Centre for Research in NanoScience and NanoTechnology, Technology Campus, University of Calcutta, JD 2, Sec III, Salt lake, Kolkata 700098, IndiaBiological Sciences Division, Indian Statistical Institute, 203 B.T. Road, Kolkata 700108, IndiaDepartment of Biotechnology, Birla Institute of Technology and Science, Vidya Vihar, Pilani, Rajasthan 333031, IndiaDepartment of Physiology, University College of Science and Technology, University of Calcutta, 92 A.P.C. Road, Kolkata 700009, India
r t i c l e i n f o
rticle history:eceived 28 October 2014eceived in revised form 26 May 2015ccepted 31 May 2015vailable online 6 June 2015
eywords:rug deliveryorous
a b s t r a c t
Targeted drug delivery with porous materials features great promise as improved therapeutic potentialfor treatment of various diseases. In the present study we have attempted a microwave synthesis ofporous hexagonal nanodisc of zinc oxide (PZHD) for the first time and its subsequent targeted deliveryto breast cancer cells, MCF7. PZHD has been fabricated suitably with 3-aminopropyltriethoxysilane toimpart additional stability and surface amines to anchor site directing ligand NHS-biotin. Biotinylatedscaffold showed targeted delivery of anticancer drug doxorubicin and pH triggered release to MCF 7 cellswith preferential distribution on specified domain. A detailed in vitro cytotoxicity study was associatedwith it to evaluate the mode of action of Dox loaded PZHD on MCF-7 cells by means of cell cycle analysis,
inc oxideytotoxicityntitumor agents
apoptosis assays, Western blot and immuno-fluorescence image analysis. The efficacy of the Dox loadedPZHD was further validated from our in vivo tumor regression studies. Finally, the whole study has beensupported by in vitro and in vivo bio-safety studies which also signified its biocompatibility with real timeapplications. To the best of our knowledge this is the first effort to use biotinylated PZHD for targeteddelivery of doxorubicin within MCF 7 cells with a detailed study of its mechanistic application. This studymight thus hold future prospects for therapeutic intervention for treatment of cancer.
. Introduction
The development of tumor-specific targeted drug deliveryTDD) systems as anticancer agents, which can recognize the inher-nt differences between normal and tumor cells, has been receivingncreasing attention in recent years [1]. To address this formidablehallenge diverse class of nanoscale targeted delivery of drugsncapsulated within nanocarriers such as drug polymer conjugates,
icelles, liposomes, dendrimers, and inorganic nanoparticles [2–4]
ave been developed to capitalize on enhanced permeability andetention (i.e., passive targeting) and endorse site-specific deliv-ry. Major advantages of a targeted drug delivery based platform
include (a) more specific targeting and delivery, (b) reduced RESuptake, (c) reduction in toxicity while maintaining therapeuticeffects, (d) greater safety and biocompatibility, (e) faster develop-ment of new safe medicines. Among all nanoscopic TDD systems,mesoporous silica nanoparticles have emerged as robust nanovec-tors for drug delivery because of their remarkable biocompatibilityand stability, among other features. Therefore, it is now well docu-mented that biocompatible porous nanomaterials are better suitedto device a targeted drug delivery platform. Although zinc oxideis regarded as one of the oldest nanostructure and is consideredas GRAS (generally recognized as safe), but only a few reports
are available to use zinc oxide nanostructures in drug delivery[5,6]. In this context Barick et al. have demonstrated 3D porousZnO nanostructures for drug delivery system (DDS) [7]. Later onZnO quantum dots based structures have been used to deviseTDD system [8,9]. However lack of availability of porous structure,
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uitable loading and release kinetics pushed researchers to drawheir attention on porous ZnO based nanostructures in TDD. More-ver, mesoporous nanospheres of ZnO have been used for deliveryf captopril [10] and also ZnO nanoparticle loaded with doxorubicinas been used for photodynamic therapy against cancer [11]. Mean-hile ZnO nanoparticles are known to be capable of selectivelyestroying cancer cells without affecting the normal cells [12,13].t the acidic lysosomal pH of cancer cells, ZnO nanostructures con-
ribute toward generation of greater reactive oxygen species (ROS)y partial dissolution. However, sporadic reports are present onDD of anticancer drugs with porous ZnO [14–16]. Here, we proposeorous hexagonal ZnO nanodisc (PZHD) for targeted drug deliverylatform with its potential utility in treatment of cancer. Advan-ages of selection of PZHD offers greater exposed surface in ordero entrap drug molecules during TDD. Furthermore, we have delib-rately selected doxorubicin (DOX) as the anticancer drug whichs known to intercalate with DNA during its action. Therefore DOXoaded targeted PZHD could be potent through site specific actionogether with selective destruction of cancer cells as mentionedarlier.
Tumor-targeting DDS consists of a tumor recognition moietynd a cytotoxic agent connected directly or through a suitable linkero form a conjugate [17]. The tumor-targeting DDS should be non-oxic, and stable in blood circulation and upon internalization intohe tumor cells the conjugate should be readily cleaved to releasehe active cytotoxic agent in specified domain. Usually cancer cellsver express many tumor-specific receptors, which can be used asargets to deliver cytotoxic agents into tumors. Cancer cells requireertain vitamins for their survival and rapid growth [18]. Conse-uently, the receptors involved in uptake of the vitamins are overxpressed on the cancer cell surface. For instance, monoclonal anti-odies [19], folic acid [14,20], aptamers [21], transferrin [22] haveeen employed as tumor-specific “guiding modules” to constructargeted drug delivery system. In this scenario, several reports have
entioned that biotin (vitamin H or B-7) is a growth promotert the cellular level, and its content in breast cancer cells is sub-tantially higher than that in normal tissues [18]. Accordingly, weave chosen biotin as the tumor-targeting molecule which bears aelf-immolative disulfide linker [18] for our targeted drug deliveryystem with the help of PZHD.
In the present study we have highlighted a systematic formationf PZHD under simple microwave assisted condition in presencef TRIS buffer. Versatility within the reaction condition guided uso the development of such unique structure. Suitable fabricationf PZHD by 3-aminopropyltriethoxysilane (APTES) was followedor its modulation. Simultaneous APTES grafting further produceddditional stealth within the structure and at the same time,endant the amine groups for anchoring our desired site direc-ing ligand biotin. Such a stable site directed scaffold favored theH stimulated sustained release of cargo (DOX) within our breastancer cell line of interest MCF-7. Associated in vitro study demon-trated potent anticancer efficacy of DOX loaded scaffold againstCF-7 cells while the pristine scaffold was fairly biocompatible
n nature. In addition fluorescent labeled targeted PZHD demon-trated the preferential distribution on cancer cells to rationalizehe anticancer efficacy. Quantitative in vitro studies delineated the
ode of action behind such targeted drug delivery. Advancementnd optimization of diverse strategies for improving the in vivoumor specific targeting efficiency of various drug delivery sys-ems is of critical importance, but very limited progress has beenchieved within in vivo models system till date. In this present
ork a series of systematic in vivo studies in murine model has
lso been done to revalidate the tumor specific targeting of biotiny-ated PZHD by TDD which has been supported by various in vitroxperiments. To best of our knowledge this is the first attempto produce unique PZHD and its successful fabrication, targeting,
Biointerfaces 133 (2015) 88–98 89
drug loading and pH triggered drug release to promote a targeteddrug delivery based platform together with detailed in vitro andin vivo study. Both in vitro as well as in vivo analysis glittered pos-itive response for a successful targeted drug delivery platform forpractical applications.
2. Materials and methods
2.1. Synthesis of porous hexagonal Zno nanodisc (PZHD)
PZHD was synthesized using our previously reported methodusing TRIS buffer as the growth directing agent [23] with somemodifications. Briefly, to a 25 mL of 0.05 M zinc acetate solution and20 mL of 15% TRIS solution was added with vigorous stirring. Themixture was then subjected to a domestic microwave heating 3 minat 300 watt. The resultant PZHD was centrifuged at 10,000 rpm andwashed several times with deionized water to remove excess ofTRIS buffer, finally the product was dried at 80 ◦C for overnight.
2.2. Amine functionalization of PZHD (PZHD-NH2)
Amine functionalization of PZHD was carried out by using APTESvia co-condensation reaction using our previously reported method[14,24]. In brief, about 0.5 g of PZHD was dispersed in about 50 mLof DMSO in a sonication bath for about 1 h. To it 400 �L of APTES wasadded and the solution was refluxed at 120 ◦C for about 3 h. Aftercompletion of the reaction the resulting amine functionalized PZHDwas centrifuged at 12,000 rpm for 15 min and washed several timeswith ethanol to remove the unreacted APTES. Finally the productwas dried at 60 ◦C for overnight to produce amine functionalizedPZHD (PZHD-NH2).
2.3. Biotinylation of amine functionalized PZHD (PZHD-BT)
Biotinylation of PZHD-NH2 was carried out following standardmethod with some modifications [25]. Briefly, 100 mg of PZHD-NH2was dispersed in 10 mL of DMSO in a sonication bath for 30 min.100 mg of NHS-biotin was dissolved in a mixture of 5 mL DMSOand 10 mL phosphate buffer (pH 8). Then PZHD-NH2 was addeddrop wise in NHS-biotin solution and stirred at room tempera-ture overnight. Finally the resultant mixture was centrifuged at10,000 rpm and washed several times with milliQ water for severaltimes to remove excess NHS-biotin from biotinylated PZHD-NH2(PZHD-BT).
2.4. RITC labeling of biotinylated PZHD (PZHD-BT-RITC)
Appropriate concentration of PZHD-BT was dispersed in 0.1 Msodium bicarbonate (NaHCO3) solution. 1 mg of RITC dissolved in2 mL of aqueous DMSO (1:1, v/v) was added instantly into it andthe reaction mixture was stirred at room temperature for 24 h ina dark condition. RITC conjugated PZHD-BT (PZHD-BT-RITC) wascentrifuged at 10,000 rpm at 4 ◦C to separate from solvent. PZHD-BT-RITC was washed in water repeatedly to remove excess of RITC[23]. A control particle was prepared using PZHD-NH2 for cellularuptake study.
2.5. Drug loading and release kinetics
Anticancer drug Doxorubicin (DOX) was loaded on to PZHD-BTto yield DOX loaded PZHD-BT (PZHD-BT-DOX) which is detailedin SI. Drug loading was evaluated UV–Vis spectrophotometri-cally by measuring the absorbance at 481 nm of a standard DOX
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olution and supernatant respectively [14]. The loading efficiencyas measured as:
(Total drug − Free drug)Total drug
× 100
Meanwhile cumulative drug release from PZHD-BT-DOX wastudied at two different pH (pH 5 and pH 7.4) sealed inside a dialy-is bag, detailed in SI. Drug release kinetics was monitored UV–Vispectrophotometrically by measuring the absorbance of the exter-al buffer at 481 nm.
.6. In vitro cytotoxicity study
.6.1. Cell viability study by MTT assay and Trypan blue assayMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
romide] assay was performed on MCF 7 cell line using threeifferent concentrations of PZHD-BT to check the toxicity of thisanocarrier [26]. Trypan blue assay was also carried out to validatehe results of MTT assay [27]. Details procedure of these two assayss described in SI.
.7. Apoptosis and cell cycle analysis using flow cytometry
Annexin-V/propidium iodide (PI) double assay was performedsing the Annexin V-FITC Conjugate Detection kit (BD biosciences)28]. Cell cycle analysis was also carried out according to previouslyeported methods [28]. Detailed procedures are described in SI.
.8. Cell migration study by wound healing assay and transwelligration assay
Breast cancer cell line MCF 7 was maintained following standardrotocol [10]. Details are described in SI. Migration was measuredy wound healing assay, in which cells were grown to 80% con-uence in 6-well plates, streaked with a sterile pipette tip, andllowed to recover in media. After 24 h, plates were visualizednder inverted microscope and migration determined by measur-
ng wound width using the image J software [29].Transwell migration assay was determined using transwell
hambers with an 8 mm-pore membrane (BD Biosciences). Cells1 × 104) were layered onto the top well in serum-free medium.he bottom chambers contained serum-supplemented mediumhat acted as a chemo-attractant. PZHD-BT-DOX treated media wassed in both the upper and lower chambers. The migration of cellsas allowed to proceed for 24 h at 37 ◦C. Cells that migrated to
he bottom of the insert were fixed, stained, and counted, and theercentage of migration was determined [30].
.9. Western Blotting and immunocytochemistry of Bcl-2, Bax,aspase 9 and p53 expression
Cells were scraped from 10-cm dishes and suspended in RIPAysis buffer (980 �L RIPA, 5 �L aprotinin, 5 �L PMSF, 5 �L EGTA and
�L Na3VO4) on ice. Collected cells were fractured by sonication once and then centrifuged at 10,000 × g, at 4 ◦C for 15 min. The proteinoncentration was determined using Bradford reagent and then0 �g of extracted protein in 4.5 �l of sample buffer (1.6 mL 1.25 Mris–HCl, 3.2 mL glycerol, 0.64 g SDS, 1.6 mL �-mercaptoethanol,.8 mL 0.5% bromophenol blue and 0.8 mL H2O) was denatured at00 ◦C for 10 min. Proteins were separated by 8% SDS-PAGE andhen electrophoretically transferred to a nitrocellulose membrane.
ubsequently, the membranes were incubated in the presence ofifferent primary antibodies at 4 ◦C overnight and then the mem-rane was incubated with different secondary antibodies at 37 ◦Cor 1 h. Finally, ECL solution was used for antibody binding andhemiluminescence of the membrane [31]. Immunocytochemistry
Biointerfaces 133 (2015) 88–98
of Bcl-2, Bax, Caspase 9 and p53 was carried out following standardprocedure (detailed in SI).
2.10. Cellular uptake study
Human breast cancer cell line MCF 7 with density of 2 × 104
cells per well were seeded into 24 well culture plate. After 24 hinterval culture medium was removed and fresh medium withPZHD-BT-RITC was added in each well. Intracellular localizationof PZHD-BT-RITC was observed on MCF 7 cells under fluorescencemicroscope (BD pathway 855) [14]. Cells were fixed by using 4%formaldehyde for 15 min. The samples were washed by PBS andthen treated with 0.1% Triton-X for permeabilisation of membrane.After that 1% bovine serum albumin (BSA) was added for 30 min;followed by staining the cells nuclei by Hoechst 33342 (1 mg mL−1)for 30 min and imaged by fluorescence microscope. In contrast,non-targeted nanoparticles PZHD-NH2-RITC was used as controlagainst the same MCF 7 cells.
2.11. Antitumor activity in vivo
The animal experiment was ethically approved by LaboratoryAnimal Ethics Committee. All experimental procedures were per-formed in conformity with institutional guidelines and protocolsfor the care and use of laboratory animals. EAC derived from breastadenocarcinoma is an aggressive and rapidly growing carcinomacommonly used for the evaluation of the effect of novel smallmolecules on tumor progression [32]. Six weeks-aged Swiss albinomice were taken and divided into three groups. Each group con-tained 5 mice. In three groups of mice EAC solid tumors weredeveloped. They were provided with standard pellet diet and tapwater under hygienic conditions. For EAC solid tumor formationmice were injected subcutaneously with 1 × 106 EAC cells in theadjacent groin region.
After 14 days of EAC injection (small size tumor was visible),mice were treated with different doses of PZHD-BT-DOX throughtail vein, at every alternate day. After 4 weeks, solid tumor volumewere measured with a slide caliper and was calculated accordingto the formula V = �/6 × D1 × D2 × D3 where D1 is the longitudinaldiameter, D2 is the anteroposterior diameter and D3 is the largestlateral diameter [33].
2.12. RNA analysis: in vitro and in vivo
Total RNA extraction and polymerase chain reaction withreverse transcription (RT-PCR) analysis were performed with theLight Cycler and SYBR Green system (Roche) and the Light CyclerSoftware 3 (Roche) was used for data analysis. The followingoligonucleotide primers were used for MCF 7 cells: P53 forward:5′-CTTTGAGGTGCGTGTTTGTG-3′, p53 reverse: 5′-AGAGGAGCT-GGTGTTGTT G-3′; GAPDH forward: 5′-CTTTGGTATCGTGGAAGG-ACTC-3′, GAPDH reverse: 5′-GTAGAGGCAGGGATGATGTTC-3′.The following oligonucleotide primers were used for mice (invivo): p53 forward: 5′-GAGTATACCACCATCCACTACAAG-3′, p53reverse: 5′-GCACAAACACGAACCTCAAAG-3′; GAPDH forward:5′-GCCTTCCGTGTTCCTACC-3′, reverse: 5′-CCTCAGTGTAGCCC-AAGATG-3′.
2.13. Biosafety study: in vitro and in vivo
2.13.1. In vitro toxicity studyIn vitro biosafety study includes haemolysis on human RBC and a
detailed analysis on human lymphocytes including WST assay, NOcontent measurement, LDH assay, ROS/RNS assay and ROS relatedanalysis. These are detailed in SI.
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.13.2. In vivo bio-safety studiesIn vivo long term toxicity study (through intravenous injection)
as carried out through a series of blood biochemical assays andistological analysis of different body organs on murine model sys-em (detailed in SI) [23].
.14. Mice brain cytotoxicity study
Mice brain mitochondria (after 24 h of treatment) were isolatedy previously described method [34,35]. Measurement of trans-embrane potential, phosphate utilization of mitochondria and
espiratory chain activity of brain mitochondria ware examined iniotinylated PZHD treated mice compare to control (detailed in SI).
. Results and discussion
.1. Physicochemical characterization of biotinylated PZHDPZHD-BT)
In the current study, a simple one step chemical syntheticethod of PZHD has been formulated from TRIS buffer and synthe-
ized PZHD has been fabricated with APTES. The entire assemblyas been characterized by different physico-chemical character-
zation tools. UV–Vis absorbance of PZHD (Fig. 1a) exhibited aignificant peak at 350 nm which resembled to that one obtainedy 20% TRIS [17]. This peak signified the formation of nanostruc-ure of ZnO and corroborated with other literatures as well [24].he position of the peak was found to be slightly shifted to 373 nm
uring amine grafting by APTES, and biotinylation [24]. PZHD wasrystalline in nature with nine crystalline peaks at 2� = 31.68◦
ig. 1. (a) UV–Vis absorbance spectra of synthesized PZHD, aminated PZHD (PZHD-NHiotinylated PZHD; (c) morphological analysis of PZHD by FESEM; (d) TEM image of PZHD
Biointerfaces 133 (2015) 88–98 91
unchanged even after amine functionalization by APTES [24]. Addi-tional peaks due to presence of impurities were avoided in thisprocess. It was worthy to mention that the crystalline patternremained unchanged with respect to that one used in 20% TRISconcentration [23]. TRIS concentration affected the nucleation pat-tern without altering their crystal structure [36]. The idea behindAPTES grafting was that it could introduce smart designing ofthe scaffold by inducing (a) acid stability to the PZHD relevantfor biomedical perspectives, (b) introduce sufficient surface aminegroups for anchoring the targeting ligand NHS-biotin, (c) at thesame time APTES itself could produce partial porous structure overthe hexagonal disc Associated study by Branuaer–Emmett–Teller(BET) isotherm revealed the surface area of 10.54 m2/g for PZHD and11.24 m2/g in its amine fabricated one (Fig. S1, SI). Both followed aregular type IV hysteresis curve of mesoporous structure for PZHDand its amine functionalized one. However porous structure andsurface area was almost unaltered in its amine functionalized formeven after post synthetic modification. Although the surface area ofour synthesized material was low in contrast to our previous report[14], but was expected with larger pore size of such hexagonal disc.In fact the surface area demonstrated for porous ZnO nanostruc-tures was found to be consistent with other literature reports aswell as [14].
Morphology of PZHD was verified by FESEM analysis (Fig. 1c),which demonstrated a hexagonal disc like patterning with a porousstructure. A simultaneous TEM analysis (Fig. 1d) also authenti-cated its porous structure. TEM analysis revealed that PZHD washexagonal in shape with a size around 200 nm. After amine func-
tionalization by APTES a thin layer of 2–4 nm over the hexagonaldisc was found over it, which gave the material a core–shell likestructure (Fig. S2, SI). This thin silica layer engendered surfaceamine for subsequent targeting, at the same time produced sta-bility of the ZnO nanostructure to some extent. PZHD was actually
2) and biotinylated PZHD(PZHD-BT); (b) X-ray diffraction pattern of PZHD and; inset illustrated a high resolution TEM image.
92 P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98
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he assemble of porous zinc oxide nanoparticles to give a hexago-al structure; when TRIS concentration was increased to 20% thisssemble of ZnO nanoparticle was broken to give spherical ZnOanoparticle. Therefore TRIS concentration played a pivotal role intructural variation of ZnO nanostructures [37]. Aggregation of suchanostructure resulted porous hexagonal nanodisc where porousoid structure with exposed surface allowed us to employ it foroading of anticancer drug required to devise DDS.
Biotinylation was confirmed by FTIR measurements (Fig. 2a) ofhe synthesized samples. PZHD exhibited surface hydroxyl groups3429 cm−1) which were consistent with our previous report [23].ZHD also exhibited the presence of carbonyl groups (1637 cm−1)nd lower band was assigned to Zn O stretching [23]. While aftermine functionalization by APTES the N H stretching (3429 cm−1)as overlapped with O H stretching frequency of hydroxyl groups.
H stretching appeared at around 2924 cm−1 of APTES frag-ent. N H scissoring vibration of primary amine appeared around
507 cm−1. Characteristic Si O and C N stretching of APTES frag-ent appeared at around 1010 cm−1 and 1126 cm−1 respectively.
his amination was deliberately used in conjugation with biotin.ZHD-BT exhibited a significant amide doublet at 1745 cm−1
amide I) and 1557 cm−1 (amide II) pertained to the binding ofiotin on to aminated PZHD (PZHD-NH2) [24]. Other significanteaks were unaltered within the biotinylated sample. Structuraltealth by APTES and subsequent pendant amine group on PZHDurface was further evaluated by EDX spectrum (Fig. S3a, SI).ncoated PZHD showed Zn, O as the chemical components whilefter amination by APTES additional peak of Si appeared alongith Zn and O. The amount of amine grafted on PZHD was quanti-ed by reversible p-nitrobenzaldehyde assay as described by Brucet al. [38,39]. p-nitrobenzaldehyde formed reversible imine withminated PZHD and released p-nitrobenzaldehyde in presence ofydrolysis buffer which was estimated accordingly. PZHD-NH2ontained 0.156 mM amine/mg of sample while PZHD-BT exhib-ted 0.02 mM amine/mg of sample. Reduction in amine contentn the later one was in accordance with the successful biotinyla-ion and corroborated with the previous results [14]. Meanwhileeta potential measurements also reiterated successful aminationnd subsequent biotinylation on PZHD. Zeta potential of PZHD wasound to be negative at both pH 5 and pH 9 (Fig. 2b), which wasttributed to the ionization of O H groups to O− resulting in a neg-
tive zeta potential. On the other hand a charge reversal was notedn PZHD-NH2, which acquired a positive zeta potential at acidic pH
due to ionization of surface amine to NH3+ groups resulting in a
ositively charged surface. The surface hydroxyl groups were com-romised to some extent as well. While in PZHD-BT the surface
T); (b) corresponding zeta potential measurements against pH. Values are mean ± SDtypical experiment. Error bars are smaller than the symbols due to small error.
amine was reduced at the expense of biotin as a result zeta poten-tial became negative; although it was low as the surface hydroxylgroups were already reduced during amination with APTES. Theseresults corroborated with previous amine quantification and FTIRresults mentioned earlier.
3.2. Drug loading and release kinetics study
Porous structure of the synthesized material allowed the load-ing of anticancer drug DOX for targeted delivery within MCF 7 cellsby biotinylation. Exposed hexagonal surface allowed its subsequenthigher loading efficiency within the pores of PZHD via hydrophilicinteraction which was monitored by measuring the absorbance at481 nm [13] using UV–Vis spectrophotometer (Fig. 3a). A sharpdrop in absorbance of the supernatant marked the loading ofDOX on to it. A high loading of around 63% was noted, whichwas attributed to its exposed surface and porous structure. Weevaluated the loading content of DOX within PZHD-BT is about0.42 mg/mg of PZHD-BT. Initial PZHD-BT was white in color butafter DOX absorption the color was changed to pink. A UV–Vis dia-gram of PZHD-BT loaded with DOX (PZHD-BT-DOX) exhibited acharacteristic peak at around 481 nm which justified required DOXloading on to it. In cumulative release profile, a pH triggered releaseof DOX was noted which established a cancer cell specific drugdelivery system (Fig. 3b). A minimum 34% release of DOX was notedunder pathophysiological pH of 7.4 for about 96 h while 83% DOXwas released at pH 5 with an initial burst release of 51% after 24 h.Therefore DOX was found to be preferentially released to MCF-7cells as revealed from the experiments carried under in vitro con-ditions. This would merit the pH triggered targeted drug deliverysystem to MCF-7 cells; this preferential release was attributed tospecific localization of DOX in acidic environment by protonation.At acidic pH of cancer cells PZHD-BT might undergo partial dissolu-tion which could also result site specific localization of DOX itself.Although the targeted platform shows a disc like structure but itssize was well retained in nano-regime in aqueous medium as evi-dent from hydrodynamic radius measurement (Fig. S3b, SI) by DLS.DLS analysis also demonstrated a good dispersity of PZHD-BT-DOXin aqueous medium. Although Zinc oxide nanoparticles have a ten-dency to undergo aggregation within solution and precipitate out,
but PZHD exhibited a stable dispersion (Fig. S4a and S4b, SI) andused for in vitro and in vivo study accordingly. Size of the PZHD wasalso within nanoregime and effectively less than the size of RedBlood Cells (Fig. S4c, SI), so it could also be injected during in vivoanalysis.
P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98 93
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.3. Anticancer activity study
Next we were prompted to check the effects of theseanocomposites on cancer cells. Cytotoxicity of the synthesizedanocomposite was tested by means of MTT assay. Cytotoxicityas determined on the basis of viability of cells in dependence of
he nanocomposite concentration against MCF 7 cells (Fig. 4a). TheC value of PZHD-BT-DOX was found to be prominently less in
50ontrast to that of DOX. Interestingly our observation revealed thatnly 11% of viable cells were present at the maximum concentra-ion of 20 �g mL−1 of PZHD-BT-DOX. A targeted drug delivery inontrast to only DOX resulted in such an overwhelming anticancer
ig. 4. (a) Relative cellular viability of MCF 7 cells against free DOX and PZHD-BT-DOX. Vaase or representative of typical experiment. (b) Cellular viability of normal MRC 5 cells aeviation) of three independent experiments in each case or representative of typical enalysis on MCF 7 cells at different doses of PZHD-BT-DOX. Values are mean ± SD (standypical experiment. (b) PZHD-BT-DOX induces apoptosis in human breast cancer cells MZHD-BT-DOX for 24 h. Representative figures showing population of viable (annexin V−
annexin V− PI+) cells.
r treatment with PZHD-NH2-BT; (b) cumulative DOX release profile from PZHD-BTndard deviation) of three independent experiments in each case or representative
efficacy. Using Trypan blue assay, we further counted the num-ber of live cells under treatments with different concentrations ofPZHD-BT-DOX for 24 h (Fig. S5, SI). Results of Trypan blue assayshowed that DOX loaded biotinylated PZHD at 20 �g mL−1 dosagesignificantly resulted in death of MCF 7 cells which was also inaccordance with the findings obtained from the MTT assay. Further-more, in order to judge the biocompatibility of PZHD-BT, cellularviability of this nanocomposite was assessed against normal human
fibroblast cell line MRC 5 (Fig. 4b). The results exhibited thatPZHD-BT delivered minimal toxicity in these cells. Three differentconcentrations like 100 �g mL−1, 250 �g mL−1 and 500 �g mL−1 ofPZHD-BT were used and it was found that nearly 68% of viable cells
lues are mean ± SD (standard deviation) of three independent experiments in eachgainst three different concentrations of PZHD-BT. Values are mean ± SD (standardxperiment. (c) Analysis of percentage of cell death by flow cytometric cell cycle
ard deviation) of three independent experiments in each case or representative ofCF 7. Flow cytometry analysis of MCF-7 cells treated with 10, 15 and 20 �g mL−1
PI−), early apoptotic (annexin V+ PI−), late apoptotic (annexin V+ PI+) and necrotic
94 P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98
Fig. 5. (a) Wound healing assay: inhibition of MCF 7 cells migration by PZHD-BT-DOX. Confluent monolayers of MCF 7 cells were wounded with a microtip (the solid yellowline indicates the edge of the wound). Control and PZHD-BT-DOX treated MCF 7 cells are showed in upper panel and lower panel respectively after 0, 12 and 24 h of treatment.( ontroe ZHD-s entatic t exp
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b) Representative bar diagram of PZHD-BT-DOX treated MCF 7 cells compare to cach case or representative of typical experiment. (c) Transwell migration assay: Ptaining of MCF 7 cells that migrated in the transwell assays is shown. (d) Represompare to control. Values are mean ± SD (standard deviation) of three independen
ere present at maximum dosage of 500 �g mL−1 with respect tohe control. Collectively these results confirmed that DOX loadediotinylated PZHD could be potent for targeted anticancer treat-ent. Furthermore we performed the flow-cytometric cell cycle
hase distribution analysis. Interestingly our observations revealedignificant death of the MCF-7 with increasing concentrations ofZHD-BT-DOX (Fig. 4c) in a dose-dependent manner.
To examine whether cells underwent apoptosis, untreated orZHD-BT-DOX treated MCF-7 breast cancer cells were stainedith annexin V and PI. Flow cytometry analysis of stained cells
an distinguish cells into four groups, namely viable (annexin V−I−), early apoptosis (annexin V+ PI−), late apoptosis (annexin V+I+) and necrotic (annexin V− PI+) cells. As illustrated in Fig. 4d,ZHD-BT-DOX exposure at different concentrations (10, 15 and0 �g mL−1) in MCF-7 cells; where dose-dependent increments of
ate apoptotic population (31± 6%, 67 ± 4%, 82 ± 5.5%) were noted.ess than 20% of population showed necrotic signs when treatedith high dosage (20 �g mL−1) of PZHD-BT-DOX.
We also examined the effect of PZHD-BT-DOX on cellular migra-ion of MCF 7 cells using in vitro wound healing assay. The assayypically measures migration of cells by measuring the closure of
standard scratch in time. We found that at 10 �g mL−1 dosage,ZHD-BT-DOX significantly retarded MCF 7 cell migration com-ared to control or untreated sets (Fig. 5a). These results wereurther reinstated from the findings of transwell migration assayFig. 5c).
.4. Targeted drug delivery study by CLSM
In spite of our physicochemical characterizations regarding suc-essful grafting of biotin on to PZHD and in vitro assays for studyingts anticancer efficacy, targeted cellular internalization study was
l. Values are mean ± SD (standard deviation) of three independent experiments inBT-DOX inhibits cellular migration of breast cancer cells MCF 7. Crystal violet dyeve bar diagram of transwell migration assay of PZHD-BT-DOX treated MCF 7 cellseriments in each case or representative of typical experiment.
very important. Confocal laser scanning microscopy (CLSM) wasused to confirm the targeting recognition capability of PZHD-BT.Fig. 6 shows the CLSM images of MCF 7 cells incubated withPZHD-BT-RITC (targeted particles) and only PZHD-NH2-RITC (nontargeted particles) after 24 h of incubation at 37 ◦C followed byHoechst staining. Interestingly it was noted that the cells incubatedwith PZHD-BT-RITC showed much higher fluorescence signal thanthat from PZHD-NH2-RITC. This result suggested that the targetedparticles were readily internalized within MCF-7 cells in contrastto the non-targeted ones. In fact this specific distribution wasattributed to successful targeting by biotin which correlated withour all previous analysis. Therefore as-prepared biotinylated PZHDcomposites could be a promising candidate for targeted anti-cancerdrug delivery.
3.5. Mode of action study
Although the mode of action was speculated by cell cycle anal-ysis and cellular internalization study, but it has been also welldocumented that anticancer activity would also affect the expres-sions of certain signaling molecules at both protein and mRNAlevels. Therefore western blot and immuno-fluorescence cellularimaging in correlation with in vitro studies would be a conclusiveevidence to establish the targeted drug delivery systems. It is wellacknowledged that apoptosis is a tightly regulated process underthe control of several signaling pathways [40] and these path-ways are complex. The Bcl-2 and Bax proteins have been shown
to be associated with anti-apoptotic and pro-apoptotic functions.Bcl-2, which is an anti-apoptotic protein, binds to Bax, therebyblocking Bax-induced apoptosis [41]. To explore the possible roleof Bcl-2 family members in the PZHD-BT-DOX induced apoptosis,the effects of PZHD-BT-DOX on the expression of Bcl-2 and Bax
P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98 95
Fig. 6. Fluorescence microscopic image of MCF 7 cells treated with PZHD-NH -RITC (control) after 24 h (a–c); MCF 7 cells under CLSM treated with PZHD-NH -BT-RITC after2 lls, mie cationr
pcdtsBaopcydscPpiPp9ocetawac
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2
4 h (d–f). Extreme left panel indicating Hoechst stained blue nucleus of MCF 7 cextreme right panel indicating the merged image (BD pathway 855 at 20× magnifieferred to the web version of the article.)
rotein was examined by western blot as well as immunocyto-hemical analysis. As shown in Fig. 7a, 20 �g mL−1 of PZHD-BT-DOXecreased the Bcl-2 protein level in contrast to the level of con-rol cells at 24 h. Bax protein expression on the other hand wasubstantially increased in MCF 7 cells after treatment with PZHD-T-DOX (Fig. 7a). Probably, Bcl-2 down regulation was caused via
p53 dependent pathway. We verified the effect of PZHD-BT-DOXn p53 protein expression in MCF 7 cells. A marked induction of53 protein was observed (Fig. 7a) in treated cells compared toontrol by both immunocytochemical as well as western blot anal-sis. This indicated that PZHD-BT-DOX induced apoptosis via p53ependent pathway, which was also consistent with other reportedtudies [42]. Results of RTPCR analysis of p53 protein of MCF 7ells also suggested that expression of p53 was increased in case ofZHD-BT-DOX treated cells compared to control (Fig. 7b). Caspaseslay a pivotal role in the terminal, execution phase of apoptosis
nduced by diverse stimuli [43]. Fig. 7a (lower panel) demonstratesZHD-BT-DOX treated MCF 7 cells resulted in cleavage of procas-ase 9 to active caspase 9 at 24 h. This result validated that caspase
was involved in PZHD-BT-DOX induced death pathway. Imagesf MCF 7 cells (Fig. 7c) treated with PZHD-BT-DOX obtained fromellular immunofluorescence study further strengthen our west-rn blot observations. Altogether it was found that PZHD-BT-DOXreatment induced the activation of tumor suppressor protein p53nd caused down regulation of anti apoptotic protein Bcl-2. Mean-hile the expression of pro-apoptotic protein Bax was increased
long with a synergistic activation of Caspase 9 in the treatedells.
.6. In vivo study in Swiss albino mice tumor
In vivo experimental studies are very much important for suc-essful anti cancer evaluation of PZHD-BT-DOX. The favorable anti
roliferation effect of DOX loaded biotinylated PZHD on cancer cellsffered us great confidence in its application in vivo for antitu-or therapy. So, in this study, we tried to employ PZHD-BT-DOX
o find out its inhibitory role in tumor growth in vivo. Firstly,AC derived from breast adenocarcinoma was injected into Swiss
2
ddle panel indicating red fluorescence of PZHD-NH2-RITC and PZHD-BT-RITC and). (For interpretation of the references to color in this figure legend, the reader is
albino mice to establish a malignant breast tumor model. Whentumor volume reached to about 150 mm3 (on the 12th day afterinoculation), PZHD-BT-DOX and only DOX were administrated totumor-bearing mice. Notable changes were observed after 7thday of injection and the tumors of treated mice were found togrow very slowly (Fig. 8a, upper panel). On the 7th day after thefinal administration, mice were sacrificed to measure the tumorweight (Fig. 8a, lower panel). This confirmed that PZHD-BT-DOXshowed the strong inhibitory effect compared to only DOX ontumor growth in vivo. Distinct histological changes were foundin treated tumor organs compared to control (Fig. 8b). The tumorexhibited small areas of necrosis in control comparable with thoseseen with PZHD-BT-DOX treatment. We also observed the differ-ences of tumor size and weight among control and treated groups(both only DOX and PZHD-BT-DOX) (Fig. 8c). These results showedthat the tumor weight and size in PZHD-BT-DOX treated groupwas significantly much lower than the control and DOX treatedgroup. A brief in vivo comparison with the non-targeted one wasalso accounted. We found that the non targeted one (PZHD-DOX)was less effective than the targeted one (PZHD-BT-DOX) (Fig. S6,SI). This result was attributed toward the cellular recognition bybiotin and targeted delivery of DOX within the tumor cells; how-ever in the non-targeted ones (PZHD-DOX) such sort of cellularrecognition is not possible therefore its effect is less. Therefore tar-geted drug delivery within the cancer cells by cellular recognitionthrough biotin resulted in the preferential release of DOX withinthe MCF7 cells over the normal cells. More importantly DOX alsohas a tendency to get release in acidic environment through proto-nation. Although unwanted release of Zn2+ was prevented howeverslight release of Zn2+ at such an acidic environment could not beruled out. We evaluated the release of Zn2+ by ICP-OES analysisand found that after 72 h (3.22 ± 0.01) ppm and (3.16 ± 0.01) ppmZn2+ was released from PZHD and PZHD-BT-DOX respectively
which might contribute to the response as well (Fig. S7, SI). Athin silica layer in PZHD-BT-DOX produced slight stability overthe PZHD. This ICP-OES analysis also confirmed that the specieswould be slowly degraded and excreted from the system in duecourse.
96 P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98
Fig. 7. (a) Expression levels of Bcl 2, Bax, Caspase 9 and p53 protein in MCF 7 cells treated with PZHD-BT-DOX were analyzed using western blot analysis. (b) p53 proteinexpression study of PZHD-BT-DOX treated MCF 7 cells by RTPCR. Values are mean ± SD (standard deviation) of three independent experiments in each case or representativeof typical experiment (� P < 0.001). (c) Cellular immunofluorescence images for the study of Bcl 2, Bax, Caspase 9 and p53 protein expressions in PZHD-BT-DOX treated MCF7 cells (BD pathway 855 at 40× magnification). Extreme left panels indicate DAPI stained blue nucleus of MCF 7 cells, middle panels denote green fluorescence of FITC taggeds erged
i
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econdary antibody of respective proteins and extreme right panel indicating the ms referred to the web version of the article.)
We also observed that the expression of p53 protein wasncreased in tumor cells of PZHD-BT-DOX treated mice compared toumor of control mice (Fig. S8, SI). So, it was concluded that PZHD-T-DOX was able to effectively inhibit the tumor growth in vivohich demonstrated its practical applications for future perspec-
ives. Although use of porous zinc oxide nanostructure in targetedrug delivery system is rare and to best of our knowledge this is therst report to use biotinylated porous hexagonal zinc oxide nan-disc for targeted drug delivery within breast cancer cells with aetailed mode of action by in vitro and in vivo studies.
.7. Biocompatibility study
Nanoparticles interacting with cells and the extracellular envi-onment can trigger a sequence of biological effects. These effectsargely depend on the dynamic physicochemical characteristics ofanoparticles, which determine the biocompatibility and efficacyf the intended outcomes. Although, no standard biocompatibil-
ty evaluation criteria have been established at present, but inur study, a series of toxicological evaluation through severalnzymatic assays were done to find out the biocompatibility ofhe synthesized material. PZHD-BT did not exhibit any significant
image. (For interpretation of the references to color in this figure legend, the reader
haemolysis under in vitro condition using human RBCs (Fig. S9, SI).Cellular cytotoxicity of human mononuclear cells (lymphocytes)treated with PZHD-BT were evaluated through WST assay, NOcontent and ROS content analysis (Fig. S10, SI). It did not show sig-nificant change at recommended dosage of PZHD-BT with respectto control. Overall PZHD-BT exhibited blood biocompatibility to beused for practical purposes.
Additionally we checked the immunochemical parameterswithin in vivo murine system during treatment of such a foreignparticle. A toxic particle would alter the immunochemical parame-ters as well as its subsequent responses. Interestingly insignificantmitochondrial membrane potential loss was observed after PZHD-BT treatment in Swiss albino mice at recommended dosage (Fig.S11a, SI). No distinct changes were found in inorganic phosphateutilization in treated samples compared to control as shown inFig. S11b (SI). Activities of Complex I, Complex II-III and Com-plex IV of treated brain mitochondria (Fig. S12a, Fig. S12b andFig. S12c respectively, SI) were almost unaltered at recommended
dosage. Only at 500 �g mL−1 dosage of PZHD-BT (which is muchmore higher than recommended dose as anticancer agent), moder-ate changes were found in brain mitochondrial activity in treatedsamples compared to control. At high concentration of PZHD-BT
P. Patra et al. / Colloids and Surfaces B: Biointerfaces 133 (2015) 88–98 97
Fig. 8. (a) Effect of PZHD-BT-DOX in mice tumor model: EAC cells (derived from breast adenocarcinoma) were injected to Swiss albino mice. After termination of experiments(4 weeks), mice were sacrificed and tumors were dissected out and photographed; upper panel – representative body images and lower panel – dissected tumor frome s werm y. Valc
(eangcpt
csirsl
xperimental mice. (b) Tumors were analyzed histopathologically and photographeasured weekly (until 4 weeks), analyzed statistically and represented graphicall
ase or representative of typical experiment (*P < 0.001).
500 �g mL−1) reactive oxygen species (ROS) was found to be gen-rated though at 50 �g mL−1 dosage (highest dose that had beenpplied for targeted drug delivery in in vivo model), there waso change with respect to the control (Fig. S13, SI). Mild ROSeneration gave no ambiguity as zinc oxide nanoparticles wereapable to produce ROS within cellular environment. Overall thesearameters demonstrated no significant alterations between con-rol and treated sets.
Finally we assessed the behavioral changes, blood biochemi-al parameters and histology of control and treated mice [23]. No
ignificant health deterioration and mortality was observed dur-ng experimental tenure; all the mice of treated set were showedegular body weight gain consistent with control. The mice wereacrificed after one months of intravenous injection of biotiny-ated PZHD. Blood and serum was collected, organs were sectioned
e taken at 40× magnification under normal microscope. (c) Tumor volumes wereues are mean ± SD (standard deviation) of three independent experiments in each
for histological analysis. Blood and serum biochemical parameterssuch as TC, DC, Platelet, LDH, creatinine, ALP, TP, cholesterol, TG,uric acid, BUN, SGOT, SGPT and phosphorous level were analyzedfollowing standard protocols [23]. Only minor rise in ALP, SGOT andSGPT was noted in the highest dose of treatment (Table 1 in SI).These results were also consistent with previous literature reportswhere zinc oxide nanoparticles exhibited ROS mediated damagein liver tissues [23]. On the other hand blood count parametersand kidney health regulating parameters such as creatinine, BUN,uric acid levels were restored to its normal value between controls
and treated sets. Histopathological analysis also corroborated withblood biochemical parameters. There were no significant changesin any of the organometric histological studies. Only mild changeswere found in liver tissue at highest dose of treatment. Althoughthat selected dose was much higher than the dose used in targeted
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rug delivery. Representative histological sections of major organsuch as brain, heart, lung, liver, spleen, kidney, testis, uterus arehown in Fig. S14 (SI). Overall, PZHD-BT is biocompatible withinurine model system with a very low cytotoxic effect at very high
oncentration.
. Conclusions
Throughout the world today, numerous researchers are explor-ng the potential use of nanoparticles as carriers for a wide rangef drugs for different therapeutic applications. In particular, thislass of carrier holds tremendous promise in the areas of cancerherapy and controlled delivery of vaccines. Cancer nano ther-peutics are rapidly progressing and are being implemented toolve several limitations of conventional drug delivery systemsuch as nonspecific bio distribution and targeting, lack of waterolubility, poor oral bioavailability, and low therapeutic indices.ere in, we demonstrated synthesis and formation of porousexagonal zinc oxide nanodisc, its fabrication and subsequent
unctionalization with biotin for targeted delivery of doxorubicinrug against biotin receptor over expressed breast cancer cell line.igh drug loading capacity was observed with a pH triggeredrug release in breast cancer cells. A detailed in vitro and in vivovaluation justified successful development of porous nano Zincxide based targeted drug delivery systems. Cell migration assays,ellular viability assays and apoptosis assay also confirmed thenhanced anticancer effect of PZHD-BT-DOX against breast cancerells than only DOX. Fluorescence imaging clearly demonstratedhat the nanoparticles deliver drug molecules into cells and havencreased specificity toward cancer cells. Possible mode of actionf this targeted drug was also evaluated by several immunoblot-ing, immunocytochemical experiments. In brief, PZHD-BT-DOXriggers p53 protein and enhances its activity to apoptotic deathf cancer cells. In vivo and in vitro toxicity evaluation verifiedhe biocompatibility of PZHD-BT as well. Even this targeted plat-orm was capable of tumor suppression within in vivo system asevealed from tumor regression studies in murine solid tumorodel. Advancement of these nanoscale targeted drug delivery sys-
ems enhances the potential for the development of smart nanoevices that may facilitate the realization of individualized can-er therapy. To best of our knowledge this is the first report tose biotinylated porous hexagonal zinc oxide nanodisc for tar-eted drug delivery of doxorubicin within MCF-7 cells associatedith a detailed in vitro and in vivo studies. Although develop-ent of such theranostic platforms are in an early stage but we
nvisioned PZHD prospect in successful targeted drug deliveryystem.
cknowledgements
We acknowledge UGC, GoI sponsored Central Instrumentalacility, Center for Research in Nanoscience and Nanotechnol-gy, University of Calcutta. P.P is grateful to UGC, GoI for Dr..S. Kothari PDF. SM is thankful to CSIR, GoI for SRF. This workas generously supported by grants from the DBT, GoI (Grant no:
T/PR15217/NNT/28/506/2011-2015; BT/BIPP0439/11/10). We arelso grateful to the ICAR, GoI (Grant no: NAIP/Comp-4/C3004/2008-014; NFBSFARA/GB-2019/2011-2015; NFBSFARA/CA-4-15/2013-6). We also grateful to Dr. Swatilekha Ghosh, Bose Institute forer kind suggestions to improve the manuscript.
[[[[[
Biointerfaces 133 (2015) 88–98
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.05.052
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