMB@OCP in Fig. 5 was larger than that from silica-MB (see Fig. 4), as a result of the steric hindrance of the network of OCP, PEG and citrate ions on the outward diffusion of MB dimers and monomers. The ratio of AM/AD decreased for MB released from silica-MB@OCP synthesized with increasing addition of PEG (Fig. 5(d)) and CiA (Fig. 5(f)). For silica-MB@OCP6000 and silica-MB@OCP2000-CiA3, the value of AM/AD of MB released at every time duration in lysosome-like buffer was higher than that released in PBS. 3.5. Degradation of samples in PBS and lysosome-like buffer In culture solution, the degradation of silica and OCP occurred after the outward diffusion of MB as shown in Fig. 6. The average degradation percent of silica-MB after immersion in PBS and lysosome-like buffer for 72 h is about 28.8% and 33.7%, respectively. Compared with that of silica-MB before degradation in Fig. 2(a),the particle size was hardly changed (Fig. 7(a)) after 72 h of culture in PBS, indicating that the degradation occurred from inside to outside. In lysosome-like buffer,the particle became swelled (Fig. 7(b)) and the outward diffusion of MB was enhanced due to the influence of protonation of silica (–Si-OH) on the interaction between silica and MB. The degradation increased once silica network was broken, which was consistent with the previous report [1]. As shown in Fig. 6, the average degradation percent of silicaMB@OCP400 is determined as 60.3% and 70.2% after 72 h of culture inPBS andlysosome-like buffer, respectively. The OCP shell remains almost intact after 72 h of incubation in PBS after the destruction of core silica and the outward diffusion of loaded MB as shown Fig. 7. SEM characterization of silica-MB (a and b) and silica-MB@OCP-PEG400 (c and d) after culture in PBS (a and c) and lysosome-like buffer (b and d) for 72 h
84C Li et al. / Colloids and Surfaces A: Physicochem. Eng.Aspects 472 (2015)78-84in Fig.7(c).Inlysosome-likebuffer,the sizeof silica-MB@OCP400[11] Amaral,V.Miguel,J.Molnar,Antimicrobial activityof phenothiazines,Vivo18(2004)725-732.became smallafter the degradation of OCP shell and the surfaceof[12]A.M.Wang.Y.Morishima,K.M.Clapp.H.M.Peng.W.B.Pratt,J.E.Gestwicki,YsilicabecamesmoothasshowninFig.7(d),indicatingthecenter-Osawa,A.P.Lieberman,InhibitionofHsp7obymethyleneblueaffectssignalingout-first releasing pattern of MB [1].The decomposition of drugprotein function and ubiquitination and modulates polyglutamine proteindegradation.J.Biol.Chem.285(2010)15714-15723carrier,especiallywithafavor ofthecoatingofocP onsilicawould[13] J.P.Tardivo, G.A Del, LH. Paschoal, A.s. 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84 C. Li et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 472 (2015) 78–84 in Fig. 7(c). In lysosome-like buffer, the size of silica-MB@OCP400 became small after the degradation of OCP shell and the surface of silica became smooth as shown in Fig. 7(d), indicating the centerout-first releasing pattern of MB [1]. The decomposition of drug carrier, especially with a favor of the coating of OCP on silica would be crucial for its clinical application. 4. Conclusions A facile sol–gel method was employed to synthesize MB-loaded silica@OCP with different shell structures composed with PEG, citrate and OCP particles. The influence of molecular weight of PEG, addition amount of PEG and citric acid on the release behaviors of MB from silica-MB@OCP was investigated in PBS and lysosome-like buffer, respectively. The sustained-release behaviors were sensitive to the shell structure and the pH value of culture solution. Relative to thatfrom silica-MB,the ratio evolution of AM/AD of MB molecules released from silica-MB@OCP was varied for samples with different shell structures. After the outward diffusion of MB from the solid matrix, the degradation behaviors of silica-MB@OCP in PBS and lysosome-like buffer were significantly enhanced, relative to those of silica-MB. The investigations of release behaviors of MB from silica-MB@OCP and their degradation properties would shed light on the potential applications of drug delivery systems for their diagnostic or therapeutic functions of tumor issues. Acknowledgements The study was supported by the doctoral research fund of Shandong University of Technology (Grant No. 406017), the Science & Technology Plan of the Education Department of Shandong Province (Grant No. J11LD52), the Science and Technology Development Plan of Shandong province, China (Grant No. 2014GZX201008), and the Shandong Provincial Natural Science Foundation, China (Grant Nos. 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