water soluble star-block copolypeptides: towards biodegradable nanocarriers for versatile and...

Communication

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Water Soluble Star-block Copolypeptides:Towards Biodegradable Nanocarriers forVersatile and Simultaneous Encapsulationa

Wensheng Zhuang, Lihui Liao, Heru Chen, Jinzhi Wang, Ying Pan,Lumian Zhang, Daojun Liu*

The synthesis of water soluble star-block copolypeptides and their encapsulation propertiesare described. The star-block copolypeptides, obtained by ring-opening polymerization ofamino acid N-carboxyanhydrides, consist of a PEI core, a hydrophobic polyphenylalanine orpolyleucine inner shell, and a negativelycharged polyglutamate outer shell. The encap-sulation study showed that these watersoluble, amphiphilic star-block copolypeptidescould simultaneously encapsulate versatilecompounds ranging fromhydrophobic to anio-nic and cationic hydrophilic guest molecules.

W. Zhuang, L. Liao, J. Wang, Y. Pan, L. Zhang, D. LiuMedical College, Shantou University, Xinling Road 22, Shantou515041, P. R. ChinaFax: +86-754-88557562; E-mail: [email protected]. ChenPharmacy College, Jinan University, Huangpu Dadao West 601,Guangzhou 510632, P. R. China

a: Supporting information for this article is available at the bottomof the article’s abstract page, which can be accessed from thejournal’s homepage at http://www.mrc-journal.de, or from theauthor.

Macromol. Rapid Commun. 2009, 30, 920–924

� 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

Dendritic polymers such as dendrimers and hyper-

branched polymers have attracted much attention in

the past decade because of their potential biological

applications, including drug delivery, gene delivery, and

imaging.[1] The shell functionalization of these branched

macromolecules has been employed to produce a wide

variety of core-shell architectures which can be used as

nanocarriers in drug delivery[2–7] and phase transfer.[8,9]

The common structures of such potential drug delivery

carriers consist of a hydrophobic interior and a hydrophilic

shell, and are mostly intended to entrap and deliver

DOI: 10.1002/marc.200800807

Water Soluble Star-block Copolypeptides: Towards Biodegradable Nanocarriers for . . .

Scheme 1. Schematic structures of star-block copolypeptides andtheir encapsulation of hydrophobic and ionic guest molecules. Rrepresents the hydrophobic side chains of L-leucine or L-phenyl-alanine. The depicted idealized structures show only 8 blockcopolypeptide arms.

non-polar guest molecules. On the other hand, the

encapsulation of polar guest molecules, such as drugs

that are sensitive to hydrolytic, enzymatic, oxidative

degradation, or toxic to normal cells, is highly demanded

for drug delivery vectors.[10] Furthermore, compound

medicines require that the drug carriers have the ability

to encapsulate two or more types of guest molecules

simultaneously. Therefore, drug delivery carriers with the

feasibility to encapsulate varied types of guest molecules

with different polarities and functionalities or even

encapsulate them simultaneously are of fundamental

interest. Nanocarriers based on amphiphilic core-shell

macromolecules that can be used to entrap both hydro-

phobic and anionic guest molecules have already been

reported.[2b] Nevertheless, the versatile and simultaneous

encapsulation property of drug carriers still remains

largely unexplored.

In this communication, we report on the design and

properties of new star-block architectures based on

hyperbranched polymeric cores surrounded by amphiphi-

lic double-layered copolypeptide blocks. This type of water

soluble and biodegradable macromolecule can simulta-

neously encapsulate versatile compounds ranging from

hydrophobic to anionic and cationic hydrophilic mole-

cules.

Results and Discussion

A cheap and low cytotoxicity hyperbranched poly(ethy-

lene imine) Mn ¼ 1 800 g �mol�1, PEI1 800) with about 15

peripheral primary amines[11] was used as a macroini-

tiator to initiate a consecutive ring-opening polymeriza-

tion of a-amino acid N-carboxyanhydrides (NCAs).[12]

Hydrophobic L-phenylalanine (Phe)-NCAs or L-leucine

(Leu)-NCAs were polymerized first, followed by the

polymerization of g-benzyl-L-glutamic acid-NCAs. Then

the g-benzyl protection groups were removed,[13] to finally

generate the designed architecture (see Scheme S1 in the

Supporting Information). It can be seen clearly that the

synthesized star-block copolypeptides consist of three

discrete domains (Scheme 1): the basic PEI1 800 core, which

could provide an environment for the incorporation of

anionic hydrophilic guest molecules via acid-base inter-

actions;[14] the inner block of hydrophobic polypeptides,

designed to create a hydrophobic microenvironment for

the encapsulation of non-polar guest molecules;[14] and

the terminal outer block of polyglutamates, which makes

the polymer water soluble and could also entrap hydro-

philic cationic guest molecules through electrostatic

interactions.

A series of star-block copolypeptides with varied

polypeptide blocks have been synthesized by tailoring

the feed ratio of initiating sites to the NCA monomer, and



they are represented as PEI1 800(Leu)m(Glu)n and PEI1 800

(Phe)m(Glu)n, where m and n refer to the numbers of

constituent units of the inner and outer blocks in one arm,

respectively. The solubility of star-block copolypeptides in

Tris buffer (pH 7.6) is dependent on the value of n and the

ratio of n/m (see Table S1 in the Supporting Information). It

turns out that polymers with large values of n and n/m

such as PEI1 800(Leu)8(Glu)16 (L2), PEI1 800(Leu)16(Glu)32 (L4),

PEI1 800(Phe)8(Glu)16 (P2), and PEI1 800(Phe)8(Glu)32 (P5),

have good solubility and hence are chosen for further

encapsulation investigation. Three kinds of model dye

compounds: pyrene and oil red O (OR) as hydrophobic

compounds, methyl orange (MO) and rose bengal (RB) as

anionic polar compounds, and crystal violet (CV) as a

cationic polar compound were employed to evaluate the

versatile encapsulation properties of these four star-block

copolypeptides.

The encapsulation study was conducted in Tris buffer

(pH 7.6) by an ultrasonication method for hydrophobic

compounds,[15] and a dialysis method for cationic and

anionic polar compounds.[5] Despite their different pola-

rities and structures, all the guest molecules could be

steadily encapsulated by all polymer solutions (Figure S3

and S4 in the Supporting Information) and the encapsu-

lated systems showed high temporal stability upon

prolonged dialysis under pH 7.6. The UV-vis spectra of

pyrene-solubilized polymer solutions exhibited a bath-

ochromic shift in the wavelength of maximum absorption

www.mrc-journal.de 921

W. Zhuang et al.

Table 1. Loading capacities, sizes of aggregates, and CACs for star-block copolypeptides. The errors of the measurements are typically in therange of �10%.

Polymers Loading capabity (nguest/nhost) CAC Radius of polymer

aggregatesa)

Pyrene OR RB MO CV 10�3 g � L�1 Radius Polydispersity

nm

L2 0.29 0.12 6.8 0.17 2.3 6.2 32 (34) 0.19

L4 1.0 0.54 7.7 0.72 3.5 2.7 64 (60) 0.15

P2 0.68 0.60 12 0.18 1.9 7.1 45 (42) 0.16

P5 0.88 0.98 11 0.20 2.4 8.7 38 (38) 0.16

aMeasured by DLS with star-block copolypeptide solutions of two concentrations 0.1 g � L�1 and 5.0 g � L�1 in Tris buffer (pH 7.6), the values

of the latter showing in the parentheses.

922

(lmax), from 336 nm in L2 and L4 solutions to 340 nm in P2

and P5 solutions. This can be explained as the result of p-p

interactions between the side phenyl rings of P2 or P5 and

the encapsulated pyrene guests, indicating that hydro-

phobic guest molecules are encapsulated and located

around the inner hydrophobic shell. The encapsulation of

cationic CV can be ascribed to electrostatic interactions

between dyes and the negatively charged side-chains of

polyglutamates under weak alkaline conditions (vide

infra). As reported earlier, the solubilization of anionic

dyes is attributed to the acid-base interactions between

dye molecules and the tertiary amines in the PEI core.[7b]

The load values of different dyes increase proportionally

with the polymer concentration ranging from 0.01 to

20 g � L�1. The loading capacities (nguest/nhost) of various

dye molecules, determined in all cases by UV-vis spectro-

scopy, are listed in Table 1. As expected, an increase in the

length of the inner hydrophobic block leads to a

corresponding growth in the loading capacity of hydro-

phobic dyes, whereas a change in the size of the outer shell

only has a limited effect. Although the loading capacity of

hydrophobic guest molecules is normally lower than one,

Figure 1. UV-vis spectra of simultaneously encapsulated dyes in L4 solutions in Trisbuffer (pH 7.6): (a) pyrene and crystal violet; (b) pyrene, methyl orange, and crystalviolet.

the solubility of pyrene could be

increased more than 100-fold relative

to pure water if the concentration of the

star-block copolypeptides was increased

to 1.0 wt.-% For cationic CV, L4 and P5

exhibited a higher loading capacity than

L2 and P2, respectively, which can be

reasonably explained by the increasing

length of polyglutamate outer blocks.

This in turn confirms that cationic guest

molecules are mainly entrapped by the

negatively charged outer domains. The

loading capacity of anionic polar guest

molecules showed a great difference



between RB and MO. The lower loading capacity of MO

can be explained as the result of the small PEI core

designed for the star-block copolypeptides. In contrast, a

much higher encapsulation capacity was detected for

higher hydrophobic RB, which can be explained by the

synergistic effect of acid-base interactions and hydro-

phobic interactions between hosts and guest molecules.[7b]

Since the synthesized star-block copolypeptides exhibit

encapsulation for versatile guest molecules, including

hydrophobic and cationic and anionic hydrophilic guest

molecules, based on the three discrete domains as well as

the specific interactions thereof, these star-block polymers

could possibly serve as hosts for simultaneous encapsula-

tion of versatile guest molecules. As evidenced by the

absorption spectrum in Figure 1(a), hydrophobic pyrene

and cationic CV can be encapsulated sequentially by

the star-block copolypeptides, and it was found that the

loading capacity of each dye was not fundamentally

affected by the other. Anionic MO and cationic CV can also

be encapsulated simultaneously by the polymers, their

respective loading capacities hardly altered. However, a

competition effect does occur for the encapsulation of a

DOI: 10.1002/marc.200800807

Water Soluble Star-block Copolypeptides: Towards Biodegradable Nanocarriers for . . .

hydrophobic dye and an anionic dye, probably due to the

steric effect caused by the small PEI core of the star-block

copolypeptides. The same effect can also be observed when

three different kinds of dyes (hydrophobic, cationic and

anionic) are entrapped simultaneously. Without optimiz-

ing the feed ratio of different dyes, a simultaneous

encapsulation of pyrene, MO and CV can nevertheless

be realized, as shown from the absorption spectrum in

Figure 1(b). The shift in lmax of respective dyes in the

polymer solution relative to that of free dyes indicates

encapsulation. To the best of our knowledge, this is the

first report that dendritic polymers can simultaneously

encapsulate versatile compounds ranging from hydro-

phobic to anionic and cationic hydrophilic molecules.

Some of the encapsulation capacities of star-block

copolypeptides are lower than a value of one, suggesting

the formation of polymer aggregates. The structural aspect

of the star-block copolypeptides in solution was investi-

gated by two independent techniques: fluorescence

measurement using pyrene as a probe[8a] and dynamic

light scattering (DLS). The excitation spectra of 6.0� 10�7M

pyrene in P5 solutions with various concentrations are

shown in Figure 2(a). A red-shift from 335 to 340 nm was

observed with increasing concentrations of P5, so it is

likely that the star-block copolypeptide molecules self-

assemble into supramolecular aggregates rather than

unimolecular micelles.[8a] The critical aggregation concen-

tration (CAC) can be obtained by plotting the intensity

ratio (I340/I335) of pyrene excitation spectra against the

logarithm of polymer concentration (Figure 2(b)). The CACs

for different star-block copolypeptides were measured

using the same method and are listed in Table 1. All the

synthesized star-block copolypeptides showed CACs lower

than 0.01 g � L�1, and these extremely low CAC values point

to a high stability of the supramolecular aggregates.

The aggregates of the star-block copolypeptides were

then characterized by DLS measurements in Tris buffer

(pH 7.6). For the polymer solution with a concentration of

0.1 g � L�1, the radius of aggregates exhibited a unimodal

size distribution within the range from 30 nm to 65 nm,

Figure 2. (a) Excitation spectra of pyrene as a function of P5concentrations (curves a, b, c, d, e, and f correspond to 0.001,0.01, 0.05, 0.1, 0.25, and 0.5 g � L�1, respectively) in Tris buffer(pH 7.6); b) Plot of I340/I335 against logarithm of P5 concentration.



depending on the structures of star-block copolypeptides

(Table 1). Almost the same size and size distribution were

detected for the polymer solution with a concentration as

high as 5 g � L�1 (Table 1), suggesting that the star-block

copolypeptides form well-defined aggregates. The inter-

molecular hydrophobic interactions, as well as the short

and sparsely-packed hydrophilic outer shell, might be

responsible for the aggregation of the synthesized star-

block copolypeptides.[2b,4] It has been reported that star-

block copolymers with long and densely packed hydro-

philic outer shells, which can completely shield the inner

hydrophobic blocks from the aqueous environment, would

exist as unimolecular micelles instead of aggregates.[4]

Conclusion

In conclusion, a novel class of water soluble, amphiphilic

star-block copolypeptides has been developed as a

nanocarrier for the simultaneous encapsulation of versa-

tile compounds ranging from hydrophobic to anionic and

cationic hydrophilic guest molecules. The incorporation of

biodegradable and biocompatible polypeptides into hyper-

branched polymers represents a new way of creating well-

defined core-shell drug delivery architectures.[16] These

polymers self-assemble into well-defined supramolecular

aggregates with extremely low CACs and are thus highly

stable upon dilution compared with conventional micelles

formed from surfactants or linear amphiphilic block

copolymers. The versatile and simultaneous encapsulation

property of star-block copolypeptides could be potentially

used for the transport of compound medicines and for

many other multifunctional applications. Further studies

to analyze the three-dimensional structures of these

supramolecular aggregates and their in vitro and in vivo

evaluation are currently underway.

Acknowledgements: This work was financially supported by theNational Natural Science Foundation of China (50643013).

Received: December 22, 2008; Revised: February 9, 2009;Accepted: February 13, 2009; DOI: 10.1002/marc.200800807

Keywords: block copolymers; core-shell polymers; drug deliverysystems; host-guest systems; ring-opening polymerization

[1] [1a] C. C. Lee, J. A. MacKay, J. M. J. Frechet, F. C. Szoka, Nat.Biotechnol. 2005, 23, 1517; [1b] S. Svenson, D. A. Tomalia, Adv.Drug Delivery Rev. 2005, 57, 2106; [1c] U. Boas, P. M. H.Heegaard, Chem. Soc. Rev. 2004, 33, 43; [1d] U. Gupta, H. B.Agashe, A. Asthana, N. K. Jain, Biomacromolecules 2006, 7,649.

www.mrc-journal.de 923

W. Zhuang et al.

924

[2] [2a] M. Kramer, J.-F. Stumbe, H. Turk, S. Krause, A. Komp, L.Delineau, S. Prokhorova, H. Kautz, R. Haag, Angew. Chem., Int.Ed. 2002, 41, 4252; [2b] M. R. Radowski, A. Shukla, H. vonBerlepsch, C. Bottcher, G. Pickaert, H. Rehage, R. Haag, Angew.Chem., Int. Ed. 2007, 46, 1265; [2c] H. Turk, A. Shukla, P. C. A.Rodrigues, H. Rehage, R. Haag, Chem. Eur. J. 2007, 13, 4187;[2d] M. Wyszogrodzka, R. Haag, Chem. Eur. J. 2008, 14,9202; [2e] R. Haag, Angew. Chem., Int. Ed. 2004, 43, 278;[2f] S. Xu, Y. Luo, R. Haag, Macromol. Rapid Commun. 2008,29, 171.

[3] S. J. Guillaudeu, M. E. Fox, Y. M. Haidar, E. E. Dy, F. C. Szoka,J. M. J. Frechet, Bioconjugate Chem. 2008, 19, 461.

[4] [4a] F. Wang, T. K. Bronich, A. V. Kabanov, R. D. Rauh, J.Roovers, Bioconjugate Chem. 2008, 19, 1423; [4b] F. Wang,T. K. Bronich, A. V. Kabanov, R. D. Rauh, J. Roovers, Bioconju-gate Chem. 2005, 16, 397.

[5] S. Chen, X.-Z. Zhang, S.-X. Cheng, R.-X. Zhuo, Z.-W. Gu, Bio-macromolecules 2008, 9, 2578.

[6] G. Kreutzer, C. Ternat, T. Q. Nguyen, C. J. G. Plummer, J.-A. E.Manson, V. Castelletto, I. W. Hamley, F. Sun, S. S. Sheiko, A.Herrmann, L. Ouali, H. Sommer, W. Fieber, M. I. Velazco, H.-A.Klok, Macromolecules 2006, 39, 4507.



[7] [7a] J. F. G. A. Jansen, E. M. M. de Brabander-van den Berg, E. W.Meijer, Science 1994, 265, 1226; [7b] M. W. P. L. Baars, R.Kleppinger, M. H. J. Koch, S.-L. Yeu, E. W. Meijer, Angew. Chem.Int. Ed. 2000, 39, 1285.

[8] [8a] H. Tian, X. Chen, H. Lin, C. Deng, P. Zhang, Y. Wei, X. Jing,Chem. Eur. J. 2006, 12, 4305; [8b] Y. Lin, X. Liu, Z. Dong, B. Li, X.Chen, Y.-S. Li, Biomacromolecules 2008, 9, 2629.

[9] A. I. Cooper, J. D. Londono, G. Wignall, J. B. McClain, E. T.Samulski, J. S. Lin, A. Dobrynin, M. Rubinstein, A. L. C. Burke,J. M. J. Frechet, J. M. DeSimone, Nature 1997, 389, 368.

[10] R. S. Dhanikula, P. Hildgen, Bioconjugate Chem. 2006, 17, 29.[11] H. Liu, Z. Shen, S.-E. Stiriba, Y. Chen, W. Zhang, L. Wei, J. Polym.

Sci., Part A: Polym. Chem. 2006, 44, 4165.[12] H. R. Kricheldorf, Angew. Chem., Int. Ed. 2006, 45, 5752.[13] K. Luo, J. Yin, Z. Song, L. Cui, B. Cao, X. Chen, Biomacromole-

cules 2008, 9, 2653.[14] M. W. P. L. Baars, E. W. Meijer, Top. Curr. Chem. 2000, 210, 131.[15] M. Liu, K. Kono, J. M. J. Frechet, J. Controlled Release 2000, 65,

121.[16] [16a] L. Crespo, G. Sanclimens, M. Pons, E. Giralt, M. Royo, F.

Albericio, Chem. Rev. 2005, 105, 1663; [16b] L. S. Nair, C. T.Laurencin, Prog. Polym. Sci. 2007, 32, 762.

DOI: 10.1002/marc.200800807

water soluble star-block copolypeptides: towards biodegradable nanocarriers for versatile and...

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