Jellyfish-Shaped Amphiphilic Dendrimers: Synthesis and Formation of

Article pubs.acs.org/Macromolecules

Jellyfish-Shaped Amphiphilic Dendrimers: Synthesis and Formation of Extremely Uniform Aggregates Shiqun Shao,† Jingxing Si,†,‡ Jianbin Tang,† Meihua Sui,*,† and Youqing Shen*,† †

Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Center for Bionanoengineering & the State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China 310027 ‡ Department of Respiratory Medicine, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China 310009 S Supporting Information *

ABSTRACT: Novel classes of jellyfish-shaped amphiphilic dendrimers composed of 7 hydrophilic poly(ethylene glycol) (PEG) arms and 14 hydrophobic polyester dendrons with β-cyclodextrin (βCD) as the core molecule were synthesized by a facile method. Seven PEG chains were first conjugated to the C-6 positions of native βCD. Subsequently, dendritic polyester architectures were constructed from the remaining 14 secondary hydroxyl groups at C-2 and C-3 positions of the βCD moiety, resulting in jellyfish-shaped amphiphilic dendrimers of different generations (7PEG-βCD-Gx) with well-defined molecular structures. The amphiphilic dendrimers self-assembled into different morphologies dependent upon the hydrophilic fraction of the dendrimers, and very surprisingly, the fourth-generation dendrimers consisting of only several percent of PEG could form aggregates with extremely narrow size distributions.

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INTRODUCTION Amphiphilic polymers can self-assemble into a wide range of nanostructures, including spherical1 or worm-like micelles,2 nanorods,1b nanotubes,3 vesicles,4 and multicompartment cylinders5 with great advantages of lower critical aggregation concentrations (CAC),6 higher thermodynamic stability,6,7 and higher capacities of loading guest molecules7 compared with those formed from low-molecular-weight amphiphiles. These nanostructures have attracted great interests because of potential applications in drug delivery,1a,b,8 bioimaging,9 catalysts,10 and fuel cells.11 Particularly, they can serve as cancer-drug delivery nanocarriers to significantly improve the solubility of anticancer drugs and promote their accumulation in tumor tissues. Amphiphilic dendrimers, while possessing the characteristic dendritic properties, primarily structural perfection, exhibit unique self-assembly properties when compared with linear amphiphilic polymers.1b,12 For instance, Percec and coworkers12a built 11 libraries containing a total of 107 amphiphilic Janus dendrimers and found that they selfassembled into a variety of morphologies in water including vesicles (dendrimersome), cubosomes, disks, tubular vesicles, and helical ribbons12a and exhibited qualitative differences from linear amphiphilic copolymers.12a Kim and co-workers12b studied the self-assembly of linear−dendritic block copolymers with fixed molecular weights and block ratios and found that the molecular area of the block copolymers increased with increasing the number of peripheral PEG chains, causing a topological transition from linear cylindrical micelles to toroid and lasso micelles. Additionally, the dense and rigid dendritic domains resulted in less chain entanglement and smaller © XXXX American Chemical Society

hydrodynamic radii than the linear analogues of identical molecular weights.13 In this study, we synthesized a series of jellyfish-shaped amphiphilic dendrimers containing polyester dendrons and seven hydrophilic poly(ethylene glycol) (PEG) arms. Most interestingly, these amphiphilic dendrimers, even with small fractions of hydrophilic segments, still could self-assemble into stable nanostructures with extremely uniform size distributions in water.

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RESULTS AND DISCUSSION Synthesis of Jellyfish-Shaped Amphiphilic Dendrimers. The synthesis procedures are reported in the Experimental Part of the Supporting Information. In brief, per-6-deoxy-6-(p-tolylsulfonyl)-β-cyclodextrin (βCD-6-(OTs)7) was prepared via tosylation of the primary hydroxyl groups of βCD with p-toluenesulfonyl chloride (TsCl) in pyridine (Scheme 1). The 1H NMR spectrum (Figure S1) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) spectrum (Figure S2) indicated that 6−9, but mostly 7, tosyl groups were introduced to each βCD. The tosylated βCD was then reacted with an excess of sodium mPEG-ethoxide to afford per6-deoxy-6-(methoxypoly(ethylene glycol))-β-cyclodextrin (7PEGβCD). The excess PEG chains were removed by dialysis against deionized water. The 1H NMR spectrum of 7PEG-βCD showed complete disappearance of the tosyl peaks in βCD-6-(OTs)7 and the Received: December 16, 2013 Revised: January 22, 2014

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Scheme 1. Synthetic Route to 7PEG-βCD-Gx Dendrimers

jellyfish-like amphiphilic dendrimers of different generations (7PEG-βCD-Gx, x = 1−4). Typically, CA (1.2/1 to the methacrylate groups) was added to the 7PEG1000-βCD-G0 in DMSO. The thiol−methacrylate Michael addition reaction was completed within an hour in DMSO at room temperature by monitoring the disappearance of the methacrylate signals in the 1 H NMR spectrum, leading to efficient production of aminefunctionalized intermediate (7PEG1000-βCD-G0.5). Without removing the slight excess of cysteamine, a slight excess of MAEA relative to the amines in the 7PEG1000-βCD-G0.5 as well as the amine and thiol groups in the unreacted CA was added to the solution and stirred at room temperature for 24 h.

appearance of PEG signals, suggesting a full substitution of tosyl groups with mPEG chains. A series of 7PEG-βCDs were obtained by varying the molecular weight of mPEG, namely mPEG550, mPEG750, and mPEG1000. 7PEG-βCD was then reacted with methacrylic anhydride in pyridine to introduce 14 methacrylate groups to the 14 secondary hydroxyl groups (7PEG-βCD-G0) (Scheme 1). With 7PEG-βCD-G0 as the core, a one-pot per generation method14 via sequential coulping of cysteamine (CA) and 2-[(methacryloyl)oxy]ethyl acrylate (MAEA), in which the CA thiol group reacted with the methacrylate group of MAEA while the CA amine group selectively reacted with the two acrylates in two MAEA molecules,15 efficiently produced the B

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Figure 1. 1H NMR spectra of 7PEG1000-βCD-Gx dendrimers (CDCl3).

The solution was further heated at 50 °C for 48 h, converting 7PEG1000-βCD-G0.5 to 7PEG1000-βCD-G1. Although CA reacted with three MAEA and produced adducts, their molecular weight was far less than the dendrimer 7PEG1000βCD-G1. Thus, the adducts could be easily removed by selective precipitation in ether, and pure dendrimers with high molecular weight were obtained with a high yield. Higher generations of 7PEG1000-βCD-Gx dendrimers were produced by repeating the above procedures (Scheme 1; Experimental Part, Supporting Information). The chemical structures of 7PEG1000-βCD-Gx dendrimers were characterized by 1H NMR spectra (Figure 1). The intensities of the terminal methacrylate groups (5.49 and 6.02 ppm), the ester bonds (4.25 ppm), the ethylene glycol units of PEG (3.63 ppm), and the methyl groups (1.12 ppm) were very consistent with the theoretical values (Table S1), which suggested a high degree of structural purity. Similarly, 7PEG-βCD-Gx dendrimers with mPEG molecular weights of 550 and 750 were efficiently prepared. It is noteworthy that during the dendrimer synthesis radical reaction inhibitors, such as 2, 6-di-tert-butyl-4-methylphenol (BHT), were needed to avoid cross-linking. The 7PEG-βCD-Gx dendrimers were further characterized by GPC. As presented in Figure 2, the GPC traces showed the increase of molecular weight with the increasing generation of dendrimers (see also Table 1). No trace of small molecules was detected, indicating that small molecules were completely removed by precipitation. Moreover, the polydispersities (PDI)

Table 1. Molecular Weight and Hydrophilic Fraction of the Dendrimers dendrimer

Mw,theora

Mw,GPCa

PDI

f hydrophilicb (%)

7PEG550-βCD-G1 7PEG550-βCD-G2 7PEG550-βCD-G3 7PEG550-βCD-G4 7PEG750-βCD-G1 7PEG750-βCD-G2 7PEG750-βCD-G3 7PEG750-βCD-G4 7PEG1000-βCD-G1 7PEG1000-βCD-G2 7PEG1000-βCD-G3 7PEG1000-βCD-G4

11 900 23 700 48 600 98 500 13 300 25 100 50 000 99 900 15 100 26 800 51 800 101 600

22 400 33 400 54 000 73 300 26 700 38 900 56 000 77 800 29 100 39 600 53 300 72 400

1.25 1.24 1.20 1.20 1.18 1.18 1.16 1.13 1.16 1.15 1.18 1.15

32.3 16.2 7.9 3.9 39.4 20.9 10.5 5.2 46.3 26.1 13.5 6.8

a

Molecular weights reported in g/mol. bf hydrophilic refers to hydrophilic fraction based on theoretic values, assuming the perfect structure.

of dendrimers were all around 1.2, owing to the variation of the tosylation degrees of βCD (Figure S2). Self-Assembly of Jellyfish-Shaped Dendrimers in Water. The jellyfish-shaped 7PEG-βCD-Gx dendrimers had hydrophobic umbrella-like dendrimer bells and hydrophilic tentacle-like PEG chains (Scheme 2A), with the latter only accounted for a few percent at higher generations. For instance, the percentages of the PEG contents (i.e., the hydrophilic fraction, f hydrophilic) in 7PEG550-βCD-G4, 7PEG750-βCD-G4, and 7PEG1000-βCD-G4 were as low as 3.9%, 5.2%, and 6.8%, respectively. It has been shown that the nanostructures of amphiphilic nonionic linear polymers in water is dependent upon the hydrophilic fraction ( f hydrophilic).16 Amphiphilic polymers with f hydrophilic > 50% are expected to form spherical micelles, and polymers with f hydrophilic values between 40% and 50% are expected to form worm- or rod-like micelles, whereas those with f hydrophilic values between 25% and 40% are expected to form vesicles, and those with f hydrophilic < 25% are expected to form inverted microstructures.16 For instance, PEG−PLA with f hydrophilic < 20% formed solid-like particles.17 Accordingly, 7PEG-βCD-G4 dendrimers theoretically could not selfassemble into micelles or vesicles in aqueous solution. However, very surprisingly, we found that the fourthgeneration dendrimers 7PEG-βCD-G4 readily formed stable aggregates in water with low PDI ranging from 0.002 to 0.063, while the first-generation dendrimers 7PEG-βCD-G1 having

Figure 2. GPC traces of 7PEG1000-βCD-Gx dendrimers. C

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Scheme 2. Schematic Representation of Jellyfish-Shaped Amphiphilic Dendrimers (A) and Proposed Aggregate Structures SelfAssembled from These Dendrimers (B, C)

Figure 3. TEM and DLS images of aggregates self-assembled from 7PEG550-βCD-G4 (A1−A3), 7PEG750-βCD-G4 (B1−B3), and 7PEG1000βCD-G4 (C1−C3) in water. Scale bars: A1−C1, 200 nm; A2−C2, 50 nm.

the highest f hydrophilic values were rather hard to form low polydisperse aggregates (Table 2, see also Experimental Part, Supporting Information). With the same length of PEG chains, the average sizes of aggregates determined by dynamic light scattering (DLS) increased with the increasing generation (Table 2). On the other hand, the sizes of aggregates assembled from same generation of dendrimers increased with decreasing the PEG chain length (Table 2). These results may be explained by that the low PEG content could not efficiently prevent the aggregation of the hydrophobic dendritic domains.18 Moreover, with increase of the dendrimer generation the PDI values (size distribution) of the resulting aggregates decreased (Table 2). Most impressively, the aggregates of all fourth-generation dendrimers were extremely uniform (Figure 3). For instance, the PDI values of

7PEG550-βCD-G4, 7PEG750-βCD-G4, and 7PEG1000-βCD-G4 determined by DLS were as low as 0.014, 0.002, and 0.063 (0 corresponds to a perfectly uniform size), respectively (Table 2). We attribute this well-defined structure to the high rigidity of these amphiphilic dendrimers.19 In fact, there are few reports on the narrowly distributed vesicles or micelles,12 as both synthetic and natural vesicles/micelles are typically polydisperse. After being stained with uranyl acetate (UAc), which is water-soluble and thus stains the hydrophilic region, these nanostructures were observed with transmission electron microscopy (TEM). The average sizes of these aggregates measured by TEM (Figures S8−S10) were comparable to those determined by DLS. Consistent with the DLS data, improved monodispersity of these dendrimeric aggregates was also D

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fluorescent dye (Supporting Information, Experimental Part). The calcein-loaded aggregates had bright fluorescent cores as indicated by confocal laser scanning microscopy (Figure 4), further confirming the vesicular structure self-assembled by 7PEG750-βCD-G4 since micelles with a hydrophobic solid core can not be loaded with such water-soluble molecules.21 Different from 7PEG550-βCD-G4 and 7PEG750-βCD-G4, 7PEG1000βCD-G4 aggregates had no staining core (Figure 3C), suggesting a micellar structure (as indicated in Scheme 2C). Interestingly, these micelles were not spherical but irregularly shaped. The above data indicate that the morphology of the aggregates is strongly dependent on the PEG length of the dendrimers. This is consistent with the contention that amphiphilic dendrimers are qualitatively different from conventional amphiphilic block copolymers12a and may be caused by the unique topological structure of the dendrimers. The hydrophobic part of higher generations of this series of amphiphilic dendrimers is rigid, with very little entanglements but just stacking of the dendrons when forming aggregates. Additionally, the actual hydrophobic volume of the dense and rigid hydrophobic structure is smaller than that of linear diblock copolymers with same molecular weight and hydrophilic fraction, while the 7 arms of flexible PEG chain occupy a larger volume. These structural characteristics promote the self-assembly of higher generations of dendrimers into monodisperse aggregates in water. However, of lower generations of dendrimers, the small hydrophobic bells and the rigid βCD cores make them difficult to entangle with each other in the solution, resulting in weak hydrophobic interaction and difficulty in forming low-polydisperse aggregates. Furthermore, the critical aggregate concentrations (CAC) of 7PEG550-βCD-G4, 7PEG750-βCD-G4, and 7PEG1000-βCD-G4 were determined as low as 4.89, 2.6, and 4.87 μg/mL, respectively (Figure 5), indicating that the aggregates formed from these amphiphilic dendrimers are rather stable in water. For example, the vesicles self-assembled from 7PEG750-βCD-G4 remained stable for even 4 months in water at room temperature (data not shown), with no significant changes in average particle size. Such stable aggregates may be useful for the medical applications such as drug delivery.22

Table 2. Sizes and Polydispersities of Dendrimeric Aggregates Determined by DLS

a

sample

size (nm)

PDI

7PEG550-βCD-G1 7PEG550-βCD-G2 7PEG550-βCD-G3 7PEG550-βCD-G4 7PEG750-βCD-G1 7PEG750-βCD-G2 7PEG750-βCD-G3 7PEG750-βCD-G4 7PEG1000-βCD-G1 7PEG1000-βCD-G2 7PEG1000-βCD-G3 7PEG1000-βCD-G4

95.72 111.9 160.3 203.8 80.70 91.57 116.2 130.4 N/Aa 49.63 68.73 107.4

0.221 0.111 0.088 0.014 0.219 0.106 0.029 0.002 N/Aa 0.323 0.129 0.063

N/A: samples were too polydisperse to be measured by DLS.

Figure 4. Confocal microscope images of calcein-loaded vesicles formed from 7PEG750-βCD-G4. Scale bar: 10 μm.

demonstrated by TEM (Figures S8−S10). Interestingly, micellar-to-vesicular morphological transition was observed between 7PEG550-βCD-G2 and 7PEG550-βCD-G3 aggregates as well as between 7PEG750-βCD-G3 and 7PEG750-βCD-G4 aggregates. Specifically, the aggregates of 7PEG550-βCD-G4 had a shadow surrounding the less gray inner core (Figure 3A), indicating a spherical vesicle with a “cave”.20 As shown in Figure 3B, 7PEG750-βCD-G4 dendrimers formed unilamellar vesicles (as indicated in Scheme 2B), as demonstrated by a clear contrast between the inner pool and the outer thin wall.4 Moreover, we fabricated aggregates of 7PEG750-βCD-G4 dendrimers loaded with calcein, a highly water-soluble

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CONCLUSIONS A series of amphiphilic jellyfish-shaped dendrimers with βCD as the core were synthesized, with 7 PEG arms and 14 polyester dendrons. The GPC and 1H NMR characterization indicated that these dendrimers had narrow molecular weight distribution and well-defined structures. These amphiphilic PEG-βCD dendrimers could self-assemble into different morphologies with extremely narrow size distribution in aqueous solution.

Figure 5. Determination of CAC of the dendrimeric aggregates by using a hydrophobic fluorescence probe pyrene (see Experimental Part, Supporting Information). The curves show the relationship of the intensity ratios (I338/I333) as a function of dendrimers concentration at room temperature. The CAC values of 7PEG550-βCD-G4 (A), 7PEG750-βCD-G4 (B), and 7PEG1000-βCD-G4 (C) were determined as 4.89, 2.6, and 4.87 μg/mL, respectively. E

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(15) Shen, Y.; Ma, Y.; Li, Z. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 708−715. (16) (a) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967−973. (b) Discher, D. E.; Ahmed, F. Annu. Rev. Biomed. Eng. 2006, 8, 323− 341. (17) Yasugi, K.; Nagasaki, Y.; Kato, M.; Kataoka, K. J. Controlled Release 1999, 62, 89−100. (18) (a) Kozlov, M. Y.; Melik-Nubarov, N. S.; Batrakova, E. V.; Kabanov, A. V. Macromolecules 2000, 33, 3305−3313. (b) Gou, P.-F.; Zhu, W.-P.; Shen, Z.-Q. Biomacromolecules 2010, 11, 934−943. (19) Guerrero-Martínez, A.; Pérez-Juste, J.; Carbó-Argibay, E.; Tardajos, G.; Liz-Marzán, L. M. Angew. Chem., Int. Ed. 2009, 48, 9484−9488. (20) Lin, S.; Zhu, W.; He, X.; Xing, Y.; Liang, L.; Chen, T.; Lin, J. J. Phys. Chem. B 2013, 117, 2586−2593. (21) Shen, Y.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M.; Zhao, J.; Wang, J.; Tang, J.; Fan, M.; Van Kirk, E.; Murdoch, W. J. J. Am. Chem. Soc. 2010, 132, 4259−4265. (22) Owen, S. C.; Chan, D. P. Y.; Shoichet, M. S. Nano Today 2012, 7, 53−65.

The applications of these amphiphilic jellyfish-shaped dendrimers in drug delivery are currently under investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Materials, instrumentation, synthesis methods and NMR characterization data for the PEG-βCD-dendrimers, preparation of the aggregates, CAC determination, and morphologies of the aggregates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax +86 571 87951493; e-mail [email protected] (M.S.). *Tel/Fax +86 571 87953993; e-mail [email protected] (Y.S.). Author Contributions

S.S. and J.S. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC21090352, 21104065, 21274125, and 21104068) and Public Programs of Zhejiang Province (2011C21055).

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REFERENCES

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