Letter pubs.acs.org/macroletters
Unimolecular Micelles from Layered Amphiphilic Dendrimer-Like Block Copolymers Yunpeng Wang, Gang Qi, and Junpo He* The State Key Laboratory of Molecular Engineering of Polymers, Collaborative Innovation Center of Polymers and Polymer Composite Materials, Department of Macromolecular Science, Fudan University, Shanghai, 200433, China S Supporting Information *
ABSTRACT: In this report, we synthesized layered amphiphilic dendrimer-like block copolymers containing a polystyrene core and poly(p-tert-butoxystyrene)/poly(p-hydroxylstyrene) shell (coded G4-PtBOS/G4-PHOS). The synthetic method is easy involving anionic polymerization, epoxidation, ring-opening reaction and hydrolysis reaction. The hydrolyzed G4-PtBOS was soluble in alkaline water and behaved as unimolecular micelle, as demonstrated by the results of DLS, cryo- and normal TEM, and pyrene entrapping experiment. The stability of the unimolecular micelles was investigated via ζ-potential measurements.
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spacers. Compared with the regular dendrimers, these copolymers may provide larger internal cavities for encapsulating more drug molecules when they possess peripheral hydrophilic functionalities or short chains. Therefore, the applications of the dendrimer-like polymers as unimolecular micelles are attractive not only for load capacity, but also for stability. Gnanou and co-workers synthesized dendrimer-like block copolymers containing a star-shaped core of six polystyrene arms and an outer shell of 12 poly(ethylene oxide) (PEO) arms through sequential cationic and anionic polymerizations.46 The resulting dendrimer-like PS/PEO block copolymer showed stronger tendency to self-organize into unimolecular micelles than the simple star-like block PS/PEO. Hirao and co-workers reported the precise synthesis of thirdgeneration, layered, dendrimer-like block copolymers of styrene and methyl methacrylate, which may be used as candidates for unimolecular micelles.47 Recently, a number of dendrimer-like copolymers have been synthesized, but their applications as unimolecular micelles were rarely reported.48−56 In this paper, we report the synthesis of fourth generation dendrimer-like copolymers with a core−shell structure that can be used to prepare unimolecular micelles. The core is composed of third generation dendritic PS and the shell is formed by multiple linear poly(p-tert-butoxystyrene) (PtBOS). The dendrimer-like copolymer, hereafter coded G4-PtBOS, is prepared through an iterative process combining anionic
nimolecular micelles were proposed by Newkome as arborescent molecules possessing both hydrophobic branched interior and hydrophilic periphery in a single molecule, thus, resembling micelles formed by conventional surfactants.1 Unimolecular micelles are usually based on covalently branched polymer structures and therefore exhibit significant stability and integration under the variation of external conditions. The unique structure and property allow unimolecular micelles to be excellent candidates as nanoreactors for fabricating uniform organic−inorganic nanocomposites2−4 and as nanocarriers for delivering anticancer drug such as etoposide and doxorubicin.5−9 The simplest case for unimolecular micelles is that derived from star-like core− shell block copolymers with the number of arm segments much larger than three which are prepared by either “arm-first” or “core-first” approach. In order to gain access to a larger number of arms, the core initiators employed were usually multifunctional compounds such as cyclodextrin bearing 21 hydroxyl groups,10−12 hyperbranched polyester (Boltorn H40) bearing ∼64 hydroxyl groups,13−22 and so on. Furthermore, polymers possessing complex branched architectures such as amphiphilic hyperbranched,23−36 brush-shaped,37 and dendrigraft copolymers (“graft-on-graft”)38 were readily used as unimolecular micelles due to multiplicity of the hydrophilic termini or outer segments. A perfect candidate for unimolecular micelle is dendrimer39−45 containing well-defined dendritic core and peripheral functional groups (usually hydrophilic). However, the synthesis is tedious and the size of the resulting micelle is limited (typically ≤15 nm). Dendrimer-like polymers possess dendritic architectures in which successive generations are linked by polymer chain © XXXX American Chemical Society
Received: March 10, 2016 Accepted: April 7, 2016
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DOI: 10.1021/acsmacrolett.6b00198 ACS Macro Lett. 2016, 5, 547−551
Letter
ACS Macro Letters
THF (0.1 mg/mL). As shown in Figure 1a, isolated spherical particles are observed with a mean diameter of 86.4 nm,
polymerization and olefin epoxidation/ring opening coupling reactions. The synthesis is relatively easy in comparison to that of regular dendrimers, which requires repeated protecting/ deprotecting processes. Extremely large molecular weight up to 4.71 × 106 Da is accessible after four steps. Unimolecular micelles were obtained by acidic hydrolysis of the precursor, G4-PtBOS, as demonstrated by the results of dynamic laser light scattering (DLS), transmission electron microscopy (TEM), ζ-potential measurements and fluorescent probe encapsulation study. The synthesis of the dendrimer-like unimolecular micelle in the present work is outlined in Scheme 1. The process involves Scheme 1. Synthesis of Amphiphilic Dendrimer-Like Copolymer Containing PS Core and PHOS Shell
Figure 1. Cryo-TEM image (a) and DLS result (b) of G4-PtBOS205 in THF (0.1 mg/mL).
ranging from 37.7 to 141.0 nm (Figure S10a). DLS results show that the hydrodynamic radius (Rh) is 36.4 nm with narrow distribution (Figure 1b). Since DLS was performed in a highly dilute solution and THF is a good solvent for both segments, it is reasonable to conclude that the measured diameter is that of unimolecule in solution. Furthermore, Rh remains approximately constant with a further increase in polymer concentration (0.01−0.5 mg/mL; Figure S10b). It is also noted that the measured diameter is much smaller than the calculated value (338 nm) assuming fully extended chain conformation, indicating that the arm segments are flexible. Hydrolysis of G4-PtBOS205 in acidic media resulted in amphiphilic dendrimer-like copolymer (G4-PHOS205) containing hydrophobic PS core and hydrophilic poly(p-hydroxystyrene (PHOS) corona. The hydrolysis reaction was confirmed by FTIR (Figure S11). The characteristic absorption at 1390 and 1365 cm−1 corresponding to the tert-butyl group58,59 disappears completely after the reaction, indicating nearly quantitative hydrolysis. The hydrolyzed product was not dried, because otherwise it could not be further redissolved. The hydrolyzed product was purified by dialysis against THF, thus a solution in THF (0.04 mg/mL) was obtained. Cryo-TEM results (Figure 2a) show scattered spherical particles with a mean diameter of
essentially two steps, that is, the preparation of a layered dendrimer-like copolymer of styrene and p-tert-butoxystyrene, followed by the hydrolysis of PtBOS. The dendrimer-like block copolymer was prepared in a similar way as previously reported.57 The interior polymer segment was derived from a living diblock copolymer, polyisoprene-b-polystyrenyllithium (PI-b-PSLi), prepared via sequential anionic polymerization of isoprene and styrene in cyclohexane to ensure that predominantly 1,4-structure in the PI segment was obtained. The coupling reaction of PI-b-PSLi with 1,3,5-tris(bromomethyl)benzene resulted in a 3-arm star-like polymer (G1). The 1,4double bonds in PI segments of G1 were efficiently epoxidized by HCOOH/H2O2. Ring-opening reaction of the epoxy groups by the same PI-b-PSLi gave the second generation dendrimerlike polymer (G2). Repeating the epoxidation and ring opening reactions led to the third dendrimer-like polystyrene (G3). The target copolymer, G4-PtBOS, was synthesized through ring opening reaction between the epoxidized G3 (G3-epoxy) and living poly(p-tert-butoxystyryl)lithium (PtBOSLi). The synthesis was monitored by various measurements. MALDI-TOF MS of PI and NMR of the diblock species, PI-b-PS, show that the number-average degree of polymerization of PI and PS segments are DPPI = 10 and DPPS = 146, respectively. GPC of the dendritic products shows shift of the narrow elution peak to shorter elution time when the generation increases. Multiangle laser light scattering results show that the molecular weights of G4-PtBOS are in the range of 2.37 × 106 to 4.71 × 106 Da (Figures S1−S9, Tables S1−S3). Unimolecular morphology of G4-PtBOS205 (the subscript 205 refers to the average degree of polymerization of PtBOS segment) was investigated by cryo-TEM from a solution in
Figure 2. (a) Cryo-TEM image of G4-PHOS205 in THF (0.04 mg/ mL). (b) TEM image of unimolecular micelles in aqueous NaOH solution at pH = 12 (0.01 mg/mL).
78.5 nm, ranging from 49.3 to 117.6 nm (Figure S12a). DLS result displays unimodal narrow distribution with Rh = 36.3 nm (Figure S12b), which is close to the cryo-TEM result. The similar sizes of G4-PHOS205 and G4-PtBOS205 obtained from DLS in THF solutions imply that the hydrolysis reaction was well-controlled without apparent cross-linking. 548
DOI: 10.1021/acsmacrolett.6b00198 ACS Macro Lett. 2016, 5, 547−551
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ACS Macro Letters Hydrophilic poly(p-hydroxystyrene) (PHOS) chain segments are soluble in alkaline solution because the phenolic groups are converted into sodium phenolate groups in aqueous NaOH solutions at pH > 9 (the conversion is complete at pH > 12).58 In order to prepare unimolecular micelle, the solution of G4-PHOS205 in THF was dialyzed against aqueous NaOH solution at pH = 12 (0.01 mg/mL). After complete replacement of THF by NaOH solution and subsequent dilution, the morphology of hydrolyzed dendrimer-like product was investigated by TEM. As shown in Figure 2b, smaller isolated spherical particles with mean diameter of 24.7 nm (Figure S13a) are observed, which are expected to be unimolecular micelles. Slight aggregation of these spherical particles are also observed, which may be attributed to small aggregates formed by the intermolecular hydrogen-bonding. DLS measurement shows a relatively larger size (Rh = 39.8 nm; Figure S13b) because DLS was performed in solution while TEM was observed in dry state. The unimodal distribution also indicated that there exists no large aggregate, which is consistent with the TEM result. The Rh of dendrimer-like copolymer became slightly larger than the same sample in THF (39.8 vs. 36.3 nm). This is a consequence of balance between chain stretching caused by the ionization of phenolic moieties in the shell and shrinkage of the hydrophobic PS core. The stability of this micellar system was assessed by ζ-potential measurement. In general, the nanoparticle system is stable if the absolute value of ζ-potential is higher than 30 mV.60,61 The ζpotential value of G4-PHOS205 in aqueous NaOH solution (pH = 12, 0.01 mg/mL) is −77.7 mV, indicating a stable dispersion. The aggregation of the unimolecular micelles was further investigated in aqueous NaOH solutions (pH = 12) using pyrene as a fluorescent probe molecule. It is well documented that the intensity ratio of the first to the third highest peaks (I1/ I3) of pyrene emission spectrum is highly sensitive to the polarity of the external environment, thus useful to measure critical aggregation concentration (CAC). The value of I1/I3 changes from 1.9 in water, 1.04 in toluene, to 0.5 in nonpolar solvents such as hexane.62,63 In the present study, I1/I3 decreases from 1.92 at low copolymer concentration (1.0 × 10−4 mg/mL or 2.7 × 10−11 M) to 1.06 at copolymer concentration of 0.5 mg/mL, and remains nearly constant with further increase in polymer concentration. A CAC value of 2.3 × 10−3 mg/mL is obtained from Figure 3a. This indicates that aggregation occurs as polymer concentration increases although dendrimer-like polymers exist as unimolecular micelles at low concentration. Nevertheless, the aggregation number may not be large because the mean diameter measured by DLS changes only slightly from 39.9 to 28.8 nm with unimodal distribution as the concentration increases significantly from 0.001 to 1.0 mg/mL (Figure 3b). The pH responsiveness of the unimolecular micelles was investigated by DLS and TEM. The hydrolyzed sample precipitated when a small amount of the THF solution was dropped into neutral water, whereas it formed stable solution when being dialyzed against alkaline water. The diameters determined by DLS increase from 50.6 to 79.6 nm, while maintaining relatively narrow distribution as the pH value rises from 9.5 to 12. This is due to higher ionization degree of PHOS chains at higher pH. TEM images show that unimolecular micelles maintain spherical particles with diameter of 24 nm, which is much smaller than that in solution (Figure S14a−f, Table S4). The ζ-potential values of the samples range from −41.9 to −77.7 mV, as shown in Figure 4, indicating an
Figure 3. I1/I3 ratio of pyrene emission vs logarithm of copolymer concentration log c (a) and DLS results (b) for unimolecular micelles in aqueous NaOH solutions at pH = 12.
Figure 4. ζ-potential vs pH for unimolecular micelles in aqueous NaOH solutions (0.01 mg/mL).
increase in stability as the pH value increases. Nevertheless, I1/ I3 values of pyrene encapsulation remain nearly constant in the range of 1.44−1.55 (Figure S15), indicating that unimolecular micelle provides similar hydrophobic environment in their core to accommodate pyrene at different pH values. This is in sharp contrast to conventional micelles formed from diblock copolymer with pH-dependent entrapping ability.58 In conclusion, amphiphilic dendrimer-like copolymers composed of dendritic polystyrene core and poly(p-hydroxystyrene) shell behave as unimolecular micelle in aqueous alkaline solution. The stability of these unimolecular micelles is ascribed to the high density of ionized peripheral chains. The unimolecular micelles appear as spherical particles with uniform size, as indicated by the TEM and DLS results. The fluorescent probe test indicates that aggregation of unimolecular micelles 549
DOI: 10.1021/acsmacrolett.6b00198 ACS Macro Lett. 2016, 5, 547−551
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ACS Macro Letters
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occurred as the copolymer concentration was increased above 2.3 × 10−3 mg/mL. However, DLS and TEM results show that the aggregates are few and not large. Therefore, we have developed a facile strategy to prepare unimolecular micelles from layered dendrimer-like copolymers with tunable size and pH responsiveness.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00198.
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Experimental details and characterization data (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We thank the financial support by the National Natural Science Foundation of China (Grant No. 21474016). J.H. thanks Prof. Daoyong Chen for helpful discussions on TEM results.
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