《材料测试技术及方法》课程教学资源（讲稿）实验四 红外光谱谱图分析 参考资料_06-FTIR silica-MB@octacalcium phosphate powders with different shell structures

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 silica￾MB@OCP. • The structure-destruction of silica￾MB was promoted after the deposi￾tion 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 silica￾MB@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 self￾destruction of drug carrier for safe excretion from the biological system [1]. The surfaces of inorganic powders, like silica and cal￾cium phosphate, are hydrophilic and favorable for cellular uptake. Silica is generally accepted as a promising carrier due to its non￾toxicity 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), lumi￾nescent 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 infec￾tion [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 under￾goes 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 antipro￾liferative 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 nega￾tively 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 forma￾tion 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 concen￾tration 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 interest￾ing 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 MB￾rich 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 octacal￾cium 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 modula￾tion 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 ammo￾nia (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 hydro￾gen 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 stir￾ring. 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 morphol￾ogy 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 spec￾trophotometer (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 silica￾MB@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 phos￾phate (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), silica￾MB@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 silica￾MB@OCP became broad (Fig. 1) and the crystallite size decreased (see Table 1), indicating that PEG and CiA affected the crystalliza￾tion 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, silica￾MB@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 stretch￾ing 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 asym￾metric (as(COO−)) and symmetric stretching (s(COO−)) models for the carboxylate group of citrate ion, respectively. The differ￾ence 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), indi￾cating 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 inter￾action 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 pro￾tonation 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 max￾imum 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 begin￾ning 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)