Dissipative Particle Dynamics Studies of Doxorubicin-Loaded Micelles

Article pubs.acs.org/JPCB

Dissipative Particle Dynamics Studies of Doxorubicin-Loaded Micelles Assembled from Four-Arm Star Triblock Polymers 4ASPCL‑b‑PDEAEMA‑b‑PPEGMA and their pH-Release Mechanism Shu Yu Nie,†,§ Yao Sun,†,§ Wen Jing Lin,† Wen Sheng Wu,†,‡ Xin Dong Guo,† Yu Qian,† and Li Juan Zhang*,† †

School of Chemistry and Chemical Engineering, South China University of Technology, Wusan Street, Guangzhou 510640, P. R. China ‡ School of Chemistry and Chemical Engineering, Zhaoqing University, Zhaoqing Avenue, Zhaoqing, 526061, P. R. China ABSTRACT: Dissipative particle dynamics (DPD) simulation was applied to investigate the microstructures of the micelles selfassembled from pH-sensitive four-arm star triblock poly(ε-caprolactone)-b-poly(2-(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (4AS-PCL-b-PDEAEMA-b-PPEGMA). In the optimized system, the micelles have a core− mesosphere−shell three-layer structure. The drug-loading process and its distribution at different formulations in the micelles were studied. The results show that DOX molecules distributed in the core and the interface between the core and the mesosphere, suggesting the potential encapsulation capacity of DOX molecules. More drugs were loaded in the micelles with the increase in DOX, and the size of micelles became larger. However, some openings start to generate on the PEG shell when the DOX reaches a certain concentration. By changing the pH values of the system, different morphologies of the micelles were acquired after the pH-sensitive blocks PDEAEMA were protonated, the mechanism of which was also analyzed through correlating functions. The results indicated that the sudden increase in solubility parameter of the pH-sensitive blocks and the swelling of the micelles were the key factors on the change of morphologies. Furthermore, with the decrease in pH value, the number and size of the cracks on the surface of the micelles were larger, which may have a direct effect on the drug release. In conclusion, 4AS-PCL-b-PDEAEMA-b-PPEGMA has great promising applications in delivering hydrophobic anticancer drugs for improved cancer therapy.

1. INTRODUCTION Because of intrinsic disadvantages most anticancer agents have, such as poor water solubility, short duration of circulation, and improper biodistribution,1,2 one promising approach to overcome these problems is the application of polymeric micelles.3 Amphiphilic polymers tend to self-assemble into structures composed of a hydrophobic core stabilized by a hydrophilic shell, and the hydrophobic inner core can carry various anticancer drugs such as cisplatin, doxorubicin, and paclitaxel.4 Star-shaped amphiphilic polymers have been developed to improve the thermodynamic stability of the polymeric micelles. Star-shaped polymer is a kind of dendritic polymer with unique properties attributed to their particular well-defined architecture with multiple polymer chains radiating from one central group.5−8 It has been proved that the star-shaped polymers possess more favorable characteristics contrasted with the linear ones,9 which allows the amphiphilic star polymers to be a promising candidate for anticancer drug delivery.10−14 In recent decades, polymers with the ability to encapsulate and release drugs in response to an acidic environment have become an exciting field of investigation.15−18 The amphiphilic polymers © 2013 American Chemical Society

modified with pH-sensitive groups can increase the selectivity for tumor cells and enhance intracellular drug delivery while reducing systemic toxicity and side effects.19−24 Dissipative particle dynamics (DPD), proposed by Hoogerbrugge and Koelman36 in 1992 and revised by Español and Warren,42 is one effective simulation method to investigate the phase behavior of complex fluids, providing a mesoscopic insight into the macroscale experimental system. The combination with the bead-and-spring model makes it very appropriate for simulations on structure and dynamics of soft matter.26 The forces applied on DPD effectively stretch the characteristic time scale of the simulated system compared with the full atomistic and molecular dynamic simulation.21,25 In addition, by establishing a relationship between a simple function form of the conservative repulsion in DPD and the Flory−Huggins parameter theory,27 DPD method has been widely applied in the study of mesoscale structures of complex Received: July 29, 2013 Revised: September 3, 2013 Published: September 30, 2013 13688

dx.doi.org/10.1021/jp407529u | J. Phys. Chem. B 2013, 117, 13688−13697

The Journal of Physical Chemistry B

Article

systems.28 For instance, Moeinzadeh et al. carried out DPD simulation to study the effect of segment length and macromonomer concentration on the distribution and size of the micelles formed by four-arm star polymers.29 Guo et al. performed some research on the pH-sensitive micelles selfassembled from cholesterol-conjugated oligopeptides for anticancer drug delivery.30 Luo et al. applied DPD simulation to investigate the pH-sensitive polymer. The result showed that the pH value affected the morphologies of micelles and the simulation results were qualitatively consistent with the experimental results.31 Although some simulation studies have worked on the phase-separation behavior of the amphiphilic molecules in the presence of solvents and the formation of drug-loaded micelles and demonstrated that the pH value had a pronounced influence on the morphology of micelles selfassembled from polymers with pH-sensitive groups, few detailed analyses were engaged on the appearance and structure of the transformed micelles at different pH environments, and the pH-sensitive mechanism was seldom explored and discussed in depth. Little research on the simulation of pHsensitive star-shaped polymer was reported. In our previous work, a new pH-sensitive amphiphilic fourarm star triblock polymer poly(ε-caprolactone)-b-poly(2(diethylamino)ethyl methacrylate)-b-poly(poly(ethylene glycol) methyl ether methacrylate) (4AS-PCL-b-PDEAEMA-bPPEGMA) was designed and successfully synthesized.32 It should be noted that the PDEAEMA blocks act as a pHsensitive part, which is hydrophobic and collapses on the core at the neutral or basic pH to prevent the premature burst drug release, but it becomes soluble in acidic solution due to the protonation of the amine groups and highly positively charged.33,34 The experimental results showed the in vitro release behavior of DOX from the three-layer micelles exhibited pH-dependent property.35 Herein, DPD mesoscopic simulation is employed to investigate the formation of drug-loaded micelles and the effect of pH values on aggregate morphologies of blank and DOX-loaded micelles. The appearance and structure of micelles were studied in both the normal state (pH 7.4) and the swelling state (pH below 7.4), and the mechanisms of these pH-induced changes are discussed extensively. This work will be helpful to understand the relationship of microstructure and macroperformances of pHsensitive star polymeric micelles for drug-controlled release.

The conservation force for nonbonded particles is defined by soft repulsion. The dissipative force corresponding to a frictional force depends on both the position and relative velocities of the beads. The random force is a random interaction between bead i and its neighbor bead j. All forces vanish beyond a certain cutoff radius rc, whose value is usually set to one unit of length in simulations. The three forces are given by the following formulas: ⎧ ⎪ aij(1 − rij)riĵ (rij < 1) FCij = ⎨ ⎪ 0 (rij ≥ 1) ⎩

FijD = −

FijR =

(4)

δt

(5)

(6)

where aii is equal to 25. χij can be obtained from the solubility parameters by χij =

(δi − δj)2 V RT

(7)

where V is the arithmetic average of molar volumes of beads i and j. δi and δj are solubility parameters, which depend on the chemical character of beads and can be obtained by moleculardynamics simulations. In addition, an extra spring force (FijS) is introduced to describe the constraint between the bonded particles in one molecule. In this work, the spring constant was set to 4, resulting in a slightly smaller distance for bonded particles than for nonbonded ones.37 2.2. Relationship between pH Values and Protonation. As the pH-sensitive chains in 4AS-PCL-b-PDEAEMA-bPPEGMA, the pKa for the protonated PDEAEMA is around 7,38 which means that it deprotonates almost completely above 8. The amine groups connected to the chains can accept protons in acidic environments, whereas they release protons in base environments. Thus owing to the protonation of the amine groups, PDEAEMA chains become soluble in acidic solution. According to related research, the protonation degree of the amine groups depends on the pH value of the solution they are presented in. In other words, the number of the protonated amine groups are various at different pH values. Moreover, at the same pH environment, the protonation degrees of the amine groups remain unchanged once the system reaches an equilibrium state. The protonation degree of the pH-sensitive groups is related to the intrinsic pKa value and the pH values of the exterior environment, whose value is given by the Henderson−Hasselbalch formula:39

(1)

∑ (FCij + FijD + FijR ) i≠j

(riĵ ·vij)riĵ

σω(rij)riĵ ζ

aij = aii + 3.27χij

where ri, vi, mi, and fi denote the position vector, velocity, mass, and total force on the particle i, respectively. For simplicity, the masses of all beads are set to 1 DPD unit. The force between each pair of beads is a sum of a conservative force (FijC), a dissipative force (FijD), and a random force (FijR): fi =

2kT

where rij = ri − rj, rij = |rij|, rij = rij/|rij|, vij = vi − vj, σ is the noise strength, ζ denotes a randomly fluctuating variable with zero mean and unit variance, δt is the time step of simulation, k is the Boltzmann constant, and T is the system temperature. The rdependent weight function ω(r) = (1 − r) for r < 1 and ω(r) = 0 for r > 1. aij is the maximum repulsion between bead i and bead j, depending on the underlying atomistic interaction, and is linearly related to the Flory−Huggins parameters(χij) as given in eq 6:

2. SIMULATION METHODS 2.1. DPD Theory. In the DPD method, a set of soft interacting particles is used to simulate a fluid system. Each particle represents a group of atoms or a volume of fluid that is large on the atomistic scale but is still macroscopically small. All beads comply with Newton’s equations of motion:36 dri dv = vi, mi i = fi dt dt

σ 2(ω(rij))2

(3)

(2) 13689



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Figure 1. Molecular structures and coarse-grained models of 4AS-PCL-b-PDEAEMA-b-PPEGMA (a), DOX (b), and water (c).

Figure 2. Topology structures of coarse-grained model of 4AS-PCL-b-PDEAEMA-b-PPEGMA: (a) before protonation and (b) after protonation. (The beads in light blue refer to DEAH.)

Table 1. Interaction Parameters aij between Different Beads in Neutral and Acidic Environment aij

center

D1

D2

D3

MAA

CL

PEG

DEA

DEAH

W

center D1 D2 D3 MAA CL PEG DEA DEAH W

25.00 31.06 29.20 28.45 34.35 28.38 45.46 25.83 297.25 136.65

25.00 25.26 25.09 25.63 25.42 31.14 27.66 219.54 104.38

25.00 25.05 26.69 25.03 34.22 26.36 247.34 121.89

25.00 26.29 25.14 33.61 25.70 248.68 126.41

25.00 27.01 27.39 30.44 173.98 76.18

25.00 34.41 25.95 244.25 117.12

25.00 40.58 116.43 49.66

25.00 183.71 135.82

25.00 25.71

25.00

αH+ =

1 × 100% 1 + 10PH − pk a

3. MODELS AND INPUT PARAMETERS In the studies, DPD simulations were employed in two systems: the neutral system and acidic systems with different pH conditions. These systems consist of water, 4AS-PCL-bPDEAEMA-b-PPEGMA, and DOX as the drug model. The neutral system is mainly conducted to study the formation of drug-loaded micelles and the distribution of DOX in micelles. In acidic systems, we further discussed the pH-induced effects on blank and drug-loaded micelles. To study the pH-induced changes on blank micelles, only water (90%) and polymers (10%) were contained, while for the drug-loaded micelles, the pH values of the solution were given in four cases: 6.9, 6.5, 6.0, and 5.0 with the same components (87% water, 10% polymers, and 3% DOX) in each simulation, relatively. The coarse-grain models are shown in Figure 1. The molecular structure of DOX was divided into three types of beads (D1, D2, and D3). One water molecule was represented

(9)

where α+H denotes the protonation degrees of the pH-sensitive groups; pKa = 6.9 is used in this study. According to eq 9, when the pH values of the solvents are various from ≥7.4, 6.9, 6.5, 6.0, and 5.0, α+H is corresponded to the values (%) 0, 50, 72, 89, and 100, relatively. The results express that at neutral environments (pH 7.4) the protonation degree of the amine groups is so small that it can be ignored. When the pH is up to 7.4, the protonation of the amine groups becomes pronounced, and with the decreasing pH value, more amine groups obtain protons and present the greatest protonation degree at pH 5.0, resulting in a state that almost each amine group accepts one proton and becomes positively charged, and thus the protonation degree reaches 100% as regarded. According to the calculation, the pH values of the simulation systems are set to 6.9, 6.5, 6.0, and 5.0, respectively. 13690



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Figure 3. Configurations of DOX-loaded polymeric micelles at different simulation steps in a neutral environment.

concentration is higher than its critical micelle concentration. Several snapshots of the formation of the DOX-loaded micelles in neutral environment are shown in Figure 3. The system includes 4AS-PCL-b-PDEAEMA-b-PPEGMA, DOX, and water with the volume ratio of 10:3:87. To show aggregate morphologies clearly, the water molecules are not displayed. As shown in Figure 3a, all components are distributed randomly in water at the beginning of the simulation. With the evolution of the simulation, some polymer molecules aggregate and form small clusters first; subsequently, the small clusters emerge and turn into larger aggregates. Hydrophobic PCLs are distributed inside the micelle forming a hydrophobic core, while hydrophilic PEG spread around the surface, forming a protective shell. In particular, the pH-sensitive chains PDEAEMA are distributed between the PEG shell and the PDEAEMA core forming a mesosphere. In the meanwhile, the DOX molecules in water gradually diffuse into the micelles (Figure 3b−i). Spherical micelles with stable structures, core (PCL)−mesosphere (PDEAEMA)−shell (PEG), are selfassembled when the system achieves balance. The aggregate morphology does not significantly change with extra simulation steps. Therefore, 20 000 steps are sufficient for achieving simulation equilibrium in this system. To show the clear structure of blank and DOX-loaded micelles, we display the cross-section views in Figure 4. It can be seen that DOX is distributed in the PCL core and the pH-sensitive mesosphere, indicating that the micelle formed by the star polymer is an appropriate carrier for DOX. 4.2. Distribution of DOX in Micelles. To show the distribution of DOX in the micelles more directly, the relative concentration profile of DOX and the blocks of 4AS-PCL-bPDEAEMA-b-PPEGMA micelles are given, as shown in Figure 5. As shown in the density profile, the distribution of DOX is

as one bead. At pH >7.4, 4AS-PCL-b-PDEAEMA-b-PPEGMA was divided into Center (neopentane), CL (caprolactone), MAA1, MAA2, DEA, and PEG. MAA1 and DEA were divided from 2-(diethylamino) ethyl methacrylate (PDEAEMA), and MAA2 and PEG were divided from poly(ethylene glycol) methyl ether methacrylate (PPEGMA). Beads in different color are corresponding to different blocks: yellow, center; dark blue, CL; pink, MAA in PDEAEMA blocks; red, DEA; dark green, MAA in PPEGMA blocks; light green, PEG; and black, DOX. At pH 6.9, the pH-sensitive DEA is ionized and represented by DEAH. The mass and volume of each bead is 96 amu and 182 Å3, respectively, and the cutoff radius, rc, between two beads is 8.1 Å. The corresponding topology structures of the coarsegrained model of the four-arm polymer is shown in Figure 2; each arm contains ten CL units, six PDEAEMA units and four PPEGMA units, which were decided by the characteristic values obtained by Synthia module in Materials Studio 5.0 (Accelrys). The interaction parameters aij were calculated according to eqs 5 and 6, as shown in Table 1. The solubility parameters were calculated by molecular dynamics simulations using the Amorphous Cell and Discovery modules in the same software. The operations of the protonation systems during the simulation should be briefly noted. The formation of the protonated micelles is based on the prior formed deprotonated micelles. As the protonated DEA is positively charged, a new force field needs to be generated according to the interaction parameters obtained by the MD simulation between DEAH and other beads. Then, the protonated system can be obtained by a new round DPD simulation on the original deprotonated system with the new force field. In particular, the number of DEAH depends on the protonation degree of the micelles, which needs to be determined in the beginning. A cubic simulation box with periodic boundary condition was applied in all directions. A box of 20 × 20 × 20 rc3 is sufficient to avoid the finite size effects, and the integration time step of 0.05 was small enough for our system to get thermodynamic equilibrium. The simulation steps of 20 000 were used. All simulations were carried out using the DPD method in Mesocite module of the commercial software package Materials Studio 5.0.

4. RESULTS AND DISCUSSION 4.1. Formation of DOX-Loaded Polymeric Micelles in Neutral Environment. It is known that amphiphilic polymers can self-assemble into micelles in aqueous solution when the

Figure 4. Cross-section views of (a) blank micelles, (b) the schematic drawing of blank micelles, and (c) DOX-loaded micelles. 13691



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the increased density of the beads and the compatibility between the beads, the D1 particles are maximally distributed in a region formed by CL particles, namely, the core of micelles; the number of D1 particles distributed in the area formed by DEA particles is considerably large, too, demonstrating that the D1 particles are distributed in the core and mesosphere of the micelles, while the capacity of the core is a little higher than that of the mesosphere. In contrast, the PEG-D1 RDF curve does not exhibit a sharp peak similar to DEA-D1 and CL-D1 curves, indicating that few D1 are distributed in the PEG shell. Similarly, the RDF curves between D2 and D3 particles of DOX molecules and polymer particles have the same trend as the D1 particle. The results reflect the compatibility between different blocks of polymer and DOX molecules: the PCL blocks have the best compatibility with the DOX molecules, which are a little larger than those of PDEAEMA blocks and DOX molecules, while a large repulsion exists between PEG blocks and DOX molecules, causing DOX to be mainly loaded in the PCL core and the interface between the PDEAEMA mesosphere and the core. 4.3. Effect of DOX Content on Morphologies of Micelles. The studies on drug loading were significant to ensure the availability and stability of the drug-loaded micelles. The aggregate morphologies of DOX-loaded micelles with different drug contents ranging from 1 to 7% are investigated. The volume ratio of 4AS-PCL-b-PDEAEMA-b-PPEGMA is fixed at 10%. The results of the simulation are shown in Figure 7. Stable microspheres are observed with all DOX molecules encapsulated into the PDEAEMA and PCL areas when the DOX content is low, as shown in Figure 7a−c. As seen from the cross-section views of the micelles, the drugs are prior to load in the interface of PDEAEMA and PCL areas in a low drug concentration, while more and more drugs distribute in the PCL area as the drug concentration increases due to the saturation of the interface area. While the DOX content increases to 5%, even though more DOX molecules diffused into the micelle, there are still some DOX molecules that cannot be entirely encapsulated. It is seen that some openings generated in the PEG shell, which results from the bigger size of the micelles caused by the overloaded DOX but without enough PEG to sustain the integrity of the shell. The size of the openings in the shell is even larger when the DOX content is up to 7%, and the structures of the micelles tend to be destroyed. The result shows that there is a maximum value for DOX content that the micelle can carry. In terms of the drug release, appropriate drug-loading efficiency is in favor of the controlled release. 4.4. Effect of pH Value on Morphologies of Blank and DOX-Loaded Micelles. 4.4.1. Morphologies of Blank Micelles at Different pH Values. The β-amino groups in PDEAEMA are capable of accepting or releasing protons in response to environmental pH changes, which can alter the solubility of the polymer in water and result in the structural changes of the micelles. Figure 8 shows the morphological changes of the blank micelles from neutral environment (pH > 7.4) to acidic environment (pH 5.0) at different simulation steps. As shown in Figure 8a, the simulation starts by forming a micelle assembled from neutral 4AS-PCL-b-PDEAEMA-bPPEGMA with 10% polymer and 90% water in the system. In a neutral environment, the β-amino groups in PDEAEMA are hydrophobic, and the micelle shows perfect core− mesosphere−shell structure. When the micelle is transferred to an acidic environment (pH 5.0) (by changing the force field

Figure 5. Relative concentration profiles of DOX and the blocks of 4AS-PCL-b-PDEAEMA-b-PPEGMA as a function of distance along the x-axis direction.

consistent with that of PCL, which indicates that DOX is mainly loaded in the PCL core of the micelles. From the profile, we can see that PDEAEMA distributes in the middle of the micelles and PPEGMA distributes outside the shell. The radial distribution function (RDF) can be used to analyze the distribution of polymer particles and drug particles in the ordered morphologies for further characterization of the miscibility of different contents.31 The relative position between different particles provides the quantitative information of the morphology of micelles, such as the average distance between polymer particles and drug particles. Via the information, the attractions between different types of beads can be analyzed. The RDF curves of PEG, DEA, and CL beads with DOX beads D1, D2, and D3 are shown in Figure 6, respectively.

Figure 6. RDF profiles of DOX and the blocks of 4AS-PCL-bPDEAEMA-b-PPEGMA.

g(r) is the distribution function, and the r values corresponding to the peaks of RDF curve reflect the distance between two kinds of particles; for CL and D1 particles, for instance, the highest peak represents the fact that most of the CL particles appear in the corresponding distance away from D1 particles. As shown in Figure 6a, the trend of the RDF curves of CL-D1 particles is similar to that of DEA-D1 particles, indicating that the relative distances of CL-D1 particles and DEA-D1 particles are very different. Overall, analyzed from the integrated area of the RDF curves, which is proportionate to 13692



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Figure 7. Morphologies of micelles with different DOX content. (The images below are the cross-section views of the micelles above.)

Figure 8. Changes on configurations of blank micelles at pH 5.0 at different time steps (DPD units). The bead in red: DEA; the bead in light blue: DEAH.

of the system), the β-amino groups accept protons to ionize and the PDEAEMA blocks become hydrophilic, resulting in the increase in the PDEAEMA solubility. Interestingly, the result shows that not all protonated PDEAEMA (namely, PDEAEMAH) chains move out from the interlayer exposing to water immediately after being protonated. At the first beginning, only a few PDEAEMAH chains stretch, forming small openings on the shell, while the other chains remain in the micelle. Upon increasing DPD steps, more PDEAEMAH chains spread outside from the openings previously formed, resulting in the formation of larger openings instead of generating new small openings. It may due to the need to overcome more resistance if the chains stretch through the shell. As more PDEAEMAH chains move out from the openings, the openings become larger and larger and turn into a cracks.40 The crack grows larger until all PDEAEMAH chains move out. Finally, the PDEAEMAH chains move out, exposing to water from one to two cracks, and are highly distributed in the same position of the surface, unlike the formation we expected: the chains may be distributed homogeneously. Besides, the core−mesosphere− shell structure is destroyed. In this case, the extended PDEAEMAH chains can be regarded as a component of the shell and the core exposes to the water to a big extent. Moreover, the micelle is less symmetrical after protonation. In terms of the previously mentioned results, we discuss the pHinduced mechanism on the morphological transition of the micelles. As the β-amino groups accept protons to ionize, they charged positively, leading to the generation of electrostatic repulsion between the groups, and the electrostatic repulsions become stronger with the increase in degree of protonation. Because of

the sudden increase in the solubility of DEA after being protonated (the repulsive parameter (25.71) between DEAH and water is much smaller than that between DEA and water (135.82), as shown in Table 1), which is even bigger than that of PEG blocks, it makes PDEAEMAH become the strongest hydrophilic block. As a result, the PDEAEMAH chains have a trend to extend to the aqueous solution and to be directly in contact with the water molecules, causing the micelles to tend to absorb a significant number of water molecules through the water molecule passages because of the much more pronounced solvation after the protonation of the amino groups, which generates a considerable pressure in the intermediate layer of micelles, powerfully acting on the PEG shell. Therefore, in the early stage of protonation, the pressure generated is so small that the PEG shell can resist. Because only a small part of the PDEAEMAH blocks transfer to the surface, the structure of micelles is hardly changed. However, even the migration of a small portion of PDEAEMAH blocks can become the driving force for more water molecules to flow into the micelles. Thus, as the pressure generated by PDEAEMAH blocks increases, the cracks are generated on the surface of the shell when the strength of PEG shell is not big enough to resist the pressure generated by PDEAEMAH blocks, leading to PDEAEMAH blocks exposed to water directly. Moreover, the number and the size of the cracks become larger over time and reach a maximum when the system is in an equilibrium. The micelle with core−shell two-layer structure is ultimately obtained. When the micelles swell, the PDEAEMAH blocks gradually extend to the surface of the micelles, turning into a part of the shell, which regard as channels for the diffusion of DOX 13693



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Figure 9. Morphologies of blank micelles at different pH values. (PEG beads in the images below are concealed to show the inner beads clearly.)

micelle, and finally the more channels of water molecules; therefore, the corresponding drug release gets faster. To reflect the changes on different layers of micelles with different pH values more intuitively, we give a schematic drawing according to the simulation shown in Figure 10. Each component represents a qualitative change rather than referring to a specific value.

molecules, especially those distribute in PDEAEMA intermediate layer can rapidly diffuse into water through the channels, leading to a quick release. Most of the DOX molecules distributed in the PCL core exposed to the shallow surface of the micelles, greatly increasing the contact area with water molecules, which is also conducive to the release of DOX. By contrast, on the basis of the drug release experimental results, the release exponents of which indicate that the drugrelease mechanism was lower than 0.43 in the first stage,35 the release of DOX from the micelles that are unprotonated or in a very low degree of protonation requires passing through an integrate shell, and a tight inner structure is primarily controlled by a combination of diffusion and erosion, so the release rate in this case is very slow.30 To further study the effect of different protonation degree on the morphologies of micelles, we first investigate the systems with only polymers and water at five pH values (pH ≥ 7.4, 6.9, 6.5, 6.0, and 5.0), corresponding to different protonation degrees (0, 50, 72, 89, 100%). The results are shown in Figure 9. As shown in Figure 9, the morphology of PDEAEMA layers and the continuity of the PEG shell have significant changes corresponding to different pH values. With the decreasing pH, the PDEAEMA blocks have a higher degree of protonation, representing the fact that the unprotonated groups (beads in red) gradually reduce while the number of the protonated groups (beads in light blue) gradually increase. Because of the hydrophilicity changes before and after the PDEAEMA blocks are protonated, the unprotonated blocks tend to get close to the hydrophobic PCL core away from the hydrophilic PEG shell, while the protonated PDEAEMA blocks tend to close to the hydrophilic PEG shell away from the hydrophobic PCL area. When the pH is up to 5.0, the amino groups of the PDEAEMA blocks are all protonated; as represented in Figure 9e, all red beads are replaced by bright blue beads. According to the result, when the protonation occurs (pH 7.4), the PDEAEMAH blocks extend through the cracks in the PEG shell and directly contact the aqueous solution. As the pH decreases, more PDEAEMA blocks are protonated and extend out. Besides, the number and size of the cracks on the PEG shell increase accordingly, and more PDEAEMAH blocks become a part of the shell exposing to water. Thus, it can be speculated that as the pH value gets lower, the higher the protonation degree of the four-arm star polymers, the larger and more prevalent the cracks formed in the surface of the

Figure 10. Schematic drawing of the morphologies of blank micelles at different pH values.

To further validate pH-induced changes on the states of PDEAEMA chains after protonation, we calculated the radius of gyrations of PDEAEMA and PDEAEMAH chains, which reflected the degree of stretch in the space, at four different pH values (pH ⩾7.4, 6.9, 6.5, 6.0, and 5.0), respectively. It turned out that there were large increases ranging from 66.6 to 71.4% on the radius of gyrations when the PDEAEMA chains protonated and turned into PDEAEMAH at different pH values, indicating that the stretch degree of the pH-sensitive chain was greatly increased after protonation. It can also be seen from the below images in Figure 9 that the PDEAEMAH chains are shown in extended states, while those unprotonated ones are shown in collapsed states. The main reason for the change in the state of PDEAEMA chains is the sudden increase in the hydrophilicity after the PDEAEMA chains are protonated; the chains tend to be in contact with the hydrophilic PEG shell and the aqueous solution, resulting in the stretch state. By contrast, because the unprotonated PDEAEMA chains have a strong contact with PCL, they show a collapsed morphology on the surface of PCL. 13694



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Figure 11. Morphologies of DOX-loaded micelles at different pH values (a) pH 6.9, (b) pH 6.5, (c) pH 6.0, and (d) pH 5.0. (The three images on the right are the cross-section views in the x, y, and z directions of the leftmost full view images.)

4.4.2. Morphologies of DOX-Loaded Micelles at Different pH Values. The aggregate morphologies of drug-loaded micelles at different pH values (pH⩾7.4, 6.9, 6.5, 6.0 and 5.0) are studied by DPD; DOX was used as the drug model. The simulation system comprises 10 vol % 4AS-PCL-bPDEAEMA-b-PPEGMA, 3 vol % DOX, and 87 vol % water. The results of DPD simulations are shown in Figure 11. The leftmost images are the whole topographies of the micelles at corresponding pH values, reflecting the morphologies of micelles in only one certain direction. Therefore, three crosssection views of the micelles in the x, y, z directions (the images on the right) are given, respectively, for clearer comparisons on the morphologies of micelles under different pH conditions. The areas DOX exposed to the water are marked out by yellow circles. As seen from the simulation results, the protonation degree of PDEAEMA blocks gradually increased as the pH value decreased, resulting in the hydrophobic PDEAEMA blocks becoming hydrophilic, and subsequently extended into the aqueous solvent after swelling, directly contacting water. The result also showed that quite a lot of DOX was distributed on the cracks of the shell, and thus it might be expected that the larger and more cracks were generated, the more drugs were exposed to the solvent, leading to the more favorable release of DOX.41 The cross-sectional views showed that as the pH value decreased, both the number and size of the cracks formed in the PEG shell became larger (more yellow circles were marked out), leading to a direct result that more DOX were dispersed

in the shallow surface of the micelles and a considerable number of drugs were even directly exposed to the surface of the micelle, which greatly increased the contact of DOX and water molecules and thus accelerated the release of DOX. By contrast, in the case that the micelle was not protonated or only under a low protonation degree, the release of DOX required to pass through the complete outer shell, which was mainly controlled by the diffusion, thus showed a much slower rate of release. The release of DOX cannot be observed due to the weak diffusion effect caused by the relatively still state of the aqueous medium in the simulation. By contrast, under the experiment condition, the flow of water molecules promotes the diffusion of drugs, especially after the water molecules transfer into micelles, which is the main difference between simulation and experiment. However, we can foresee the release trend of DOX from the micelles and predict the release mechanism according to simulation results. To improve our research, we will perfect the simulation system by considering the flow of water. Besides, further research will be carried out on the protonation mechanism of the pH-sensitive micelles combining with simulation on atomic level.

5. CONCLUSIONS DPD method was employed to investigate the aggregate behavior of the pH-sensitive blank and DOX-loaded micelles self-assembled from triblock amphiphilic four-arm star polymer 4AS-PCL-b-PDEAEMA-b-PPEGMA. The formation of DOXloaded micelles was studied, and microspherical micelles with 13695



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(7) Iatridi, Z.; Tsitsilianis, C. Water-Soluble Stimuli Responsive StarShaped Segmented Macromolecules. Polymers 2011, 3, 1911−1933. (8) Jin, S.; Higashihara, T.; Jin, K. T.; Yoon, J.; Rho, Y.; Ahn, B.; Kim, J.; Hirao, A.; Ree, M. Synchrotron X-ray Scattering Characterization of the Molecular Structures of Star Polystyrenes with Varying Numbers of Arms. J. Phys. Chem. B 2010, 114, 6247−6257. (9) Lapienis, G. Star-Shaped Polymers having PEO Arms. Prog. Polym. Sci. 2009, 34, 852−892. (10) Zhu, W.; Nese, A.; Matyjaszewski, K. Amphiphilic PEG-grafted Poly (ester-carbonate)s: Synthesis and Diverse Nanostructures in Water. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 1942−1952. (11) Jiang, G.; Xu, H. Synthesis and Evaluation of a Star Amphiphilic Block Copolymer from Poly(ε-caprolactone) and Poly(ethylene oxide) as Load and Release Carriers for Guest Molecules. J. Appl. Polym. Sci. 2010, 118, 1372−1379. (12) Liu, J.; Huang, W.; Pang, Y.; Zhu, X.; Zhou, Y.; Yan, D. SelfAssembled Micelles from an Amphiphilic Hyperbranched Copolymer with Polyphosphate Arms for Drug Delivery. Langmuir 2010, 26, 10585−10592. (13) Nabid, M. R.; Tabatabaei Rezaei, S. J.; Sedghi, R.; Niknejad, H.; Entezami, A. A.; Oskooie, H. A.; Heravi, M. M. Self-Assembled Micelles of Well-defined Pentaerythritol-Centered Amphiphilic A4B8 Star-Block Copolymers Based on PCL and PEG for Hydrophobic Drug Delivery. Polymer 2011, 52, 2799−2809. (14) Cao, W.; Zhu, L. Synthesis and Unimolecular Micelles of Amphiphilic Dendrimer-like Star Polymer with Various Functional Surface Groups. Macromolecules 2011, 44, 1500−1512. (15) Shen, Y. Q.; Zhan, Y. H.; Tang, J. B.; Xu, P. S.; Johnson, P. A.; Radosz, M.; Van Kirk, E. A.; Murdoch, W. J. Multifunctioning pHResponsive Nanoparticles from Hierarchical Self-Assembly of Polymer Brush for Cancer Drug Delivery. AIChE J. 2008, 54, 2979−2989. (16) Sutton, D.; Nasongkla, N.; Blanco, E.; Gao, J. Functionalized Micellar Systems for Cancer Targeted Drug Delivery. Pharm. Res. 2007, 24, 1029−1046. (17) Chen, D. Q.; Song, P.; Jiang, F.; Meng, X. Y.; Sui, W. P.; Shu, C. Y.; Wan, L. J. pH-Responsive Mechanism of a Deoxycholic Acid and Folate Comodified Chitosan Micelle under Cancerous Environment. J. Phys. Chem. B 2013, 117, 1261−1268. (18) Yue, Z. G.; Wei, W.; You, Z. X.; Yang, Q. Z.; Yue, H.; Su, Z. G.; Ma, G. H. Iron Oxide Nanotubes for Magnetically Guided Delivery and pH-Activated Release of Insoluble Anticancer Drugs. Adv. Funct. Mater. 2011, 21, 3446−3453. (19) Giacomelli, F. C.; Stepanek, P.; Giacomelli, C.; Schmidt, V.; Jager, E.; Jager, A.; Ulbrich, K. pH-Triggered Block Copolymer M i c e l l e s B a s e d on a p H - R e s p on si v e P D P A ( p o l y [ 2 (diisopropylamino)ethyl methacrylate]) Inner Core and a PEO (poly(ethylene oxide)) Outer Shell as a Potential Tool for the Cancer Therapy. Soft Matter 2011, 7, 9316−9325. (20) Wang, W.; Cheng, D.; Gong, F.; Miao, X.; Shuai, X. Design of Multifunctional Micelle for Tumor-Targeted Intracellular Drug Release and Fluorescent Imaging. Adv. Mater. 2012, 24, 115−120. (21) Lin, S. L.; Xu, M. Y.; Yang, Z. R. Dissipative particle dynamics study on the mesostructures of n-octadecane/water emulsion with alternating styrene-maleic acid copolymers as emulsifier. Soft Matter 2012, 8, 375−384. (22) Huang, X. J.; Xiao, Y.; Lang, M. Synthesis and Self-Assembly Behavior of Six-Armed Block Copolymers with pH- and ThermoResponsive Properties. Macromol. Res. 2011, 19, 113−121. (23) Chen, J.; Qiu, X.; Ouyang, J.; Kong, J.; Zhong, W.; Xing, M. M. Q. pH and Reduction Dual-Sensitive Copolymeric Micelles for Intracellular Doxorubicin Delivery. Biomacromolecules 2011, 12, 3601−3611. (24) Zhu, W.; Nese, A.; Matyjaszewski, K. Thermoresponsive Star Triblock Copolymers by Combination of ROP and ATRP: from Micelles to Hydrogels. J. Polym. Sci., Part A: Polym. Chem. 2011, 49, 1942−1952. (25) Groot, R. D.; Madden, T. J. Dynamic Simulation of Diblock Copolymer Microphase Separation. J. Chem. Phys. 1998, 108, 8713− 8724.

stable structure core (PCL)−interlayer (PDEAEMA)−shell (PEG) were formed. The DOX distribution of micelles and the morphologies of the micelles with different DOX content showed that the micelles assembled from 4AS-PCL-bPDEAEMA-b-PPEGMA had a good loading performance on DOX, which were distributed in the core and interlayer of the micelles. Besides, there is a maximum value for DOX content that the micelle can carry. In addition, the effect of pH value on morphologies of blank and DOX-loaded micelles was studied. The morphologies of micelles at different pH value were different due to the protonation degree of PDEAEMA blocks and finally resulted in the difference in the swollen state of the micelles. The results showed that the PDEAEMA chains transited from the collapsed state into the extended state under acidic condition, resulting from the protonation of amino groups, and the PDEAEMA blocks became hydrophilic, leading to a part of DOX modules exposed outside to the surface of micelles, which promoted the release of DOX. The pH-induced release mechanism was also discussed, in which the cracks generated in the surface of micelles might mainly attribute to the release of DOX. These conclusions showed that 4AS-PCLb-PDEAEMA-b-PPEGMA has potential applications in cancer drug delivery and would guide the experimental preparation of the drug delivery system with desired properties.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-20-87112046. E-mail: [email protected]. Author Contributions §

S.Y.N. and Y.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (nos. 21176090, 21136003, 21206045), Team Project of Natural Science Foundation of Guangdong Province, China (no. S2011030001366), and Science and Technology Foundation of Guangdong Province, China (no. 2012B050600010).

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