extfieldsofstudy: Medicine Biology

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Long range recognition and selection in IDPs: the interactions of the C-terminus of p53

The C-terminal domain of p53 is an extensively studied IDP, interacting with different partners through multiple distinct conformations. To explore the interplay between preformed structural elements and intrinsic fluctuations in its folding and binding we combine extensive atomistic equilibrium and non-equilibrium simulations. We find that the free peptide segment rapidly interconverts between ordered and disordered states with significant populations of the conformations that are seen in the complexed states. The underlying global folding-binding landscape points to a synergistic mechanism in which recognition is dictated via long range electrostatic recognition which results in the formation of reactive structures as far away as 10 Å, and binding proceeds with the steering of selected conformations followed by induced folding at the target surface or within a close range.

Proteins generally adopt well-defined tertiary structures under physiological conditions, and this structure largely determines their functions. However there is a class of proteins called intrinsically disordered/unstructured proteins (IDPs/IUPs) that do-not-fold-into well-defined structures and yet are biologically functional [1][2][3] . These IDPs remain highly conformationally dynamic under native conditions and play important biological roles. IDPs are a large class of proteins. On the basis of their amino acid content and sequences, which is different from that of globular proteins, IDPs are now estimated to represent a significant fraction of many genomes 1,4 . For example, roughly three-quarters of the proteins in mammals are predicted to contain disordered segments or are fully disordered. The disorder is thought to result from the reduced contacts among the hydrophobic amino acids. The structural plasticity of IDPs allows them to interact with numerous different targets. These interactions are thought to be mediated by rapid conformational disorder-order transitions 5,6 , enabling these regions to interact with a variety of partner proteins with low affinities but high specificities 7,8 . Interestingly, although IDPs have low hydrophobic content overall, the motifs they employ to interact are generally short stretches of hydrophobic residues (6 residues) 9,10 . The conformational diversity of these interactions has been revealed by numerous structural, biophysical and computational studies [11][12][13][14][15][16][17] .
Experimental and computational studies have revealed two major mechanisms that can be used to describe the binding of IDPs to their targets [11][12][13][14][15][16][17] : conformational selection, resulting from selection of the bound-state-like structure present in the ensemble of uncomplexed IDPs 16,17 ; induced folding, resulting from an initial encounter between the IDP and the target followed by conformational changes yielding the final shape of the IDP, referred to as the fly casting mechanism 13,14 . The link between flexibility, specificity and mechanisms that couple folding and binding of such rapidly interconverting conformations are of great interest. This is further underscored by the finding that IDPs are implicated in diseases such as cancer, neurology and metabolism 15,18,19 . With increasing attention being paid to the disruption of protein-protein interactions in therapy this assumes an even greater importance. While it may appear that the structural heterogeneity of these regions makes them unlikely targets of small molecules, two recent reports have opened new windows of opportunity: the exploitation of entropic expansion of IDPs with small molecules 20 and the discovery that a tin(IV) oxochloride cluster selectively targeted a disordered region of the TFIID transcription complex 21 .
p53 is a transcription factor consisting of ordered and disordered regulatory regions engaged in multiple interactions, and is one of the most extensively studied IDPs 22,23 . The p53 protein consists of 393 residues and can be divided into three functional regions: (i) an N-terminal domain (residues 1-93) containing a transcriptional activation domain and a proline-rich domain; (ii) a core DNA-binding globular domain (residues 102-292), which contains most of the mutations found in cancers; and (iii) a C-terminal domain (CTD) consisting of a tetramerization domain (residues 320-356) and a regulatory domain (residues 363-393). The extreme CTD is reported to regulate specific DNA-binding activity of p53 24 , either by altering the conformation of p53 or by interfering sterically with the ability of the protein to bind DNA [24][25][26] . Deletion of this regulatory region, binding of antibodies, post-translational modifications including phosphorylation and acetylation abolish this effect 25,26 . The CTD has also been reported to modulate the stability and cellular localization of p53 27 .
Given the diversity of functional interactions of the p53CTD, we decided to investigate its conformational dynamics in the context of the structural information available. The crystal structures of the p53CTD bound to five different globular proteins, adopting four different conformations 28-32 is available (Fig. 1). The p53CTD adopts an α -helix (residues 377-388) when bound to S100 calcium-binding protein B (S100B(β β )), (PDB 1DT7 28 ); a β-strand (residues 379-387) when bound to Sirtuin, (PDB 1MA3 29 ); a β-turn (residues 380-386) when bound to cAMP response element-binding (CREB) binding protein CBP bromodomain, (PDB 1JSP 30 ); no secondary structure (residues 369-374) when bound to the histone methyltransferase Set9 (PDB 1XQH 31 ) or (residues 378-386) when bound to the cyclin A/cyclin-dependent protein kinase 2 complex (PDB 1H26 32 ). These complexes reveal a diversity of interactions: mostly hydrophobic when bound to S100B(β β ) or to CBP, a combination of hydrophobic and hydrogen bonds (hbonds) when bound to Cyclin A and electrostatics complemented by hbonds when complexed to Sirtuin or to Set9. Overlapping regions of the CTD (sequence 363 RAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD 393 Fig. S1) are stabilized in different binding pockets through such diverse interactions, emphasizing the inherent plasticity of this disordered region. Given the paucity of experimental data on the details of how these interactions are mediated at the atomic level, we address this issue using molecular simulations in this current study. Molecular simulations have been used in earlier studies to interrogate the p53CTD-protein complexes, providing valuable insights [33][34][35][36] : p53CTD adopts transient α -helices in solution 34,35 ; the binding of p53CTD to S100B(β β ) induces secondary structure in p53 through a fly casting like process 34 . We now build upon this work by exploring the binding between the p53CTD and five of its partners using detailed atomistic simulations. We explore the binding process using a two-pronged strategy: we first use steered molecular dynamics simulations (SMD) to examine the process of unbinding of the peptide from its complexed state and further complement this with approximating the process of binding, whereby we place the peptide in different conformations at different distances from the binding pocket and carry out umbrella sampling molecular dynamics (USMD) simulations. The stability of the complexes is examined using a series of standard molecular dynamics (sMD) simulations. The conformational space sampled by the peptide is explored using replica exchange molecular dynamics (REMD) simulations (and are outlined in the supplementary material). The five binding partners chosen for this study represent diversity in size, function, and nature of interactions with the p53CTD, and crucially, are available in crystallographically/NMR resolved complexes.

Materials and Methods
System preparation. The experimental structures of five p53CTD -receptor complexes were obtained from the protein data bank: (a) For the p53CTD-S100B(β β ) complex, the first structure of the NMR ensemble 28 was chosen; in the NMR models, S100B(β β ) exists as homodimer with a 22-mer fragment (residues 367-388) of p53CTD bound to each unit of S100B(β β ) but for simplicity, only one monomer complex was used in the current study. Only a short fragment (residue 377-388) of p53CTD was used as the rest of the peptide is disordered, highly flexible and doesn't interact with the S100B(β β ) in any of the NMR models. Two calcium ions that were bound to S100B(β β ) were retained. (b) In the case of the p53CTD-Sirtuin complex, a 9-mer fragment (residue 379-387) of p53CTD co-crystallized with Sir2 was used 29 . Lys382 of p53CTD was acetylated as seen in the original structure. A zinc ion coordinated by Sir2 was retained. (c) The crystal structure of p53CTD-Cyclin A complex contains a 9-mer (residue 378-386) p53CTD bound to a complex of cyclin A and phosphorylated cyclin-dependent protein kinase 2 (cPDK2) 32 . Since the p53CTD only interacts with cyclin A, cPDK2 was not included. Only one copy of the complex from the crystallographic asymmetric unit is considered here. (d) In the NMR models of p53CTD-CBP bromodomain, a 20-mer fragment of p53CTD is complexed with the CBP bromodomain 30 . However only a short fragment (residues 380-386) of p53CTD is bound to the receptor protein and the rest of the peptide is disordered, highly flexible in all the NMR models and doesn't interact with the CBP bromodomain. Therefore only a shorter (residues 380-386) fragment of p53CTD was used in the p53CTD-CBP complex and the first model of the NMR ensemble was chosen for the current study; Lys382 of p53CTD is monoacetylated as is necessary for binding. (e) The crystal structure of p53CTD-methyltransferase (Set9) complex contains a 6-mer fragment (residue 369-374) of the p53CTD peptide 31 . Although the crystallographic asymmetric unit contained two copies of the complex, only one was considered here. Lys372 of the p53CTD was monomethylated as it is in the original structure. For simulations starting from fully extended conformations of p53CTD, an extended p53CTD (residue 370-389) structure was generated using the Xleap module of the Amber11 package 37 .

MD simulations.
The five p53CTD-receptor structures chosen above were used as starting structures for the sMD simulations of the complexes, and for the apo p53CTD and receptor simulations corresponding structures extracted from the complexes were used. In each structure the N-and C-termini of the p53CTD fragment was capped with acetyl and amide groups respectively. Hydrogen atoms were added to the experimental structures using the Xleap module of the Amber11 package. All the systems were neutralized by the addition of counter ions. The neutralized systems were solvated with TIP3P 38 water molecules to form a truncated octahedral box with at least 10 Å separating the solute atoms and the edges of the box. MD simulations were carried out with the Sander module of the Amber11 package in combination with the parm03 force field 39 . Force field parameters for acetylated, monoacetylated Lysine and monomethylated Lysine were used as described elsewhere 40 . All the systems were first subjected to 1000 steps of energy minimization. This was followed by MD simulations, for which  the protein was initially harmonically restrained (25 kcal mol −1 Å 2 ) to the energy minimized coordinates, and the system was heated up to 300 K in steps of 100 K followed by gradual removal of the positional restraints and a 1 ns unrestrained equilibration at 300 K was carried out. The resulting system was used for the MD simulations. A total of 45 (5 complexes, 5 receptors, 5 peptides in triplicates) MD simulations were carried out for 100 ns each, totaling 4.5 μs (Table S1A).

Steered Molecular Dynamics.
To investigate in some detail the process of (un)binding of the peptides to their receptors, the equilibrated structures of the five p53CTD-receptor complexes were subjected to SMD simulations. SMD is a biasing method that utilizes time-dependent external forces to induce structural changes in biomolecules 41,42 . SMD is rooted in single molecule pulling experiments, and forces the system to evolve away from its initial equilibrium condition, thus accelerating the transitions between different energy minima. In recent years SMD has become very popular and has been extensively applied to several biological processes, including folding/unfolding, transport of ions and organic compounds through membrane channels, and has provided insights into the ligand (un)binding pathways/mechanisms [43][44][45] . In the current work, to facilitate peptide unbinding, a set of distance restraints between receptor and p53CTD peptide atoms that are derived from the crystal structures of the p53CTD-receptor complex were used as the pulling variable during SMD. A spring constant (5 kcal/mol/Å 2 ) and pulling velocity (0.00005 Å/timestep ) were chosen as pulling parameters to prevent distortions to the receptor as a consequence of pulling. For each model, five independent SMD simulations, all starting with the same structure but different initial velocities, were carried out for 10 ns each (Table S1C) using the collective variables (colvar) module in NAMD 46 .
Umbrella Sampling. To obtain a detailed understanding of the free energies along the pathways of (un) binding of the p53CTD and their binding partners, USMD simulations were carried out, with the peptide placed at different distances from its binding site/pocket and restrained by an external harmonic force. The distance restraint involved the centers of mass of the binding site atoms of each protein and peptide and were imposed using the collective variables (colvar) module in NAMD 46 with a force constant of 50 kcal/mol. USMD was carried out with the p53CTD in its bound conformation placed at the binding site as well as at distances of 10 Å, 20 Å and 30 Å from the binding pockets, with 25 ns simulations carried out at each distance. Similarly for each binding partner, the simulations were repeated with the p53CTD in its non-native bound conformations at the binding pocket as well at the three different distances (Table S1D). Only three of the five p53CTD-receptor complexes (p53CTD-S100B(β β ), p53CTD-Cyclin A, p53CTD-Sirtuin) were chosen for the USMD as the bound p53CTD (approximately the same length) in these three cases represent both the ordered (different secondary structures) and disordered states. The other two systems (p53CTD-CBP, p53CTD-Set9) were not considered here because (a) the region of p53CTD co-crystallized with Set9 is very different from the others and (b) the p53CTD binding site in the CBP is very narrow and replacement of the native bound p53CTD with nonnative bound peptide resulted in huge steric clashes. In all the three USMD simulations, the same region of p53CTD was used ( 378 SRHKKLMFK 386 ).

Binding Energy calculations. Molecular Mechanics Generalized Born Surface Area (MMGBSA) and
Poisson Boltzmann Surface Area (MMPBSA) methods were used for the calculation of binding free energies between the peptides and their partner proteins 47 . The molecular mechanics energy term used in these calculations represents the internal bonded energy (energy of bonds + angles + dihedrals) as well as the non-bonded van der Waals and Coulomb energies. Two sets of MMPBSA/MMGBSA calculations were carried out for each system (complexed state and apo states). For the calculation of binding energies from the complex and apo simulations the last 50 ns of the sMD trajectories were used. For the USMD simulations, snapshots from the last 15 ns of USMD trajectories of both the native and non-native complexes with the peptide placed at different distances were used for binding energy calculations.
Analysis. RMSD, RMSF and secondary structure calculations were all carried out with the PTRAJ module in AMBER. Native binding contacts are defined as contacts between protein and peptide residues in their crystal structures when their Cα atom are closer than 6.5 Å. Native binding contacts at 10 Å are also calculated similarly by moving the peptide 10 Å away from the centre of mass (COM) of the binding site in its corresponding experimental structure. Cluster analysis was based on the pair-wise cartesian root mean squared deviation of only the heavy atoms between conformations, with an rmsd cutoff of 2 Å and using the kclust program in the MMTSB-tools 48 . The VMD (Visual molecular dynamics) program 49 and Pymol 50 was used for visualization of trajectories and preparation of figures.

Results
Standard MD simulations (multiple copies) of the complexes show that the simulations are stable and the peptides remain close to their bound states (rmsd < 2.5 Å, Fig. 2). In contrast the simulations of the peptides in their apo states (either starting from the bound states or from a fully extended state) shows that the peptides are highly flexible and adopt multiple conformational states (rmsd > 4 Å, Figs 2 and S2). The apo peptides adopt collapsed conformations (Figs 2 and S2) and sample the bound state conformations with varying probabilities (Figs 2, S3 and S4).
Coupled binding and folding from unbinding simulations. Simulations of the peptides in their apo states show that the peptides adopt the bound state conformations and also collapsed conformations (Figs 2, S3 and S4). Hence to examine whether binding occurs through conformational selection, induced fitting or some combination, we need to explore the actual process of binding of the peptide. We first study the technically easier process of unbinding using SMD. A key assumption is that binding/folding is largely a reverse of unbinding/ unfolding. To monitor the conformational flexibility of the p53CTD peptide during pulling, peptide rmsd was measured relative to its starting experimental bound structures. Changes in receptor-peptide interactions upon peptide unbinding were monitored by recording the fraction of native binding contacts (fnbc), together with the distance between the center of mass (COM) of the binding site and the peptide fragment. Although with increasing COM, an increase in peptide rmsd and a decrease in the fnbc was observed during SMD, interesting trends were observed. In the case of S100B(β β )-p53CTD and CBP-p53CTD complexes, where the complex is stabilized mainly by hydrophobic interactions, rapid loss in fnbc was already observed (> 60%) at COM of ~10 Å (Fig. S7). In other cases where the bound conformation of the peptide is stabilized by numerous h-bond interactions (Sir2, Cyclin A and Set9 complexed with p53CTD) a lag in reduction of fnbc was observed, with more than 50% of the native contacts existing even at COM of ~15 Å. Overall, the changes in peptide conformations appear to follow a common trend, with a rapid increase in rmsd (~3 Å) (Fig. S8) for all the peptides by ~10 Å from the binding site. However further changes in the peptide conformations appear to be less rapid, with the conformations of the bound states such as helix or sheet existing partially even at distances of ~20 Å from the receptor. The disordered bound conformations show rapid fluctuations and become partially ordered. The 2 D free-energy landscape (Fig. 3) revealed varied populations in several minima for the different complexes. A minima at the upper left corner of each graph corresponds to the complexed state (with the peptide in its folded/bound state with rmsd < 1.5 Å and fnbc > ~60%) for the complexes of p53CTD with Cyclin A, Sirtuin and Set9, all of which are characterized by a number of hydrogen bonds mediating interactions between the proteins and the peptides. Such a native minima is less populated and slightly shifted (rmsd < 1.5 Å and fnbc ~40-60%) for the complexes between p53CTD with S100B(β β ) and p53CTD with CBP, where the protein-peptide interactions are governed mainly by hydrophobic interactions. The energy landscapes are also decorated with other minima that correspond to partially unfolded and unbound states, with rmsd > 3.0 Å or fnbc < 40%. There are several sparsely populated minima that are separated by low energy barriers that are rarely/transiently populated. Comparison of the landscape of all the five combined SMD simulations for each system shows that in all the simulated systems the bound/folded peptide follows two different pathways to reach its unfolded/unbound conformation. Along path1 (white arrow Fig. 3), the pc53CTD fragment rapidly loses its bound conformation (rmsd > 3 Å) but retains most of the native binding contacts (fnbc > 60%), suggesting that the peptide becomes disordered upon exiting from the binding pocket. This suggests that order must be induced in the peptide by the receptor upon binding, and is seen for the p53CTD complexes with Sirtuin and Cyclin A -this is the induced folding mechanism 13,14,51 . Along path2 (Black arrows Fig. 3), the bound conformation (alpha helix or beta sheet or disordered state) is retained (rmsd < 2.5 Å) or at least partially retained, with more than 70% of the fnbc lost already. This suggests that the peptide adopts the bound conformation even before it binds to its partner, and the native binding contacts play a role in selecting such a folded/bound conformation of the peptide, a scenario referred to as the conformational selection mechanism 16,17 and appears to characterize complex formation between p53CTD and S100B(β β ), CBP or Set9.
In contrast to the increased conformational fluctuations/changes of p53CTD peptides, all the corresponding receptor conformations except two remained relatively stable (rmsd < 3 Å, Fig. S8) upon pulling the peptide from its binding pocket. Increased receptor fluctuations were observed for S100B(β β ) and CBP proteins. In the case of S100B(β β ) it is known that the protein undergoes conformational changes upon peptide binding [33]. The p53CTD binding site in CBP is made up mostly by loops and it is clear that the binding of the peptide stabilizes the loop conformations.
p53CTD-receptor initial encounter and complex formation. The SMD yields a trajectory of the conformational changes associated with the process of unbinding. However it has its limitations, particularly at larger distances of the peptide from the binding site, where the sampling is insufficient. To overcome this and explore the conformational landscapes of the peptides in detail, we position the peptides at specific distances (0, 10, 20 and 30 Å) from the binding site/pocket and restrain them by an external harmonic force and subject the protein -peptide complexes to USMD (each peptide was placed at each location in 3 different conformations as outlined in Methods).
Irrespective of the conformation used (native or non-native), when the peptides are placed at larger distances (20 Å and 30 Å) from the binding sites, their dynamics are characterized by increased flexibility and rapid unfolding and refolding, suggesting that the receptors do not influence their behaviour. However, at 10 Å, the corresponding native bound (alpha helical, beta sheet and disordered) conformations of the p53CTD fluctuated significantly with some unfolding (Fig. 4) yet significantly populated in their corresponding native folded states with the associated native binding contacts (fnbc ~60%). When the native bound conformations of the peptides are replaced by the native conformations seen in the other 2 complexes, folding and unfolding of secondary structures are seen and surprisingly, partial formation of native bound conformations (alpha helix in the case of S100B(β β ) and beta sheet in the case of Sir2) are also observed (Fig. 4). This clearly reflects the long range influence of the receptor on modulating the conformational dynamics of the peptides; native and non-native contacts govern these dynamics.
When the peptides are placed in the binding sites in their experimentally bound states, the simulations were stable as expected and no major conformational changes were observed. The 2D energy landscape of the fnbc relative to the rmsd of the peptides shows a single minima with rmsd < 2 Å and fnbc > 70% for all the three native complex simulations (Fig. 5). However, when the peptides are placed in the binding sites in their non-native conformations, unsurprisingly, increased fluctuations were observed at the binding interface. For example in the case of the S100B(β β ) protein, where the binding site is highly hydrophobic, when the alpha helical conformation of p53CTD was replaced by a beta sheet or a disordered conformation, increased fluctuations were observed for both the peptide as well as S100B(β β ), especially in the binding site residues. Despite the binding site of S100B(β β ) protein being large and highly hydrophobic and highly suitable for the binding of an alpha helix, when the peptides were placed in non-helical states, they could not be induced into the alpha helical states (Fig. 5); this of course could also result from insufficient sampling. Although the binding pocket is hydrophobic, there are charged residues on both sides of the binding pocket and these residues interact with complementary charged residues of p53CTD, and such non-native contacts appear to limit the formation of the alpha helical state. Similar trends were observed for the other two proteins, Sir2 and Cyclin A, when the non-native conformation of the peptide was located at the binding interface (Fig. 5). When simulations were carried out with the peptide bound in a non-native alpha helical conformation, increased fluctuations and partial unfolding was observed with an rmsd of ~3 Å. As both these enzyme either exhibit a narrow and slightly charged binding interface (in the case of Sir2), which is not suitable for the binding of alpha helices (due to the absence of stabilizing hydrophobic interactions in the case of Sir2), increased fluctuations as well significant absence of native binding contacts were observed. However both Sir2 and cyclin A tolerated disordered non-native conformations of the peptides as non-native h-bond interactions with the binding site residues were sufficient to stabilize the peptides. But, in none of the cases, formation of native bound conformation or native binding contacts was observed when the simulations were initiated from non-native bound conformations. This contrasts with the induced folding mechanism, where the peptide binds before it folds. While it is possible that longer simulation times may witness such transitions, analysis of data across different timescales in our simulations shows convergence (Fig. S9).
Although the receptors remain stable overall, irrespective of the bound conformations of the peptides, and of their separation from the peptides (10, 20 and 30 Å), varying patterns of fluctuations of residues in and around the binding sites were observed. When the peptide is bound, the binding pocket residues do not fluctuate much, and if the peptide is in its native conformation, then the fluctuations of the binding site residues are the lowest  (Fig. 6). However surprisingly, these fluctuations increase when the peptide is between 10 and 20 Å after which they get attenuated. This pattern is observed for residues in the binding pocket that either make direct contacts with the peptide in its bound state or are not specific, thus suggesting that recognition begins to occur at distances between 10-20 Å.
To characterize the energetics associated with the process of coupled binding and folding, we carried out MMPBSA and MMGBSA type binding energy calculations on the ensembles, generated by the USMD simulations. In all the simulated complexes, when the peptide was positioned at distances far from the binding site (20 Å and 30 Å), irrespective of the conformation that the peptide adopts, the total binding energy was low suggesting little influence of the two molecules on each other (Fig. 7). However at a distance of 10 Å from the binding sites, the total binding energy favored the association/complexation process in all the simulations (Fig. 7). Again at the binding interface, the formation of receptor-peptide complex is highly energetically favored, with the native complexes clearly favored. Interestingly such a clear preference for the native conformations of the peptides was apparent even at 10 Å (Fig. 7). Both the electrostatics and van der Waals components begin to contribute at 10 Å from the binding site (Fig. 8) although, as expected, the long range component i.e. the electrostatics dominated (however when we factor in the desolvation penalties (Fig. S10), the magnitude of electrostatic contributions is attenuated); and this component was strongest only when the peptide was in its native conformation. At the binding site, the complexation is energetically more favored with the native bound conformation of the peptide with both electrostatics and VdW making up the binding energy; as expected, the contribution of VdW is much more than it is at 10 Å. This is not surprising since the binding is accompanied by conformational rearrangements for better/tight packing and stabilization of the complex structure. Our USMD simulations of the receptor-peptide complexation suggest that each receptor preferentially binds to p53CTD in which structural elements that resemble the bound state exist, ie, through an extended conformational selection mechanism. A point to note is that our electrostatic interactions were evaluated without including the effects of salt; however estimates of the salt effects (data not shown) show that the patterns of changes across the systems remain similar to the no-salt case; the interactions are attenuated only slightly.
To test the above hypotheses, we next decided to carry out in silico mutagenesis studies. We chose the S100B(β β ) and Cyclin A systems since both receptors exhibit a wide hydrophobic cavity (partially charged in the case of Cyclin A), yet p53CTD binds S100B(β β ) as a helix while it binds Cyclin A in an extended disordered conformation. We identified Lys381 and Phe385 from p53CTD as contributing the most to the binding with Cyclin A (Fig. S11) and therefore mutated them to Ala. The double mutant p53CTD K381A,F385A shows increased helicity as compared to the wild type in solution, although the simulations were initiated from both, the helical (as in the complex with S100B(β β ) as well as disordered (as in the complex with Cyclin A) states. A loss in the binding energy to Cyclin A (ΔΔG WT-MUT = + 9.8 kcal/mol) was seen; wildtype p53CTD binds to Cyclin A in the disordered state and increased helicity disfavors the binding. In contrast, a slight increase in the binding energy was observed for binding to S100B(β β ) (ΔΔG WT-MUT = − 2.3 kcal/mol). Once again, these effects were pronounced already at ~10 Å from the binding site, with increase in binding energy to S100B(β β ) (ΔΔG WT-MUT = − 4.8 kcal/mol) and decrease in binding energy to Cyclin A ((ΔΔG WT-MUT = + 7.4 kcal/mol). Our simulation suggests that increasing the population of the ordered state (in this case the alpha helical) results in improved binding when conformational selection operates (as it appears to occur in the case of p53CTD binding to S100B(β β )). However such an increase in the ordered state results in a reduction in the intrinsic flexibility of the p53CTD and therefore disfavors the binding when induced folding operates (as it appears to occur in the case of binding to Cyclin A).

Discussions
A major characteristic of IDPs is their ability to fluctuate among several alternative conformations. This enables them to easily bind to multiple receptors. However this makes understanding mechanisms of their specificity difficult. In this context MD simulations have been increasingly used to detail their folding and binding. One such IDP, the C-terminal domain of the tumour suppressor protein p53, interacts with several proteins [28][29][30][31][32] . In the current work we have combined equilibrium and non-equilibrium atomistic explicit solvent MD simulations to investigate the binding of this IDP with a subset of its binding of partners which include S100B(β β ), Cyclin A, CBP, Sirtuin and Set9.
MD simulations reveal that the free p53CTD peptide is quite flexible in its apo state adopting multiple conformations including those observed in the experimentally resolved complexes. A particular stretch of the sequence tends to be more ordered compared to the rest of the p53CTD; this is the region that is common in binding to the various partners. Recent observations have demonstrated that such persistent residual structure in disordered ensembles have important roles in initial recognition and binding 52,53 . Existence of such folded-like/bound-like conformations hints at the existence of the conformational selection mechanism in recognition/binding. However recent NMR and coarse grained molecular simulations on other protein-peptide interactions suggest that increasing the amount of structure in the unbound states can lead to attenuations in the binding rates [54][55][56] .
To study the process of recognition and complexation, we first pull the peptides slowly out of their binding pockets. We see that the peptides lose their order and become disordered/unfolded. They sample a wide variety of conformations, including the folded/bound conformation, even at large distances from their target proteins. This transition can occur along two distinct pathways: (a) along path1 the rate of unfolding (ordered to disordered transitions) is faster than the rate of unbinding (loss of binding contacts). (b) along path2, the rate of peptide unbinding is faster than of its unfolding, and the peptide still retains its folded/bound conformation despite losing most of its native binding contacts.
Thus, the free peptide simulations support the conformational selection mechanism, but the peptide unbinding simulations suggest that both conformational selection and induced folding mechanisms are employed depending upon the receptor. These simulations also indicate that the ordered/structured state of p53CTD is less stable in the absence of its binding partners. The peptide rapidly interconverts between several sub-states that are probably separated by small free energy barriers. These conformational fluctuations also likely contribute to efficient IDP recognition by allowing the peptide to fold rapidly upon binding through fly-casting 57 . However in recent years the magnitude of this effect has been questioned, with suggestions that it may at most result in ~1.6 fold acceleration [57][58][59] . Indeed, this mechanism was also based on the assumption that unstructured regions/ peptides adopt extended conformations which in turn increase the capture radii, thus resulting in higher k on . But recent studies have shown that the unbound IDPs tend to be much more compact than previously assumed (as is also seen in our simulations), further reducing the proposed fly-casting affects [57][58][59] .
We additionally simulate the initial encounter during complex formation by placing the peptides at increasing distances and in varying conformational states from the receptors. We find a marked attenuation in flexibility (rapid folding and refolding) of the peptide as the peptide approaches its binding partner concomitant with the formation of contacts with the receptor proteins. While only the native complex is favoured at the binding site, we also see structural and energetic (largely electrostatic) bias towards the native conformations of the peptides even at separations of ~10-15 Å. It appears that electrostatic interactions between charges on IDPs and those near the binding sites are responsible for the long range steering as well as catalyzing efficient folding after the initial encounter. This underpins the regulations of these interactions by post-translational modifications that add or remove charges on IDPs. After the initial steering, the initial contacts facilitate the folding of peptide on the receptor surface and finally the packing (van der Waals interactions) between the receptor and peptide stabilizes the ordered form of the peptide. Thus the driving force required for coupled folding and binding is provided by favorable interactions between the peptide and the receptor and reduced entropic penalty which results from the binding of pre-formed structural elements.
The above mechanisms were further examined by simulations of a postulated double mutation which suggests that increasing the population of the ordered state results in a reduction in the intrinsic flexibility of the p53CTD. In the case of S100B(β β ) which binds an ordered helical state of p53CTD (conformational selection), this increased order resulted in an improvement in affinity. In contrast, the binding to Cyclin A, which prefers a disordered conformation (induced fit), binding was reduced. A similar observation has been made for the E3 rRNase domain-Im3 interactions 60 , where an increase in disorder brought about by a mutation in E3 rRNase, resulted in reduced binding.
Our studies suggest that for the complexes mediated by van der Waals interactions, conformational selection appears to dominate the process of recognition, whereas for the complexes where h-bonds mediate interactions, induced folding appears to be the dominant mode of recognition. Similar features have been reported for complexation involving proteins with ligands and with DNA 61 . The authors of this study show how systems that are electrostatically driven, such as negatively charged DNA and positively charged regions of DNA binding proteins, are likely dominated by induced fit mechanisms. Similarly, we find that the interactions between positively charged p53CTD and the negatively charged Sir2 are dominated by induced fit. In contrast, the interactions between the largely hydrophobic S100B(β β ) and positively charged p53CTD, characterized by short range interactions are dominated by conformational selection mechanisms. Based on our simulation results we propose that p53CTD binding occurs synergistically via a combination of preformed structural elements (conformational selection) and coupled folding and binding (induced folding). Of course, both mechanisms must operate synergistically, with conformational selection being important for the initial recognition followed by the reactive complex relaxing into the final bound state through multiple induced folding processes. Similar synergism has also been proposed for the binding of various IDPs to their targets. These include interactions between the c-Myb and the KIX domain 62 and between the transactivation domain binding of nuclear co-activator binding domain (NCBD) and the transcription coactivator CBP activation domain of p160 steroid receptor coactivator (ACTR) 63 .
Although current evidence appears to suggest one or the other mechanism governs the interactions between the several IDP-protein interactions such as phosphorylated kinase-inducible domain (pKID) of the cAMP-response element binding (CREB) protein and the KIX domain of the transcriptional coactivator CREB-binding protein (CBP) 64,65 , we hypothesize that similar synergism will characterize these interactions. Of course IDPs vary greatly in their conformational propensities, thereby resulting in mechanisms that combine the two processes to different extents. This manifests at one extreme in the KIX domain employing these two mechanisms to different degrees in interacting distinctly with two different IDPs (pKID 64 and TAD of c-Myb 62 ). In contrast, we find in our study, that the same IDP utilizes these mechanisms to varying degrees as it flirts with different receptors, each resulting in distinct biological outcomes.
Implications for oncogenic mutations: What implications might this have in biology given that p53 is highly mutated in cancers? Analysis of the mutations deposited in the (a) IARC database 66 and in the (b) COSMIC database 67 of both the N-and C-termini of p53 show low mutability, similar to that reported for PTEN 68 ; in contrast there is some evidence that missense mutations in disordered regions do have a functional impact in diseases 69 . Do these regions engage in certain kinds of protein-protein interactions that are essential for maintaining the general health of cells? It has been noted that ordered proteins engage in metabolism, catalysis etc while disordered regions are engaged in regulation and signalling 8,70 . In our search for mutations seen in cancers in this region, we have come across only one mutation, G398E. Earlier simulation studies 71 on the binding of this mutant to the receptor S100B(β β ) suggested that it likely binds with higher affinity, thus leading to disabling of the transcription function of p53. A quick look at the electrostatic potential of two receptors of CTD, S100B(β β ) and CBP suggest that there must be some kinetic attenuation of the interactions with the former as a very anionic potential of S100B(β β ) is approached by a peptide now bearing a negative charge on G398E (Fig. S12) In contrast the cationic potential of CBP must enhance the uptake of the negatively charged mutant. If this mutation alleviates binding of p53 to S100B(β β ), then it certainly must enhance transcription as it would do if CBP bound the mutant with higher affinity 30 . It is clear that detailed experimental studies will be required to unpack the role of this promiscuous segment of p53.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Chemistry
Materials Science

text:
Size-Dependent Photodynamic Anticancer Activity of Biocompatible Multifunctional Magnetic Submicron Particles in Prostate Cancer Cells

In this study, newly designed biocompatible multifunctional magnetic submicron particles (CoFe2O4-HPs-FAs) of well-defined sizes (60, 133, 245, and 335 nm) were fabricated for application as a photosensitizer delivery agent for photodynamic therapy in cancer cells. To provide selective targeting of cancer cells and destruction of cancer cell functionality, basic cobalt ferrite (CoFe2O4) particles were covalently bonded with a photosensitizer (PS), which comprises hematoporphyrin (HP), and folic acid (FA) molecules. The magnetic properties of the CoFe2O4 particles were finely adjusted by controlling the size of the primary CoFe2O4 nanograins, and secondary superstructured composite particles were formed by aggregation of the nanograins. The prepared CoFe2O4-HP-FA exhibited high water solubility, good MR-imaging capacity, and biocompatibility without any in vitro cytotoxicity. In particular, our CoFe2O4-HP-FA exhibited remarkable photodynamic anticancer efficiency via induction of apoptotic death in PC-3 prostate cancer cells in a particle size- and concentration-dependent manner. This size-dependent effect was determined by the specific surface area of the particles because the number of HP molecules increased with decreasing size and increasing surface area. These results indicate that our CoFe2O4-HP-FA may be applicable for photodynamic therapy (PDT) as a PS delivery material and a therapeutic agent for MR-imaging based PDT owing to their high saturation value for magnetization and superparamagnetism.

Introduction
During the past few decades, spinel ferrite (MFe 2 O 4 ) materials have been widely investigated because of their excellent magnetic and electrical properties [1] and their potential applications in different technological fields [2] and in biomedicine [3]. Among the various spinel ferrite materials, CoFe 2 O 4 has received particular attention owing to its excellent chemical stability, good mechanical hardness, high coercivity, moderate saturation-magnetization, positive anisotropy constant, large magnetostrictive coefficient, wear resistance, and electrical insulation [4][5][6][7]. These properties, along with their great physical and chemical stability, make CoFe 2 O 4 nanoparticles suitable for magnetic recording and biomedical applications. Importantly, CoFe 2 O 4 nanoparticles are among the most promising candidates for biomedical applications, including magnetic resonance imaging (MRI), magnetic fluid hyperthermia, magnetic separation, biosensors, and targeted and controlled drug delivery [8][9][10][11][12][13][14].
Although CoFe 2 O 4 nanoparticles have various practical applications, recent studies in biomedical fields have focused on the fabrication of multifunctional nanoparticles with various enhanced properties. To improve the properties of these nanoparticles, many research groups have reported various synthetic methods, such as modification of the surface with specific functional groups that enable conjugation and specific uptake of circulating nanoparticles by organs and subsequent delivery of the drugs into the targeted lesions [15][16][17][18]. Modifications have also been applied to provide strong luminescence or MRI signal in a visible range to enable clinicians to track the nanocarrier system [19][20][21]. Among these various biofunctional groups, hematoporphyrin (HP) is one of the most popular photosensitizers (PSs) and photodynamic therapy (PDT) agents due to its unique properties, including high photo-activity, strong photodynamic efficiency, low toxicity, and fast clearance [22]. These novel properties allow HP to be widely used in medical, biological, and environmental applications. Therefore, the emerging technology of bonding of PDT agents to magnetic materials has become important because these multifunctional materials may have applications in the treatment of various types of cancer cells in vitro and in vivo. In this regard, we previously reported the development of multifunctional magnetic submicron particles conjugated with PSs and vancomycin as photodynamic inactivation agents for targeting of bacteria and capturing or removing pathogens from sites of contamination [23]. However, studies performed to date have not sufficiently investigated photodynamic cancer therapy using magnetic particles. In addition, it is important to confirm the anticancer activity of multifunctional particles of different particle sizes to determine the optimal particle size and dose.
Therefore, in this study, we focused on comparing the photodynamic anticancer efficiencies of various sizes of CoFe 2 O 4 particles conjugated with HP and folic acid (FA) molecules (CoFe 2 O 4 -HP-FA). To this end, we fabricated CoFe 2 O 4 -HP-FA with well-defined sizes by a simple surface modification process that functionalized the CoFe 2 O 4 particles by coating them with HP and conjugation with FA as previously described [24,25]. We then examined the capacity of the CoFe 2 O 4 -HP-FA for specific targeting and killing of cancer cells and evaluated their particle size-dependent cytotoxicity and photodynamic anticancer activity. In addition, we performed in vitro MR-imaging with and without cells and analyzed apoptotic death in prostate cancer cells to confirm the potential clinical applications of CoFe 2 O 4 -HP-FA.

Characterization of CoFe 2 O 4 -HP-FA
The morphologies and crystal structures of CoFe 2 O 4 submicron particles were investigated using field emission scanning electron microscopy (FE-SEM; SU-70, Hitachi, Tokyo, Japan) and X-ray diffractometry (XRD; X Pert Pro MPD, PANalytical, Almelo, The Netherlands). FE-SEM images of CoFe 2 O 4 particles with controlled external sizes are shown in Figure 1A. All particles exhibited spherical morphology with rough surfaces and were remarkably uniform with a narrow size distribution. The external size of the CoFe 2 O 4 particles was precisely controlled from 60 to 335 nm by simply increasing the volume ratio of V EG /V DEG . The mean diameter of the prepared CoFe 2 O 4 particles was directly proportional to the volume ratio of V EG /V DEG during the solvothermal reaction. When the ratio of V EG /V DEG was varied from 5/15 to 10/10, 15/5, and 20/0, the diameter of the resulting superstructured CoFe 2 O 4 particles was 60, 133, 245, and 335 nm, respectively. Moreover, high-magnification FE-SEM images clearly showed that the individual spheres were composed of irregular nanograins with a size of approximately 15 nm (bottom images of Figure 1A). To obtain monodispersed CoFe2O4 particles with tunable grain sizes, we designed a modified synthetic route by controlling the NaOAc/Na acrylate ratio (w/w in grams). The size variation of CoFe2O4 particle grains was confirmed by XRD pattern analysis ( Figure 1B). The positions and relative intensities of all diffraction peaks were well matched with the cubic spinel structure of CoFe2O4 particles, which is in good agreement with the values reported in the literature (JCPDS Card No. 22-1086). The diffraction peaks from CoFe2O4 occurred at Bragg angles of 30.2°, 35.6°, 43.3°, 53.6°, 57.1°, and 62.7°, representing reflections from the (220), (311), (400), (422), (511), and (440) lattice planes, respectively. To estimate the grain size according to the mass ratio of NaOAc/Na acrylate, the observed diffraction peak profiles (2θ = 35.6°) were reasonably well fitted by a convolution of Lorentzian functions (middle graph of Figure 1B). The average grain size was calculated based on Scherrer's equation. With the increase in the mass ratio of Na acrylate, the average size of the primary crystallites decreased from 25.7 to 17.5, 13.5, and 11.9 nm, respectively. Therefore, the grain size of CoFe2O4 particles decreased gradually with increasing volume ratio of Na acrylate. Additionally, the crystalline characteristics of CoFe2O4 particles increased as the primary grain size increased. These two experimental results suggest that both the external size and grain size of CoFe2O4 particles could be adjusted easily by controlling the VEG/VDEG and NaOAc/Na acrylate ratios.
The magnetic properties of monodispersed CoFe2O4 particles with tunable grain size were investigated using a superconducting quantum interference device (SQUID; MPMS-5S, Quantum Design, San Diego, CA, USA), as shown in Figure 2A. The temperature characteristics of the magnetization at a constant field of 5 kOe were typical of superparamagnetic materials. The field-cooled (FC) magnetization curves were nearly constant, whereas the curves for the samples cooled in the To obtain monodispersed CoFe 2 O 4 particles with tunable grain sizes, we designed a modified synthetic route by controlling the NaOAc/Na acrylate ratio (w/w in grams). The size variation of CoFe 2 O 4 particle grains was confirmed by XRD pattern analysis ( Figure 1B). The positions and relative intensities of all diffraction peaks were well matched with the cubic spinel structure of CoFe 2 O 4 particles, which is in good agreement with the values reported in the literature (JCPDS Card No. 22-1086). The diffraction peaks from CoFe 2 O 4 occurred at Bragg angles of 30.2 • , 35.6 • , 43.3 • , 53.6 • , 57.1 • , and 62.7 • , representing reflections from the (220), (311), (400), (422), (511), and (440) lattice planes, respectively. To estimate the grain size according to the mass ratio of NaOAc/Na acrylate, the observed diffraction peak profiles (2θ = 35.6 • ) were reasonably well fitted by a convolution of Lorentzian functions (middle graph of Figure 1B). The average grain size was calculated based on Scherrer's equation. With the increase in the mass ratio of Na acrylate, the average size of the primary crystallites decreased from 25.7 to 17.5, 13.5, and 11.9 nm, respectively. Therefore, the grain size of CoFe 2 O 4 particles decreased gradually with increasing volume ratio of Na acrylate. Additionally, the crystalline characteristics of CoFe 2 O 4 particles increased as the primary grain size increased. These two experimental results suggest that both the external size and grain size of CoFe 2 O 4 particles could be adjusted easily by controlling the V EG /V DEG and NaOAc/Na acrylate ratios.
The magnetic properties of monodispersed CoFe 2 O 4 particles with tunable grain size were investigated using a superconducting quantum interference device (SQUID; MPMS-5S, Quantum Design, San Diego, CA, USA), as shown in Figure 2A. The temperature characteristics of the magnetization at a constant field of 5 kOe were typical of superparamagnetic materials. The field-cooled (FC) magnetization curves were nearly constant, whereas the curves for the samples cooled in the demagnetized state showed marked maxima, corresponding to the blocking temperature above which superparamagnetism occurred. The blocking temperatures for the smallest nanograins sized 11.9 and 13.5 nm were 245 and 275 K, respectively, which precludes them from being used in medical applications. However, the blocking temperatures for the particles built from larger grains (17.5 and 25.7 nm) were close to RT and above. These results also provide evidence for good separation of nanograins inside the super-structured particles (clusters), as direct contact between aggregating grains would increase the magnetic moment of the effectively larger grains and thus prevent thermal excitation from randomizing magnetic moment directions. Notably, superparamagnetism of magnetic particles is critical for medical applications because this feature prevents aggregation of large magnetic clusters, which occurs for particles with permanent magnetic moments.
demagnetized state showed marked maxima, corresponding to the blocking temperature above which superparamagnetism occurred. The blocking temperatures for the smallest nanograins sized 11.9 and 13.5 nm were 245 and 275 K, respectively, which precludes them from being used in medical applications. However, the blocking temperatures for the particles built from larger grains (17.5 and 25.7 nm) were close to RT and above. These results also provide evidence for good separation of nanograins inside the super-structured particles (clusters), as direct contact between aggregating grains would increase the magnetic moment of the effectively larger grains and thus prevent thermal excitation from randomizing magnetic moment directions. Notably, superparamagnetism of magnetic particles is critical for medical applications because this feature prevents aggregation of large magnetic clusters, which occurs for particles with permanent magnetic moments. Using a wet chemical process, size-controlled CoFe2O4 particles were easily bonded with HP molecules after plasma treatment. As shown in Figure 2B, the photoluminescence (PL) and photoluminescence excitation (PLE) spectra of CoFe2O4-HP-FA exhibited the same characteristics as pure HP molecules in tetrahydrofuran (THF). The peak at 400 nm in both PLE spectra was attributed to the Soret band of HP, and the peaks at 500, 532, and 568 nm was attributed to the Q bands of HP. At the excitation wavelength of 500 nm, pure HP produced two strong emission peaks at 623 and 690 nm, and CoFe2O4-HP-FA yielded slightly blue-shifted peaks at 619 and 685 nm. The blue-shifted emission peaks were attributed to strong bonding between HP and the magnetic CoFe2O4 particles. To understand the bonding property of the carboxyl groups of HP and FA and the metal ions of the CoFe2O4 particle, the FT-IR spectra of all samples were compared as shown in Figure S1. Based on this result, we concluded that the carboxyl terminal groups of HP formed cation-carboxylate complexes as Using a wet chemical process, size-controlled CoFe 2 O 4 particles were easily bonded with HP molecules after plasma treatment. As shown in Figure 2B, the photoluminescence (PL) and photoluminescence excitation (PLE) spectra of CoFe 2 O 4 -HP-FA exhibited the same characteristics as pure HP molecules in tetrahydrofuran (THF). The peak at 400 nm in both PLE spectra was attributed to the Soret band of HP, and the peaks at 500, 532, and 568 nm was attributed to the Q bands of HP. At the excitation wavelength of 500 nm, pure HP produced two strong emission peaks at 623 and 690 nm, and CoFe 2 O 4 -HP-FA yielded slightly blue-shifted peaks at 619 and 685 nm. The blue-shifted emission peaks were attributed to strong bonding between HP and the magnetic CoFe 2 O 4 particles. To understand the bonding property of the carboxyl groups of HP and FA and the metal ions of the CoFe 2 O 4 particle, the FT-IR spectra of all samples were compared as shown in Figure S1. Based on this result, we concluded that the carboxyl terminal groups of HP formed cation-carboxylate complexes as a result of chemical coordination bonding between the cation and the carboxylate; some molecules would also be expected to exist in the protonated carboxyl state. The IR spectrum of FA bonded to CoFe 2 O 4 particles showed similar results after the interface bonding reaction, demonstrating that the protonated carboxyl group disappeared and that new bands appeared at 1570 and 1430 cm −1 ( Figure S1B).
The concentration of HP molecules bonded to the surfaces of the CoFe 2 O 4 particles depended on the weight and size of the CoFe 2 O 4 particles as a substrate. As the size of the particles decreased, the number of HP molecules bonded to the surfaces of the CoFe 2 O 4 particles increased under the same weight conditions due to the increase in surface area of the whole particles. When the density of CoFe 2 O 4 particles was fixed (ρ = 5.3 g/cm 3 ), approximately 50 µg of CoFe 2 O 4 particles with various sizes (60, 133, 245, and 335 nm) would contain 7.4 × 10 10 , 6.9 × 10 9 , 1.1 × 10 9 , and 4.5 × 10 8 primary particles with surface areas of 8.4, 3.8, 2.1, and 1.6 cm 2 , respectively. Practically, the number of calculated HP molecules was inversely proportional to the size of the CoFe 2 O 4 particles determined by analysis of the UV-VIS absorption spectra. When the particle size varied (60, 133, 245, and 335 nm), the weight of the HP molecules bonded to the surfaces of the CoFe 2 O 4 particles was 5.52, 5.24, 4.84, and 3.17 µg, respectively, under the same weight conditions (50 µg of CoFe 2 O 4 -HP particles). Similarly, the weight of the FA molecules bonded to the surfaces of the CoFe 2 O 4 particles was 2.64, 2.23, 2.07, and 1.68 µg according to particle sizes (60, 133, 245, and 335 nm), respectively, under the same weight conditions (50 µg of particles). Therefore, the smallest particle complex was expected to exhibit the highest generation efficiency for singlet oxygen due to the increase in dose.
Singlet oxygen generation from CoFe 2 O 4 -HP-FA was confirmed by indirect detection of DPBF photodegradation. As a specific singlet oxygen quencher, DPBF readily undergoes a 1,4-cycloaddition reaction with singlet oxygen to form endoperoxides. DPBF then further irreversibly decomposes into 1,2-dibenzoylbenzene [26]. The reaction can easily be followed by measuring the decrease in the optical density of DPBF absorption at 424 nm [27]. Figure 2C illustrates the changes in the absorption spectra of DPBF, which depended on the reaction time with irradiation and the size of the CoFe 2 O 4 -HPs-FAs particles. Notably, under excitation with the Xe lamp, the absorption intensity decreased as the irradiation time increased according to the UV-VIS spectra of DPBF in the THF solution with CoFe 2 O 4 -HPs-FAs. Moreover, the photodegradation efficiency of DPBF increased as the particle size of CoFe 2 O 4 -HPs-FAs decreased to 335, 245, 133, and 60 nm. These results were consistent with the change in the number of conjugated HP molecules as the particle size decreased.

In Vitro Magnetic Resonance (MR) Imaging Measurement
To evaluate the potential applications of CoFe 2 O 4 -HP-FA as a contrast agent for MR-imaging, in vitro MR-imaging contrasts were detected in CoFe 2 O 4 -HPs-FAs samples and PC-3 cells treated with various concentrations (0, 3.13, 6.25, 12.5, 25, and 50 µg/mL) of 60 nm CoFe 2 O 4 -HP-FA using a 3.0 T human clinical scanner at RT as previously described [24,25]. Figure 2D shows that all CoFe 2 O 4 -HPs-FAs efficiently shortened the transverse relaxation time (T 2 ) and significantly decreased signal intensity in T 2 -weighted MR images of PC-3 cells, even at low concentrations. Additionally, the T 2 phantom changed significantly in signal intensity with increasing CoFe 2 O 4 -HP-FA concentration in PC-3 cells. The strong T 2 shortening effect, which likely resulted from stronger magnetic susceptibility caused by agglomeration, led to spin dephasing and a substantial decrease in the MR-imaging signal, thereby generating a "darkening" contrast in T 2 -weighted MR images.

Biocompatibility of the Multifunctional Magnetic Submicron Particles
The biocompatibility of biomaterials is critical for clinical applications. Therefore, we evaluated the cytotoxicity of different sizes and concentrations of CoFe 2 O 4 -HP-FA in fibroblasts (L-929 cells) and prostate cancer cells (PC-3 cells) to confirm their biocompatibility as recommended in International Standard ISO10993-5 [24,25,28]; we repeated this analysis because it is essential for clinical application. Figure 3A,B show the viability of L-929 and PC-3 cells after treatment with CoFe 2 O 4 -HP-FA of different sizes for 24 h. All sizes of CoFe 2 O 4 -HP-FA did not exhibit cytotoxicity in L-929 and PC-3 cells under our experimental conditions at concentrations of 0 to 50 µg/mL. As shown in Figure 3A,B, the viability of both cell types was higher than 95%. The cytotoxicity tests demonstrated that CoFe 2 O 4 -HP-FAs were not cytotoxic and exhibited good biocompatibility in fibroblasts (L-929) and prostate cancer cells (PC-3 cells), regardless of their size; therefore, all of our CoFe 2 O 4 -HP-FAs could be safely applied for in vivo experiments and clinical applications in photodynamic cancer therapy.

In Vitro Photodynamic Anticancer Activities
To compare the photodynamic anticancer activity of different sizes of CoFe2O4-HP and CoFe2O4-HP-FA in prostate cancer cells, we used PC-3 cells, which are commonly used for studies of metastatic prostate cancer and are known to express prostate-specific membrane antigen (PSMA) as previously described [24,25,[27][28][29][30]. Figure 3C,D show the photodynamic anticancer activity of these particles according to the viability of PC-3 cells normalized to that of the control. PC-3 cell viability was lower than 3% in the presence of 50 μg/mL CoFe2O4-HP, regardless of particle size, and lower than 5% in the presence of 25 μg/mL CoFe2O4-HP for all particle sizes except 335 nm (29.4% ± 4.8%). At a concentration of 12.5 μg/mL CoFe2O4-HP, the viability was 18.2% ± 5.2% for particles measuring 60 nm (p < 0.003), 41.1% ± 2.2% for particles measuring 133 nm (p < 0.004), 51.5% ± 6.2% for particles measuring 245 nm (p < 0.02), and 80.4% ± 3.5% for particles measuring 335 nm (p < 0.01). These results demonstrate that CoFe2O4-HP had significantly different anticancer activity depending on the concentration in inverse proportion to particle size, despite the lack of targeting molecules for cancer cells.

In Vitro Photodynamic Anticancer Activities
To compare the photodynamic anticancer activity of different sizes of CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA in prostate cancer cells, we used PC-3 cells, which are commonly used for studies of metastatic prostate cancer and are known to express prostate-specific membrane antigen (PSMA) as previously described [24,25,[27][28][29][30]. Figure 3C,D show the photodynamic anticancer activity of these particles according to the viability of PC-3 cells normalized to that of the control. PC-3 cell viability was lower than 3% in the presence of 50 µg/mL CoFe 2 O 4 -HP, regardless of particle size, and lower than 5% in the presence of 25 µg/mL CoFe 2 O 4 -HP for all particle sizes except 335 nm (29.4% ± 4.8%). At a concentration of 12.5 µg/mL CoFe 2 O 4 -HP, the viability was 18.2% ± 5.2% for particles measuring 60 nm (p < 0.003), 41.1% ± 2.2% for particles measuring 133 nm (p < 0.004), 51.5% ± 6.2% for particles measuring 245 nm (p < 0.02), and 80.4% ± 3.5% for particles measuring 335 nm (p < 0.01). These results demonstrate that CoFe 2 O 4 -HP had significantly different anticancer activity depending on the concentration in inverse proportion to particle size, despite the lack of targeting molecules for cancer cells.
Additionally, the viability of PC-3 cells in the presence of 25 µg/mL CoFe 2 O 4 -HP-FA was lower than 1.2%, regardless of particle size. In particular, the viability of cells in the presence of 12.5 µg/mL CoFe 2 O 4 -HP-FA was 0% ± 0% for particles measuring 60 nm (p < 0.002), 3.7% ± 0.9% for particles measuring 133 nm (p < 0.001), 10.4% ± 3.5% for particles measuring 245 nm (p < 0.004), and 16.6% ± 6.6% for particles measuring 335 nm (p < 0.007). Moreover, the viability of cells in the presence of 6.25 µg/mL CoFe 2 O 4 -HP-FA was 0% ± 0.4% for particles measuring 60 nm (p < 0.002), 30.8% ± 5.9% for particles measuring 133 nm (p < 0.01), 58.0% ± 7.3% for particles measuring 245 nm (p < 0.04), and 73.6% ± 3.4% for particles measuring 335 nm (p < 0.02). For particles with a size of 60 nm, the cell viability in the presence of 3.13 or 1.56 µg/mL CoFe 2 O 4 -HP-FA was 0.3% ± 0.1% (p < 0.002) and 19.0% ± 0.1% (p < 0.006), respectively. These results demonstrate that CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA had significantly different anticancer effects in inverse proportion to particle size. As the concentration of CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA increased, both overall and as the size decreased, the viability of PC-3 cells decreased significantly. These results showed that smaller CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA particles exhibited stronger phototoxic effects than larger particles in PC-3 cells. Furthermore, these results were also consistent with the observed effects of different concentrations of HP in CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA having different sizes, as described above. High concentrations of HP on the surface of CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA could easily generate a large amount of singlet oxygen, which acts as a key molecule in photodynamic anticancer activity by inducing apoptotic cell death during LED irradiation [29,30].
In contrast, comparison of the photodynamic anticancer activity of CoFe 2 O 4 -HP and CoFe2O4-HP-FA indicated that the anticancer activity of 6.25 µg/mL CoFe 2 O 4 -HP-FA was 4.4-11.5-fold higher than that of CoFe 2 O 4 -HP and that CoFe 2 O 4 -HP-FA exhibited more potent phototoxic effects than CoFe 2 O 4 -HP in PC-3 cells when used at relatively low concentrations (1.56 and 3.13 µg/mL). These results suggest that enhanced cellular uptake of CoFe 2 O 4 -HP-FA into PC-3 cells occurred through PSMA expressed by these cells, leading to FA receptor-mediated endocytosis based on the increased phototoxic activity compared with that of CoFe 2 O 4 -HP [31].

Cellular Uptake and Intracellular Localization
To compare the cellular uptake and intracellular distribution of our particles without (CoFe 2 O 4 -HP) and with the FA targeting molecule (CoFe 2 O 4 -HP-FA) in prostate cancer cells (PC-3 cells), we used TEM to visualize the cellular uptake and intracellular distribution of the particles, as shown in Figure 4. The images clearly showed differences in cellular uptake and intracellular localization between CoFe 2 O 4 -HP and CoFe 2 O 4 -HP-FA. As shown in Figure 4C, CoFe 2 O 4 -HP-FA particles were located mostly in the cytoplasm of PC-3 cells and had very strong photodynamic anticancer effects. However, CoFe 2 O 4 -HP particles without FA were located only on the surface of the cell membrane ( Figure 4B) and therefore exhibited lower photodynamic anticancer activity than CoFe 2 O 4 -HP-FA. These TEM images suggest that FA could enhance the cellular uptake and intracellular localization of CoFe 2 O 4 -HP-FA via binding to PSMA because PC-3 cells, which are known to be negative for the FA receptor (FR), express PSMA, thus leading to FR-mediated endocytosis [31]. This may explain the observed phototoxic effects of CoFe 2 O 4 -HP-FA in PC-3 cells, consistent with the photodynamic anticancer activity shown in Figure 3C,D. for particles measuring 335 nm (p < 0.007). Moreover, the viability of cells in the presence of 6.25 μg/mL CoFe2O4-HP-FA was 0% ± 0.4% for particles measuring 60 nm (p < 0.002), 30.8% ± 5.9% for particles measuring 133 nm (p < 0.01), 58.0% ± 7.3% for particles measuring 245 nm (p < 0.04), and 73.6% ± 3.4% for particles measuring 335 nm (p < 0.02). For particles with a size of 60 nm, the cell viability in the presence of 3.13 or 1.56 μg/mL CoFe2O4-HP-FA was 0.3% ± 0.1% (p < 0.002) and 19.0% ± 0.1% (p < 0.006), respectively. These results demonstrate that CoFe2O4-HP and CoFe2O4-HP-FA had significantly different anticancer effects in inverse proportion to particle size. As the concentration of CoFe2O4-HP and CoFe2O4-HP-FA increased, both overall and as the size decreased, the viability of PC-3 cells decreased significantly. These results showed that smaller CoFe2O4-HP and CoFe2O4-HP-FA particles exhibited stronger phototoxic effects than larger particles in PC-3 cells. Furthermore, these results were also consistent with the observed effects of different concentrations of HP in CoFe2O4-HP and CoFe2O4-HP-FA having different sizes, as described above. High concentrations of HP on the surface of CoFe2O4-HP and CoFe2O4-HP-FA could easily generate a large amount of singlet oxygen, which acts as a key molecule in photodynamic anticancer activity by inducing apoptotic cell death during LED irradiation [29,30].
In contrast, comparison of the photodynamic anticancer activity of CoFe2O4-HP and CoFe2O4-HP-FA indicated that the anticancer activity of 6.25 μg/mL CoFe2O4-HP-FA was 4.4-11.5-fold higher than that of CoFe2O4-HP and that CoFe2O4-HP-FA exhibited more potent phototoxic effects than CoFe2O4-HP in PC-3 cells when used at relatively low concentrations (1.56 and 3.13 μg/mL). These results suggest that enhanced cellular uptake of CoFe2O4-HP-FA into PC-3 cells occurred through PSMA expressed by these cells, leading to FA receptor-mediated endocytosis based on the increased phototoxic activity compared with that of CoFe2O4-HP [31].

Cellular Uptake and Intracellular Localization
To compare the cellular uptake and intracellular distribution of our particles without (CoFe2O4-HP) and with the FA targeting molecule (CoFe2O4-HP-FA) in prostate cancer cells (PC-3 cells), we used TEM to visualize the cellular uptake and intracellular distribution of the particles, as shown in Figure  4. The images clearly showed differences in cellular uptake and intracellular localization between CoFe2O4-HP and CoFe2O4-HP-FA. As shown in Figure 4C, CoFe2O4-HP-FA particles were located mostly in the cytoplasm of PC-3 cells and had very strong photodynamic anticancer effects. However, CoFe2O4-HP particles without FA were located only on the surface of the cell membrane ( Figure 4B) and therefore exhibited lower photodynamic anticancer activity than CoFe2O4-HP-FA. These TEM images suggest that FA could enhance the cellular uptake and intracellular localization of CoFe2O4-HP-FA via binding to PSMA because PC-3 cells, which are known to be negative for the FA receptor (FR), express PSMA, thus leading to FR-mediated endocytosis [31]. This may explain the observed phototoxic effects of CoFe2O4-HP-FA in PC-3 cells, consistent with the photodynamic anticancer activity shown in Figure 3C,D.

Apoptotic DNA Fragmentation and Caspase-3 and -7 Enzyme Activity Assays
Internucleosomal DNA fragmentation is a hallmark of apoptosis in cells, resulting from activation of the caspase-3/7 cascade [32]. Therefore, we examined apoptotic cell death resulting from DNA fragmentation after irradiation. As shown in Figure 5A, no DNA ladder formation was observed in the untreated control; however, a typical DNA ladder pattern was progressively observed in PC-3 cells treated with 6.25 µg/mL CoFe 2 O 4 -HPs- FAs (measuring 60, 133, 245, and 335 nm). DNA fragmentation was also inversely proportional to the size of the CoFe 2 O 4 -HP-FA, and this result was consistent with the observed anticancer activity of 6.25 µg/mL CoFe 2 O 4 -HP-FA. Thus, the increased anticancer activity observed in Figure 3D resulted from induction of apoptotic DNA fragmentation, as shown in Figure 5A.

Apoptotic DNA Fragmentation and Caspase-3 and -7 Enzyme Activity Assays
Internucleosomal DNA fragmentation is a hallmark of apoptosis in cells, resulting from activation of the caspase-3/7 cascade [32]. Therefore, we examined apoptotic cell death resulting from DNA fragmentation after irradiation. As shown in Figure 5A, no DNA ladder formation was observed in the untreated control; however, a typical DNA ladder pattern was progressively observed in PC-3 cells treated with 6.25 μg/mL CoFe2O4-HPs- FAs (measuring 60, 133, 245, and 335 nm). DNA fragmentation was also inversely proportional to the size of the CoFe2O4-HP-FA, and this result was consistent with the observed anticancer activity of 6.25 μg/mL CoFe2O4-HP-FA. Thus, the increased anticancer activity observed in Figure 3D resulted from induction of apoptotic DNA fragmentation, as shown in Figure 5A. Caspase enzymes are cytoplasmic aspartate-specific cysteine proteases that play a central role in apoptosis in response to pro-apoptotic signals. In particular, caspase-3 and -7, known as the effector caspases, act further downstream and direct cellular breakdown through cleavage of structural proteins. Activation of caspase-3 and -7 is a hallmark of apoptosis [30,32]. Therefore, we evaluated Caspase enzymes are cytoplasmic aspartate-specific cysteine proteases that play a central role in apoptosis in response to pro-apoptotic signals. In particular, caspase-3 and -7, known as the effector caspases, act further downstream and direct cellular breakdown through cleavage of structural proteins. Activation of caspase-3 and -7 is a hallmark of apoptosis [30,32]. Therefore, we evaluated the effects of 60-nm CoFe 2 O 4 -HP-FA on photo-induced apoptosis after irradiation using caspase-3 and -7 enzyme activity assays. Figure 3C,D show concentration-dependent increases in photodynamic anticancer activity in PC-3 cells after irradiation, mainly as a result of significantly increased caspase-3 and -7 enzyme activity. Figure 5B shows the caspase-3 and -7 activity before and after irradiation followed by treatment with various concentrations of 60-nm CoFe 2 O 4 -HP-FA particles. These results indicate that irradiation after treatment with CoFe 2 O 4 -HP-FA effectively activated caspase-3 and -7 in PC-3 cells, leading to apoptotic cell death.

Morphological Detection of Apoptotic Cell Death
Next, we morphologically detected apoptotic cells using Annexin V-FITC and PI staining to evaluate the predominant morphological changes through which CoFe 2 O 4 -HP-FA caused apoptotic cell death; this assay can easily evaluate apoptotic cell death using Annexin V-FITC, which binds to the translocated membrane phosphatidylserine of apoptotic cells, and PI, which reveals the nuclear deformation of apoptotic cells. Figure 5C shows early-and late-stage apoptosis by double-staining with Annexin V-FITC and PI after irradiation for 30 min. Importantly, cells stained by Annexin V-FITC and PI were not detected in the untreated control, demonstrating that cell death induced by light irradiation after treatment with CoFe 2 O 4 -HP-FA may occur through apoptosis for all sizes of CoFe 2 O 4 -HP-FA.
We also observed nuclear fragmentation in PC-3 cells using Hoechst 33342 dye, as shown in Figure 5D. CoFe 2 O 4 -HP-FA-treated PC-3 cells showed more condensed and granular apoptotic nuclear bodies as compared with the control, demonstrating that light irradiation after treatment with CoFe 2 O 4 -HP-FA induced apoptosis in prostate cancer cells (PC-3 cells).
Taken together, findings were consistent with the observed anticancer activity ( Figure 3D). These effects and the resulting cell death may be mediated via apoptosis induced by irradiation after CoFe 2 O 4 -HP-FA treatment.

Preparation of CoFe 2 O 4 -HP-FA Submicron Particles with Various Particle Sizes
CoFe 2 O 4 submicron particles were prepared using a method similar to that reported previously [33]. In a typical procedure for synthesis of CoFe 2 O 4 particles, FeCl 3 ·6H 2 O (4/3 mmol), CoCl 2 ·2H 2 O (2/3 mmol), NaOAc, and Na acrylate were dissolved in a mixture of ethylene glycol (EG) and diethylene glycol (DEG) in a beaker under magnetic stirring. After 30 min, the as-formed viscous slurry was transferred into a Teflon-lined stainless-steel autoclave with a capacity of 80 mL. The autoclave was heated to and maintained at 200 • C for 10 h and then allowed to cool to room temperature (RT). The ratio of NaOAc/Na acrylate (w/w in grams) controlled the grain size of the CoFe 2 O 4 particles. For example, ratios of 1.4/0.1, 1.2/0.3, 1.0/0.5, and 0.5/1.0 led to synthesis of CoFe 2 O 4 particles with average grain sizes of 25.7, 17.5, 13.5, and 11.9 nm, respectively. Similarly, the volume ratio of EG to DEG (V EG /V DEG ) controlled the external particle size of the CoFe 2 O 4 particles.
To introduce a large amount of PS molecules, the surfaces of CoFe 2 O 4 particles having different sizes were treated with microdielectric barrier discharge (DBD) plasma for 30 min under an electrical discharge power of less than 3 W (0.7 kV, 5 mA, and phase angle of~1 radian). After plasma treatment, the optical functionality on magnetic particles was provided by a wet chemical process with HP, similar to the method described in our previous report [27]. The concentration of HP and FA molecules bonded to the surfaces of CoFe 2 O 4 nanoparicles was estimated by using UV-visible absorption spectroscopy. The comparative absorption ODs at 500 nm (HP: 2.02 × 10 −4 M) and 283 nm (FA: 1.1 × 10 −4 M) of the reference solution and the remaining solution obtained after removing the magnetic aggregation in the magnetic particle solution result the reacted concentration of HP and FA molecules with the particles.

Detection of Singlet Oxygen
Degradation of 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen quencher was studied in the presence of CoFe 2 O 4 -HP-FA. In the photochemical experiment, 3.0075 mL of THF solution containing CoFe 2 O 4 -HP-FA (1 mg) and DPBF (4.61 × 10 −8 M) was introduced into a 1-cm quartz cell under dark conditions. The experiments were performed by irradiating the samples with a Xe lamp (150 W, Abet Technologies, Milford, CT, USA). A 480-nm glass cut-off filter was used to filter out ultraviolet light such that only the HP Q band was irradiated, thereby preventing direct photodegradation of DPBF by UV light. After every 10 min of irradiation, the absorption spectra of the samples were monitored using a UV-Vis spectrophotometer. We then compared the photodegradation of DPBF in solutions of CoFe 2 O 4 -HP-FA particles of different sizes to evaluate the relative capacity for 1 O 2 production.

Biocompatibility Assessment
To evaluate the biocompatibility of different sizes of CoFe 2 O 4 -HP-FA, cytotoxicity tests were performed using L-929 and PC-3 cells as previously described [27,28,30]. Precultured cells were plated in 24-well plates (Costar Corp., Greenwich, CT, USA) at 1.0 × 10 5 of 2.0 × 10 5 cells/well for PC-3 and L-929 cells, respectively. The cells were then incubated at 37 • C in an atmosphere containing 5% CO 2 for 24 h to obtain confluent monolayers of cells prior to use. After incubation, all cells were treated with various concentrations (0, 6.25, 12.5, 25, or 50 µg/mL) of CoFe 2 O 4 -HP-FA having different sizes and incubated at 37 • C in an atmosphere containing 5% CO 2 for 24 h under dark conditions. The cells were then washed three times with PBS and incubated with Cell Counting Kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) for 30 min to determine the cytotoxicity of each concentration of CoFe 2 O 4 -HP-FA. The cytotoxicity of each sample was calculated by measuring the absorbance of each well at 450 nm using a multimode microplate reader (Synergy HT; BioTek Instruments, Inc., Winooski, VT, USA). The relative cell viability is presented as the percentage of surviving cells in relation to that of untreated control cells.

Assessment of Photodynamic Anticancer Activity in Vitro
To evaluate the in vitro photodynamic anticancer activity of different sizes of CoFe 2 O 4 -HP-FA in cancer cells, we used PC-3 prostate cancer cells as previously described [24,25,29,30]. Briefly, the cells were incubated with different concentrations of differently sized CoFe 2 O 4 -HP-FA at 37 • C with 5% CO 2 for 2 h to facilitate uptake of the particles into the cells under dark conditions. After incubation, the cells were irradiated using a customized green light-emitting diode (LED) equipped with a UV filter for 30 min. The LED had a wavelength range of 480-580 nm, a maximum wavelength of 515 nm, and a spot area of 20 mW. The cells were then incubated for another 24 h at 37 • C in an atmosphere containing 5% CO 2 after irradiation with the LED. The following day, cell viability was measured using CCK-8 solution as described above to determine photodynamic anticancer activity.

Assessment of Cellular Uptake and Intracellular Localization of CoFe 2 O 4 -HPs-FAs
To confirm the cellular uptake and intracellular localization of CoFe 2 O 4 -HPs-FAs in prostate cancer cells, we used a transmission electron microscope (TEM) with 60 nm CoFe 2 O 4 -HPs-FAs. All samples were observed using the TEM (JEM-1011; JEOL, Tokyo, Japan) for acquisition of high-magnification images as previously described [24].

Apoptotic DNA Fragmentation Analysis
For DNA fragmentation analysis, apoptosis was induced by irradiation for 30 min after treatment of PC-3 cells with differently sized CoFe 2 O 4 -HP-FA at a concentration of 6.25 µg/mL. After irradiation for 30 min, cellular DNA was prepared using a Quick Apoptotic DNA Ladder Detection kit (K120-50; BioVision Inc., Milpitas, CA, USA) according to the manufacturer's instructions. The cells were rinsed with PBS, and total DNA was extracted after the cells were lysed with Tris-EDTA lysis buffer. Total DNA was then purified with enzyme and ammonium acetate solution. Purified DNA was electrophoresed on 1.2% agarose gels at 5 V/cm for 90 min. After electrophoresis, DNA stained with Midori Green Advanced DNA stain (MG03; Nippon Genetics Europe GmbH, Duren, Germany) was analyzed using a gel documentation system (Quantum ST4; Vilber Lourmat, Eberhardzell, Germany).

Caspase-3 and -7 Enzyme Activity Assay
To confirm the cell death mechanism in prostate cancer cells, the activity of caspase-3 and -7 was measured using a Caspase-Glo 3/7 assay kit (G8091; Promega, Madison, WI, USA) according to the manufacturer's instructions before and after light irradiation. Briefly, precultured PC-3 cells were incubated with different concentrations of 60-nm CoFe 2 O 4 -HP-FA at 37 • C in 5% CO 2 for 2 h under dark conditions and were then irradiated for 30 min. Before and after 0 or 1 h of irradiation, cells were removed from the incubator and allowed to equilibrate to RT for 10 min. Next, 100 µL of Caspase-Glo reagent was added to each well, and the content of the well was gently mixed using a plate shaker at 400 rpm for 30 s. The plate was then incubated at RT for 30 min. The luminescence of each sample was measured in a multimode microplate reader (Synergy™ HT-; BioTek, Winooski, VT, USA).

Morphological Detection of Apoptotic Cell Death
To detect apoptotic cell death, we used an EzWay Annexin V-FITC apoptosis detection kit (K29100; Komabiotech Inc., Seoul, Korea) for detection of cell membrane inversion after irradiation. Briefly, PC-3 cells were irradiated with the LED for 30 min after treatment with 60-nm CoFe 2 O 4 -HP-FA at a concentration of 3.13 µg/mL, and the cells were stained with Annexin V-FITC reagent to stain the cell membranes and PI to stain the dead cells. The morphology of the cell membrane was observed under a laser-scanning confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany) with a 20× objective lens and fluorescence optics (excitation at 488 nm for FITC and 530 nm for PI; emission at 518 nm for FITC and 620 nm for PI).
To evaluate apoptotic cell nuclei, Texas Red C2-maleimide (30 ng/mL in PBS; Invitrogen, Carlsbad, CA, USA) was used to stain the cell membrane, and Hoechst 33342 (1 µg/mL in PBS; Sigma-Aldrich Co., Seoul, Korea) was used to stain the nuclei. Cell morphology was analyzed using a laser-scanning confocal microscope (LSM 700) with a 20× objective lens and fluorescence optics (excitation at 595 nm for Texas Red C2-maleimide and 352 nm for Hoechst 33342; emission at 615 nm for Texas Red C2-maleimide and 620 nm for PI). Images of the cell membranes and nuclei were analyzed using ZEN imaging software (ZEN 2009; Carl Zeiss MicroImaging GmbH, Oberkochen, Germany).

Statistical Analysis
The data shown in this study were obtained from two independent experiments (n = 6) and expressed as the mean ± standard deviation (SD) for quantitative data. Statistical comparisons were performed with Student's t-tests, and differences with p < 0.05 were considered significant.

Conclusions
In this study, we successfully designed and fabricated novel, biocompatible CoFe 2 O 4 -HP-FA particles based on PS-coated magnetic submicron particles with variable sizes for application as PDT agents in the treatment of cancer. The anticancer activity of these particles was confirmed in PC-3 cells in vitro. Additionally, we demonstrated that our CoFe 2 O 4 -HP-FA particles had high water solubility, good MRI compatibility in PC-3 cells, biocompatibility (i.e., without cytotoxic effects), and marked photodynamic anticancer activity via induction of apoptotic cell death in PC-3 cells. At the critical particle size and concentration, anticancer activity was in linear proportion with particle concentration and inversely proportional to the size of the particles. The particle size and dose effects were affected by the surface area of each particle because the number of HP molecules conjugated to the particles increased as the size decreased and the surface area increased. These results indicate that CoFe 2 O 4 -HP-FA particles may be useful in the field of PDT and could have potential as therapeutic agents for MR-imaging based on PDT due to their high saturation value for magnetization and superparamagnetism.
However, this study only used a green light to activate the HP molecules, which generated singlet oxygen to kill the cancer cells; HP molecules also have a peak in the red light band. Thus, further studies, such as in vitro and in vivo anticancer studies using red light to confirm the anticancer activity and to improve better tissue penetration for MR-imaging-based PDT, will be needed to verify the potential of these particles for clinical applications.