Amrita center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Center, Amrita Vishwa Vidyapeetham(University), Kochi, India
Received 2 May 2013; accepted 21 October 2013
nanomedjournal.com
Abstract
A multifunctional core-shell nanomedicine capable of inhibiting the migratory capacity of metastatic cancer cells followed by impartingcytotoxic stress by photodynamic action is reported. Based on in silico design, we have developed a core-shell nanomedicine comprising of~80 nm size poly(lactic-co-glycolic acid) (PLGA) nano-core encapsulating photosensitizer, m-tetra(hydroxyphenyl)chlorin (mTHPC), and~20 nm size albumin nano-shell encapsulating tyrosine kinase inhibitor, Dasatinib, which impair cancer migration. This system wasprepared by a sequential process involving electrospray of polymer core and coacervation of protein shell. Cell studies using metastatic breastcancer cells demonstrated disruption of Src kinase involved in the cancer migration by albumin–dasatinib nano-shell and generation ofphotoactivated oxidative stress by mTHPC-PLGA nano-core. This unique combinatorial photo-chemo nanotherapy resulted synergisticcytotoxicity in ~99% of the motility-impaired metastatic cells. This approach of blocking cancer migration followed by photodynamic killingusing rationally designed nanomedicine is a promising new strategy against cancer metastasis.
Metastasis is the dissemination of cancer cells from itsprimary locations to distant organs and this unique phenomenonis a devastating aspect of cancer. Despite clinical advancementsin the management of cancer, metastasis stays as one of the prime
All sources of support for research.MK thanks European Commission for support through Marie Curie
International Incoming Fellowship (Return phase, project No: 9085507).Authors thank Department of Science and Technology (DST), India throughthe project “Theragnostics, Regenerative Medicine and Stem Cell Researchusing Cell-Targeted Nanomaterials” (SR/NM/NS-99/2009). Giridharan L Mis thankful to Indian Council of Scientific and Industrial Research (CSIR) forSenior Research Fellowship.
⁎Corresponding author at: Amrita Center for Nanosciences andMolecular Medicine, Amrita Institute of Medical Sciences and ResearchCenter, Kochi, India.
Please cite this article as: Malarvizhi G.L., et al., A rationally designed photo-breast cancer cells followed by photodynamic killing. Nanomedicine: NBM 20
reasons for cancer associated death. In order to metastasize,cancer cells must detach from its primary location, migrate intothe surrounding stroma, intravastate into circulation, extravas-tate, and successfully re-establish growth at different sites.1
Interplay of numerous signal transduction events and celladhesion molecules conduce to cancer migration and metastasis.For example, E-cadherin is a transmembrane protein that ensuresand mediates cell–cell contacts within tissues.2–5 Thus, intactE-cadherin functions as a negative regulator of cancer migration.However, when Src kinase is hyperphosphorylated, E-cadherinis internalized and the junctions of cell–cell contacts are lost.This can provide essential motility for cancer cells to initiatemigration.6 Similarly, Src controls integrins and focal adhesionkinases (FAK) which are critically involved in cancer migration,because the autophosphorylation site of FAK at Y397 residue isalso an SH2 domain for Src. Hence, hyperactivated Src results
chemo core-shell nanomedicine for inhibiting the migration of metastatic14;10:579-587, http://dx.doi.org/10.1016/j.nano.2013.10.006
Figure 1. (A1)Modeled structure of Src inhibitor, dasatinib. (A2) Dasatinib docked with active site of albumin nano-shell. (A3-A4) Closer view of dasatinib–albumin interaction revealing the interacting amino acid residues. (B1)Modeled structure of photosensitizer, mTHPC. (B2) mTHPC docked with active site ofalbumin. (B3-B4) Closer view of mTHPC-albumin interaction revealing the interacting amino acid residues. (C1)Modeled structure of PLGA nano-core. (C2)PLGA nano-core interacting with active site of albumin nano-shell. (C3-C4) Closer view of PLGA-albumin core-shell interaction revealing the interactingamino acid residues. Atoms color code: red, oxygen; green, carbon; blue, nitrogen; yellow, chlorine; white, hydrogen. Docked drug in albumin is shown asmagenta sphere (A2, B2, C2). Docking simulations were performed using AutoDock 4.2.
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in the phosphorylation of additional tyrosine residues on FAKthrough phospshotyrosine–SH2 interactions, leading to aggres-sive cancer cell migration and invasion.7 Furthermore, Srccontrols the formation of invadopodia, the actin rich protrusionsin cancer cell–substratum contact points that function in thedegradation of ECM in metastatic breast cancer.8 All theseevidences suggest that inhibiting hyperactive Src kinase is apromising strategy to impair cancer migration by re-stabilizingE-cadherin, down regulating FAK and integrins, and inhibitingthe polymerization of invadopodia.
However, inhibition of migration alone is not sufficient tocontrol metastasis. Being a transient phenomenon, inhibition ofcell migration may need to be substantiated with killing ofmetastatic cells. This means, imparting cytotoxic stress to themigration-impaired cells either by chemotherapy or radiationtherapy is essential to yield desired therapeutic outcome. Thisdemands a combinatorial approach for: a) stopping cancer cellmigration by inhibiting the underlying molecular mechanismsand, b) imparting cytotoxic stress using a suitable method.Accordingly, in this paper, we are probing the possibility ofdeveloping a multifunctional nanomedicine that can addresssuch challenging multi-targeted approaches. Here, we report anovel core-shell nanomedicine construct that is rationallydesigned to impair the migratory capacity of metastatic breastcancer cells (MDA-MB-231) by the action of albumin nano-shell loaded with anti-migration drug, dasatinib, followed byimparting cytotoxicity by photo-triggered release of reactiveoxygen stress from PLGA nano-core loaded with photosen-sitizer, mTHPC.
Methods
The detailed methodology of the experiments is given in thesupplementary information.
Results
In silico docking analysis
In order to rationally select the appropriate carrier for en-capsulating drugs, prior to wet chemical synthesis, we haveperformed in silico docking simulations and studied possiblechemical interactions between the selected chemodrugs and drugcarriers. Figure 1, A1-A4 shows the interaction of Src inhibitor,dasatinib, with human serum albumin nano-shell. Enlarged viewof interacting residues (amino acids) can be seen in Figure 1, A3and A4. The results indicate strong hydrophobic interaction ofdasatinib with albumin resulting into a binding-energy (B.E.) −10.76 kcal/mol through the combination of van der Waals forces,electrostatic and intermolecular interactions. The best bindingenergy structure conformation suggested that nitrogen andoxygen atoms of dasatinib could closely interact with Lys 195,Lys 199, Arg 218, Arg 222 and Ala 291 residues of albuminthrough hydrogen bonds, and Trp 214, His 242, Ile 290, and Asp451 through hydrophobic interactions, which further stabilizesthe binding of dasatinib with albumin. In contrast, the photo-sensitizer drug, mTHPC (Figure 1, B1-B4) showed relativelyless interaction with albumin (B.E. −7.5 kcal/mol). To study thenature of interaction between PLGA nano-core and albumin
Figure 2. (A) Schematic on the processing steps of core-shell nanomedicine. (B) AFM image of mTHPC-PLGA nano-core. Inset: DLS graph showing thehydrodynamic diameter of nano-core (~70 nm). (C) SEM image showing the morphology of core-shell nanomedicine. Inset: DLS graph showing thehydrodynamic diameter of core-shell nanomedicine (~95 nm). (D) TEM image of core-shell nanomedicine. (E) TEM image showing the morphology of anisolated core-shell nanoparticle revealing the distinct nano-core and shell structure. (F) FT-IR spectra of mTHPC-PLGA nano-core, dasatinib–albumin nano-shell, and core-shell nanomedicine.
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nano-shell at core-shell interface, we have docked these twomolecules (Figure 1, C1-C4) and found moderately stronghydrophobic interactions with B.E. −7.2 kcal/mol. This indicatesthe possibility for a stable interaction between PLGA core andalbumin shell at the core-shell interface. The list of interactingatoms in dasatinib, mTHPC, and PLGA with the hydrophobicdrug-binding resides of albumin is shown in supplementarytables S1, S2, and S3, respectively.
Preparation and characterization of core-shell nanomedicine
Schematic representation of the core-shell nanomedicinesynthesis is depicted in Figure 2, A. We have first preparedmTHPC loaded polymer nano-core using electrospray tech-nique and the nano-shell of dasatinib–albumin was formed byethanol coecervation9. Figure 2, B shows AFM image ofelectrosprayed mTHPC-PLGA nano-core having size of~80 nm. Figure 2, B inset presents DLS measurement showinghydrodynamic diameter of nano-core ~80 nm, which is inconsistence with the AFM data. Figure 2, C shows SEMimage of core-shell nanomedicine indicating a size of~100 nm. This suggests the formation of a nano-shell withthickness ~20 nm over the core. This was verified by TEMimaging (Figure 2, D and E) which clearly showed theformation of a unique polymer nano-core having sphericalmorphology decorated with the protein nano-shell as originallydesigned. Zeta potential of PLGA nano-core was ~−15.5 mVand that of nano-shell was ~−12.4 mV. Drug loading andencapsulation efficiency of mTHPC in PLGA was found to be2.3% and 44% respectively, and that of dasatinib in albuminwas 1.8% and 94% respectively.
FT-IR studies of core-shell nanomedicine
FT-IR spectra of the nano-core, nano-shell, and core-shellsystem are separately shown in Figure 2, F. Presence of infraredpeaks at 3430 cm−1 and 1647 cm−1 in the FTIR spectrum ofmTHPC-PLGA nano-core indicated hydroxyl groups andprimary/secondary amines of mTHPC respectively.10 Aromaticamines in porphyrin structure of mTHPC were also evident fromthe peak at 1288 cm−1. This peak might also overlap with car-boxylic acid and ester linkages in PLGA matrix. The presence ofketone groups in PLGA was represented by peak at 1121 cm−1.In the case of nano-shell, the peak at 1642 cm−1 indicated C = Ostretching vibrations from RCONHR ring of amide class indasatinib and albumin.11,12 In core-shell nanomedicine, additiveIR peaks of both the core and shell could be clearly seen.
Photophysical properties of core-shell nanomedicine
Figure 3, A shows fluorescence emission spectrum of 2.5 μMmTHPC-PLGA nano-core exhibiting ~38 fold enhancement inemission intensity (λ652 nm 2.75 × 106) compared to equimolarconcentration of free mTHPC in aqueous phase (λ652 nm
7.2 × 104). The inset photograph also showed excellent brightred fluorescence from 2.5 μMmTHPC-PLGA upon U.V. illumi-nation (365 nm), whereas equimolar concentration of freemTHPC showed very less fluorescence. Fluorescence basedphotobleaching studies (Figure 3, A inset) under laser irradiationat 652 nm yielded little useful data for free mTHPC, possibly dueto aggregation in aqueous phase. In contrast, core-shell nano-formulation containing 2.5 μM mTHPC in PLGA core showedtypical photobleaching characteristics of monomeric mTHPC,even though the sample was dispersed in aqueous phase. The
Figure 3. (A) Comparison of fluorescence emission of mTHPC-PLGA nano-core (red), and free mTHPC (blue) in the aqueous phase. Inset photograph showsenhanced red fluorescence from mTHPC-PLGA nano-core upon U.V. illumination (365 nm). Inset plot: Photobleaching characteristics of mTHPC-PLGA nano-core (red) and free mTHPC (blue) in the aqueous phase. (B) Comparison of singlet oxygen emission of mTHPC-PLGA nano-core (red) and free mTHPC (blue)in the aqueous phase. Inset shows integrated photoluminescence (PL) intensity of singlet oxygen emission from mTHPC-PLGA nano-core (red) and freemTHPC (blue) over 50 min under light irradiation. (C) Comparison of UV-visible absorption characteristic of dasatinib–albumin nano-shell (red) and freedasatinib (blue) in the aqueous phase, indicating the co-existence of protein (280 nm) and drug (325 nm). (D) Controlled release of dasatinib from albuminnano-shell in acidic (pH4), neutral (pH7), and basic (pH10) conditions in cell-free aqueous medium (PBS).
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complete dataset of the photophysical characterization is given insupplementary information (Figure S1, A1 and A2).
Singlet oxygen release properties of core-shell nanomedicine
Figure 3, B shows singlet oxygen (SO) emission spectrum ofmTHPC-PLGA nano-core and free mTHPC in the aqueousphase. Core-shell nanomedicine containing 2.5 μM mTHPC inPLGA core exhibited ~3 fold enhancement in SO intensity (SOλ526 nm~9 × 105) compared to equimolar concentration of freemTHPC in aqueous phase (SO λ526 nm~3 × 105). Fluorescencebased SO emission studies at 526 nm (Figure 3, B inset) showedvery weak SO emission from 2.5 μM free mTHPC in theaqueous phase, whereas equimolar concentration of mTHPC-PLGA nano-core showed a steady state increase in SO emissionintensity with light irradiation, indicating the release of SO fromthe nano-core to the aqueous phase. The complete dataset ofsinglet oxygen studies is given in supplementary information(Figure S1, B1 and B2).
Physicochemical characterization of core-shell nanomedicine
The interaction of hydrophobic drug dasatinib with albuminnano-shell of core-shell nanomedicine was studied using UV-visible absorption spectrum (Figure 3, C). While 20 μM free
dasatinib showed absorbance at 325 nm with intensity ~2.0,core-shell nanomedicine containing equimolar concentration ofdasatinib-bound-albumin showed ~2 fold increase in the ab-sorbance value (3.8). A separate absorbance at 280 nm in nano-shell indicates the presence of albumin together with dasatinib.
Drug release studies in core-shell nanomedicine
The drug release studies performed in dasatinib–albuminnano-shell of core-shell nanomedicine are shown in Figure 3, D.The results indicate that dasatinib is released from albumin shellin a controlled manner, regardless of variations in the pH ofdispersion medium. Also, there was only ~8% burst release ofthe drug in all the three (acidic, neutral, and alkaline) pH ranges,probably due to the strong hydrophobic interactions betweendasatinib and albumin, as predicted by the docking studies.~20% release of dasatinib was observed from the protein shell incell-free aqueous medium (PBS) after 48 h incubation, althoughmore amount of dasatinib might be released in intracellularregions upon incubation with cells due to drug diffusion coupledwith proteolytic degradation in albumin shell in the cellularmicroenvironment. mTHPC, being a highly hydrophobicphotosensitizer, did not show detectable amount of release inthe cell-free aqueous medium.
Figure 4. Comparison of cell migration in (A)MCF-7 cells and (B)MDA-MB-231 cells before and after core-shell nanomedicine treatment, as determined bycell migration (scratch) assay. Comparison of morphological alteration (actin cytoskeleton) in MCF-7 and MDA-MB-231 cells before and after nanomedicinetreatment. MCF-7: (A1) before, and (A2) after treatment; MDA-MB-231: (B1) before, and (B2) after treatment.
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Cell migration and cytoskeleton impairment activity ofcore-shell nanomedicine
In cell studies, at first we have enquired the effect ofdasatinib–albumin nano-shell in cancer cell migration and cyto-skeleton alterations. Figure 4, A, B shows cell migration (scratch)assay performed in core-shell nanomedicine treated cancer cells.While untreated cancer cells (Figure 4, A, B, left side) pro-liferated quicker, migrated and sealed up to ~80% of the scratchwithin 24 h, core-shell nanomedicine (containing 5 μM dasati-nib in albumin shell) treated cells (Figure 4, A, B, right side)showed substantial reduction in the migratory potential with only~15% of the scratch being sealed up in 24 h. Core-shellnanomedicine containing 2.5 μM mTHPC in nano-core alone(without dasatinib in shell) showed no effect in the migrationof MDA-MB-231 cells (supplementary information, Figure S2,A1-A3) indicating that the inhibition of cell migration is effectedby dasatinib–albumin nano-shell. Although MDA-MB-231treated with 5 μM free dasatinib initially showed inhibition incell migration, the free drug treated cells eventually regained themigratory capacity in 24 h (supplementary information, FigureS2, B1-B3). Figure 4, A1, B1 presents untreated cancer cellsshowing intact actin cytoskeleton, and prominent filopodialextensions. In contrast, core-shell nanomedicine (containing5 μM dasatinib in albumin shell) treated cancer cells (Figure 4,A2, B2) showed significant impairment in cytoskeleton withdisrupted actin filaments. Immunoblotting experiment conductedin the core-shell nanomedicine (5 μM dasatinib–albumin nano-
shell) treated cells showed effective inhibition of phospho-Srcin MDA-MB-231, while the level of total Src remained unaltered(Supplementary information, Figure S2, C).
In vitro cytotoxicity studies of core-shell nanomedicine
In the next step, we have studied the cytotoxicity of 2.5 μMfree mTHPC (M), core-shell nanomedicine containing 2.5 μMmTHPC-PLGA nano-core (MP) alone, and two separate formu-lations of core-shell nanomedicine: 2.5 μM mTHPC in PLGAcore, and, a) 1 μM, and b) 5 μMdasatinib in albumin shell (DA),in MCF-7 cells under dark (dark toxicity) as well as light con-ditions (light toxicity). Interestingly, IC50 value of bothdasatinib and mTHPC in the core-shell nanoformulation wasfound to be significantly improved compared to that of therespective free drug combination (Supplementary information,Figure S3, S4, S5). As noticed from Figure 5, A1, equimolarconcentrations of both free mTHPC and core-shell containingmTHPC-PLGA in nano-core has produced only slight darktoxicity (~20%) in MCF-7 cells. Despite combining dasatinib–albumin (DA: 1 μM and 5 μM) with 2.5 μM mTHPC-PLGAnano-core in the core-shell formulation, in the absence of lightirradiation, only a marginal improvement (~20%) in cytotoxicitywas observed in MCF-7 cells, and ~60% of MCF-7 cells werestill metabolically active. However, when the core-shellnanomedicine was photoactivated (Figure 5, A2), mTHPC inthe nano-core killed ~80% of MCF-7 cells, leaving ~20% of thecells viable, when dasatinib concentration in the nano-shell (DA)
Figure 5. MTT assay shows the cell viability of core-shell nanomedicine treated MCF-7 cells (A1) before, and (A2) after light irradiation. Flow cytometrydata show enhanced intracellular ROS in (B3) core-shell nanomedicine treated MCF-7 cells compared to (B2) free mTHPC treated, and (B1) untreated cells.Optical microscope images show the cell morphology of MCF-7 cells (C1) before core-shell nanomedicine treatment, (C2) after treatment, but before lightirradiation, and (C3) after light irradiation of nanomedicine. (M: free mTHPC, MP: mTHPC-PLGA nano-core, MP/DA: core-shell nanomedicine containingmTHPC in PLGA core and dasatinib in albumin nano-shell).
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was 1 μM. When dasatinib in albumin nano-shell (DA) wasincreased to 5 μM, the photoactivated core-shell nanomedicine(containing 2.5 μMmTHPC in PLGA core) exerted a synergisticcytotoxicity, killing ~98% of the motility-impaired MCF-7 cells.Flow cytometry analysis (Figure 5, B1-B3) performed in core-shell nanomedicine (containing 2.5 μM mTHPC in PLGA core,and 5 μM dasatinib in albumin shell) treated cells indicated thegeneration of intracellular ROS in 61.2% of MCF-7 cells afterlight irradiation of the nano-core (Figure 5, B3). We have alsostudied the alterations in cell morphology in response to core-shell nanomedicine treatment using optical microscopy. Figure 5,C1 shows the intact cytoskeleton of untreated MCF-7 cells andFigure 5, C2 shows the morphology of cytoskeleton impairedMCF-7 cells before light irradiation of the core-shell nanomedi-cine. When mTHPC-PLGA nano-core was photoactivated, thecytoskeleton impaired cells showed membrane blebbing, indi-cating apoptotic cell death (Figure 5, C3) In MDA-MB-231 cells,as seen from Figure 6, A1, equimolar concentration of both freemTHPC (M) and mTHPC-PLGA nano-core (MP) has producedonly ~15% dark toxicity. However, when the concentration ofdasatinib in albumin nano-shell was increased to 5 μM (DA:5 μM), ~80% of the cytoskeleton impaired MDA-MB-231 cellsregistered toxicity even before light irradiation of nano-core.May be these metastatic cells are more prone to migration-impairment related stress. When the nano-core (of core-shellnanomedicine) was photoactivated (Figure 6, A2), a synergistic
cytotoxicity led to the killing of ~99% of migration impairedcells. Flow cytometry data (Figure 6, B1-B3) on the generationof intracellular ROS indicated high ROS in ~62.4 % of MDA-MB-231 cells after light irradiation (Figure 6, B3). Figure 6,C1 shows the normal morphology and intact cytoskeleton ofMDA-MB-231 cells before the nanomedicine treatment, whereasFigure 6, C2 shows distinct change in the morphology of MDA-MB-231 cells before light irradiation, due to the effect of thenano-shell. After photoactivation of the nano-core, almost all thecore-shell nanomedicine treated cells showed classic microscop-ic features of apoptotic cell death (Figure 6, C3).
Discussion
Metastatic breast cancer utilizes multiple factors for its survivaland invasion, and even with all possible treatment options, themedian survival rate remains at ~18 months. Metastasis is one ofthe major reasons for more than 90% of cancer-associatedmortality13 and hence, one of the promising therapeutic strategiesis to impair the migratory capacity of metastatic cancer cells andthen destroy the cells. Cancer migration is a tightly regulatedpathophysiological event, and it is controlled by numerousintracellular signaling kinases such as Src and FAK. FAK is akey protein responsible for the regulation of cell motility, andhence knock-down of activated FAK significantly impairs the
Figure 6. MTT assay shows the cell viability of nanomedicine treated MDA-MB-231 cells (A1) before, and (A2) after light irradiation of core-shellnanomedicine. Flow cytometry data show enhanced intracellular ROS in (B3) nanomedicine treated MDA-MB-231 cells compared to (B2) free mTHPCtreated, and (B1) untreated cells. Optical microscope images show the cell morphology of MDA-MB-231 cells (C1) before core-shell nanomedicine treatment,(C2) after treatment, but before light irradiation, and (C3) after light irradiation of nanomedicine. (M: free mTHPC, MP: mTHPC-PLGA nano-core, MP/DA:core-shell nanomedicine containing mTHPC in PLGA core and dasatinib in albumin nano-shell).
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migratory capacity in cancer cells. However, activation of FAK ispositively regulated by hyperactivated Src kinase, which upregulates integrins, leading to accelerated migratory potential forcancer cells.14–17 Furthermore, Src directly drives cancer migra-tion by inducing the breakage of E-cadherin, which otherwisefunctions as an adhesion molecule for maintaining cell–cellcontacts. Moreover, hyperactive Src also aggravates metastasis byextensively polymerizing the invadopodia in cancer cells. Inessence, either by direct hyperphosphorylation of Src or by theactivation of downstream signaling, cancer cells acquire essentialmigratory signals that ultimately trigger the cells to detach fromprimary tumor site and migrate to other organs. Considering thiscritical role of elevated Src in driving cancer metastasis, wehypothesized that, targeting hyperactive Src kinase to controlcancer migration followed by imparting cytotoxic stress to thespatially confined metastatic cells will be a promising therapeuticstrategy. To implement this plan, we have developed a core-shellnanomedicine, wherein a protein nano-shell was targeted againstSrc kinase and a polymer nano-core was assigned to release lethaldose of reactive oxygen species under light activation.
In order to study the feasibility of loading two drug moleculesin appropriate nanocarriers (core/shell), initially, we havenumerically studied the stability of interaction between theselected drugs with carrier molecules using in silico docking
simulations. At first, the interaction of mTHPC and dasatinibwith human serum albumin was studied with the aim of load-ing both these drugs together in a single carrier. However,with albumin, mTHPC showed relatively weaker interaction(BE: −7.5 Kcal/mol) compared to dasatinib (BE: −10.76 Kcal/mol). This may lead to fast release of mTHPC in serum, whichcan cause undesirable phototoxicity effects. Hence, we havedecided to load dasatinib alone in albumin, while a polymericsystem, PLGA, was considered for loading mTHPC. Beinghydrophobic, both mTHPC and PLGA will have strong inter-actions, and very recently, Low et al. reported that photo-activated mTHPC-PLGA nanoparticles showed selectivephototoxicity to cancer cells, with negligible dark toxicity tonormal cells.18 However, our major emphasis was to deliverthese two drug molecules (dasatinib and mTHPC) together usinga single nanocarrier, because, two separate nano-carrier systemsmay not simultaneously reach the target tumor tissue or cell atpharmacologically relevant concentration for achieving a syner-gistic effect. Considering the experimental design of sequentialinhibition of cancer migration followed by photo-triggered cellkilling, the best option was to form a core-shell system, where,the polymer with mTHPC forms a nano-core, and protein withdasatinib forms a nano-shell, and both these drugs can bedelivered intracellularly in a sequential manner.
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Preparation of the core-shell system was achieved by a two stepprocess. First, mTHPC loaded PLGA nano-core was formed byelectrospray method and then a nano-shell of dasatinib loadedalbuminwas formed by ethanol coacervation. The unique formationof ~20 nm size nano-shell of dasatinib–albumin over ~80 nm sizemTHPC-PLGA nano-core with distinct core-shell interface wasevident from TEM image. Monodispersed core-shell nanoparticlesdisplayed excellent stability (30 days at room temperature) andaqueous phase dispersibility, as indicated by the DLS measure-ment. Zeta potential measurements showed appreciable negativecharge (−12.4 ± 0.2 mV), indicating that the core-shell nanopar-ticles are stabilized by steric repulsive force.
FT-IR vibration spectroscopy indicated that, despite theimplementation of high voltage during electrospray process,mTHPC has retained its unique vibration features within thePLGA nano-core. This was evident from the peaks correspond-ing to hydroxyl groups of phenyl rings, secondary, and tertiaryaromatic amines of porphyrin ring in mTHPC. Similarly, in thenano-shell, peak corresponding to the ketone group of amide ringrepresented dasatinib and albumin. FT-IR spectrum of core-shell nanomedicine revealed additive peaks of mTHPC-PLGAnano-core and dasatinib–albumin nano-shell, indicating that thechemical structure integrity of the core and the shell is retainedwithout any significant modification in the functional groupsafter the formation of core-shell structure.
Fluorescent studies revealed ~38 fold enhanced fluorescenceby mTHPC-PLGA nano-core compared to equimolar concen-tration of free mTHPC, when both dispersed in aqueous phase.This means, not only mTHPC has retained its intact monomericform in PLGA core, but also the polymer shields the photo-sensitizer from hydration attack in aqueous phase. Fluorescencebased photobleaching characteristics of mTHPC-PLGA nano-core also showed typical bleaching profile of monomericmTHPC, whereas free mTHPC dispersed in water showed nocharacteristic photophysical features due to its aggregation andfluorescence quenching. All these experimental results indicatedthat the photophysical property of mTHPC loaded into PLGAnano-core is significantly improved in the aqueous mediumcompared to that of free mTHPC.
Singlet oxygen (1O2) release from mTHPC is the mostimportant requirement for achieving desired cytotoxic effectupon photoactivation of the nano-core. We found ~3 fold increasein the 1O2 efficiency of mTHPC-PLGA nano-core compared tothat of equimolar concentration of free mTHPC in the aqueousmedium. This is an important datum that indicates improveddissolution and stability of monomeric mTHPC in PLGA, whichotherwise tends to aggregate in water as free drug. In the case ofnano-shell, the absorption property of dasatinib was also founddoubled in albumin-bound-state compared to that of free dasatinibin aqueous medium. Clearly, the solubility of dasatinib in albuminis higher than that of the same in aqueous phase. Further more,dasatinib showed only a minimal burst release of ~8% in albumin-bound-state, and thereafter a controlled release was observed inaqueous medium, possibly due to the strong hydrophobicinteractions between the drug and protein shell.
In cell studies, cancer migration assay revealed the capabil-ity of dasatinib–albumin nano-shell to effectively inhibit themotility of both metastatic (MD-MB-231) and non-metastatic
(MCF-7) cancer cells almost equally. This effect was not seen inthe case of cells treated with core-shell nanomedicine containingmTHPC-PLGA nano-core alone. Changes in the cell morphologysubstantiated this observation wherein apparent changes in actincytoskeleton with distinct rounded-up morphology, loss offilapodium, and weak attachment on the substratum were seenin the core-shell nanomedicine treated cells. Immunoblot studieson phospho-Src also confirmed its inhibition in dasatinib–albumin treated cells, suggesting that the anti-migratory effectof the nano-shell is mediated by the inhibition of aberrant Srcsignaling. All these results indicate that, although in 24 h, only10%-12% of dasatinib is released from the nano-shell in cell-freeaqueous medium, at intracellular regions, more dasatinib mightbe released due to proteolytic degradation of albumin carrier bycellular enzymes apart from diffusion-aided drug release.Therefore, we expect higher release of dasatinib in intracellularthan in extracellular. Moreover, since the dissolution of dasatinibin albumin-bound form was significantly improved in aqueousmedium, this could have also contributed to the enhancedavailability of dasatinib to cancer cells. In contrast, free dasatinibbeing hydrophobic precipitated prematurely in the aqueousmedium and thus limited its availability to the cancer cellsleading to decreased anti-proliferative and anti-migratory activitycompared to that of albumin-bound nanodasatinib.
Although the nanomedicine showed ~94% intracellularuptake by MDA-MB-231 cells (supplementary information,Figure S6, A3), and IC50 value of dasatinib is improved in core-shell nanomedicine treated cancer cells, it may be noticed thateven after successful inhibition of migration, ~60% of MCF-7cells and ~20% of MDA-MB-231 remained metabolically ac-tive (Supplementary information, Figure S7, A and B). Thismeans that Src inhibition alone is not sufficient enough to impartcytotoxicity, and it is possible that after transient inhibition ofmigration, metabolically active cells may regain the motility onprolonged incubation by the re-activation of Src kinase or cellsignaling cross-talks. Hence, immediately after impairing thecell migration using dasatinib–albumin nano-shell, we have im-parted additional cytotoxic stress by photo-activating themTHPC-PLGA core of core-shell nanomedicine. In photody-namic therapy (PDT), high concentration of cytotoxic ROS isburst released when light of appropriate wavelength excites aphotosensitizer drug. Our results showed that combined effect ofSrc inhibition and light triggered ROS stress could exert syner-gistic cytotoxicity, killing ~98% of MCF-7 cells, and ~99% ofmetastatic MDA-MB-231. Although it appears that a significantpercentage of MCF-7 and MDA-MB-231 cells experience darktoxicity, this effect is predominantly due to the presence ofdasatinib in the nano-shell, because, the controls, free mTHPC orcore-shell nanomedicine containing mTHPC-PLGA nano-corealone, under dark conditions, could not produce significanttoxicity, and only after light irradiation of the nano-core, the cellswere killed. The low dose light irradiation (net energy delivered:5.4 J/cm2) ensured that laser illumination as such did not pro-duce any light toxicity to cancer cells (Figures 5, A2, and 6, A2control cells: green bar). Also, the drug-free core-shell nanopar-ticles prepared from biocompatible nanocarriers, PLGA andalbumin by itself showed no nanotoxicity to cancer cells afterlight irradiation, indicating that the cancer cells underwent
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apoptosis exclusively due to the synergistic activity of mTHPCand dasatinib in core-shell nanomedicine (Supplementary infor-mation, Figure S8). Our flow cytometry data showed significantgeneration of intracellular ROS in core-shell nanomedicinetreated cells after light irradiation, suggesting that intracellularROS generation was the mechanism behind cell death. Thissuggests that the release ofmTHPC from PLGA core will bemoreefficient intracellularly and necessary singlet oxygen is releasedfrom mTHPC to kill the cancer cells. An incubation period of 4 hwas sufficient enough for themetastatic cells to effectively uptakecore-shell nanomedicine (containingmTHPC in PLGA core), andto achieve synergistic cytotoxicity under light irradiation.
Usually, PDT exerts cytotoxic ROS and kills cancer cellsmainly by necrosis.19 However, in our case, the core-shellnanomedicine triggered mostly apoptosis in almost all of themigration-impaired cells. This result is in consistence with therecent report which shows that apoptosis can be well achieved inPDT if the nanoparticle containing photosensitizer is localized ina suitable intracellular organelle.20 It appears that together withSrc inhibition, a very low dose of light and photosensitizer issufficient to induce apoptosis in migration-impaired cancer cells.This synergism was possible mainly due to the sequential act ofdasatinib and photosensitizer mTHPC from the core-shellnanomedicine, which also aided in the independent working ofboth the drugs in the desired manner.
In conclusion, we have shown that inhibiting metastatic dis-semination in cancer cells followed by imparting photodynamicROS stress using rationally designed core-shell nanomedicine isa promising approach for managing metastatic cancers.
Acknowledgment
The authors thank Mr. Sajin P Ravi and Mrs. Sreerekha P Rfor their technical support. We also thank Amrita VishwaVidyapeetham (University) for providing all infrastructuralsupport for the successful accomplishment of this research work.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2013.10.006.
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