Supramolecular Assembly of Block Copolypeptides with

Dec 11, 2008 - Nanocharacterization Laboratory, McCullough Building, 476 Lomita Mall, Stanford, California 94305. ReceiVed June 12, 2008. ReVised ...
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Langmuir 2009, 25, 707-715

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Supramolecular Assembly of Block Copolypeptides with Semiconductor Nanocrystals Bayu Atmaja,† Jennifer N. Cha,‡,| Ann Marshall,§ and Curtis W. Frank*,† Department of Chemical Engineering, Stanford UniVersity, 381 North-South Mall, Stanford, California 94305, IBM Almaden Research Center, 650 Harry Road, San Jose, California 95120, and Stanford Nanocharacterization Laboratory, McCullough Building, 476 Lomita Mall, Stanford, California 94305 ReceiVed June 12, 2008. ReVised Manuscript ReceiVed August 8, 2008 We report the analogy between the self-assembly properties of amphiphilic phospholipids and the similar behavior observed for quantum dot (CdSe/CdS)-diblock copolypeptide hybrid systems, and the effect of the self-assembly on secondary structures of the polypeptides. At neutral pH, the diblock copolypeptide, poly(diethyleneglycol-Llysine)-poly(L-lysine), comprises a positively charged poly-L-lysine (PLL) block and a hydrophilic and uncharged poly(diethyleneglycol-L-lysine) (PEGLL) block. By itself, the copolypeptide is not amphiphilic. However, when the polymers are mixed with water-soluble, negatively charged, citrate-functionalized quantum dots (QDs) in water, shell-like structures or dense aggregates are spontaneously formed. Electrostatic and hydrogen-bonding interactions between the positively charged PLL residues and the negatively charged ligands on the QDs lead to charge neutralization of the PLL block, while the PEGLL block remains hydrophilic. As a result, a pseudo “amphiphilic” molecular unit is formed in which the “hydrophobic” and hydrophilic sections constitute the charge-neutralized PLL residues together with the associating QD and the remaining polypeptide residues that are not neutralized, respectively. The generation of these “amphiphilic” molecular units in turn drives the formation of the QD-polypeptide assemblies. Support for this analogy comes from the observed transition in the shape of the assembly from a shell-like structure to a dense aggregate that is very much analogous to the vesicle-to-micelle transition observed in lipid systems. Furthermore, this shape transition can be explained qualitatively using a concept that is analogous to the surfactant number (N ) ahc/ahg), which has been applied extensively in amphiphilic lipid systems. Specifically, as the ratio of the “hydrophobic” area (ahc) to the hydrophilic area (ahg) decreases, a shape transition from the shell-like structure to the dense aggregate occurs. In addition, the size of the shell-like structure changes as a function of the dimensions of the “amphiphilic” molecular unit in a manner that is similar to how the size of the lipid vesicle changes with the dimensions of the lipid molecule. Circular dichroism (CD) measurements have shown that the PEGLL-PLL molecule has a well-defined secondary structure (R-helical PEGLL block and random coil PLL block) that remains virtually unchanged after reacting with the QDs. This finding is consistent with the hypothesis that it is the electrostatic interaction between the amines on the PLL block and the citrate ligands on the QDs that drives the self-assembly.

Introduction There has been tremendous interest recently in the use of semiconductor nanocrystals quantum dots (QDs) as fluorescent markers in biological imaging/assays1-6 and for optical-device components.7-9 Liposomes made from phospholipids and block copolymers have also garnered much interest in their use for * To whom correspondence should be addressed. E-mail: curt.frank@ stanford.edu. Telephone: (650)723-4573. † Stanford University. ‡ IBM Almaden Research Center. § Stanford Nanocharacterization Laboratory. | Present address: Department of Nanoengineering and Materials Science, 9500 Gilman Drive, M/C 0448, University of California at San Diego, La Jolla, CA 92093-0448. (1) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759–1762. (2) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (3) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (4) Wu, X. Y.; Liu, H. J.; Liu, J. Q.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N. F.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41–46. (5) Chan, W. C. W.; Maxwell, D. J.; Gao, X. H.; Bailey, R. E.; Han, M. Y.; Nie, S. M. Curr. Opin. Biotechnol. 2002, 13, 40–46. (6) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434–1436. (7) Dabbousi, B. O.; Bawendi, M. G.; Onitsuka, O.; Rubner, M. F. Appl. Phys. Lett. 1995, 66, 1316–1318. (8) Ho, J. C.; Levchenko, I.; Ostrikov, K. J. Appl. Phys. 2007, 101, 094309. (9) Song, H.; Lee, S. Nanotechnology 2007, 18, 255202.

drug delivery, catalysis, and chemical storage.10-13 Our selfassembled QD-diblock copolypeptide (QD-polypeptide) assemblies, including the shell-like structures and dense aggregates (Figure 1) can potentially be used as simultaneous targeted drugdelivery vehicles and in vivo imaging agents that are highly tunable,stable,andbiocompatible.Inthiswork,theQD-polypeptide assembly consists of citrate-functionalized CdSe/CdS core-shell QDs that are synthesized in water14 and diblock copolypeptides, poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL)15 (Figure 2). Compared to conventional liposomes, the QD-polypeptide assemblies may have the capability not only to deliver drugs but also to allow us to visually track where the drugs have been delivered and their relative concentrations through excitation with a single wavelength. In addition, because of the sizedependent optical properties of the QDs,16-18 multicolor QD-polypeptide assemblies can be used to monitor the delivery (10) Scrimin, P.; Tecilla, P. Curr. Opin. Chem. Biol. 1999, 3, 730–735. (11) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967–973. (12) Discher, D. E.; Ahmed, F. Annu. ReV. Biomed. Eng. 2006, 8, 323–341. (13) Antonietti, M.; Forster, S. AdV. Mater. 2003, 15, 1323–1333. (14) Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater. 2000, 12, 2676–2685. (15) Yu, M.; Nowak, A. P.; Deming, T. J.; Pochan, D. J. J. Am. Chem. Soc. 1999, 121, 12210–12211. (16) Li, L. S.; Hu, J. T.; Yang, W. D.; Alivisatos, A. P. Nano Lett. 2001, 1, 349–351. (17) Alivisatos, A. P. Science 1996, 271, 933–937. (18) Weller, H. Angew. Chem., Int. Ed. Engl. 1993, 32, 41–53.

10.1021/la801848d CCC: $40.75  2009 American Chemical Society Published on Web 12/11/2008

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Figure 1. (a) Schematic of the idealized molecular arrangements of the QDs (spheres) and the poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) molecules in a shell-like structure (top) and dense aggregate (bottom). In either structure, a pseudoamphiphilic molecular unit is created through charge neutralization of the PLL residues (random coil) by the QD and the presence of the hydrophilic PEGLL block (see Discussion, section I). Each PEGLL residue carries a diethylene glycol side chain, and the block adopts an R-helical conformation. A transition in shape from shell-like structure to dense aggregate is observed as the ratio of the sizes of the “hydrophobic” section to the “headgroup” is decreased. A multilayer of QDs within the “membrane” of the shell-like structure instead of the single layer as depicted here is also plausible. (b) Schematic of the molecular interactions between a poly-L-lysine (PLL) residue and the carboxylate moiety of the citrate ligand that functionalizes the QD (sphere). The region within the dashed line depicts an enlargement of these molecular interactions. Electrostatic interaction between the positively charged PLL residue and one of the negatively charged carboxylate moieties (pKa ) 6.4) is responsible for the chargeneutralization effect. H-bonding may also occur between the PLL residue and carboxylate moiety.

Figure 2. (a) Chemical structure of poly(diethylene glycol-Llysine)-poly(L-lysine) diblock copolypeptide (PEGLL-PLL). Nε moieties of the poly-L-lysine backbone are functionalized with diethylene glycol (EG2) side chains to synthesize the PEGLL block. (b) Idealized depiction of the secondary structure of the PEGLL-PLL molecule. At the pH of self-assembly (pH ∼ 8.2), the PLL block is positively charged and adopts a random coil conformation; the PEGLL block is hydrophilic and uncharged, and it adopts an R-helical conformation represented by a rigid rod.15 Here, the multiple diethylene glycol side chains along the length of each R-helix are idealized to constitute a “sheath”.

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of drugs to different receptors/organs simultaneously.3-6,19 Owing to the high luminescence and photostability of the QDs,1-3,5,20 these multicolor assemblies can be used to track long-term changes in cellular interactions that may occur due to the efficacy of the drugs. To enable targeted drug delivery, the polypeptide residues or QDs can be functionalized with moieties2-4,20,21 that recognize and bind target organs or receptors, and the pH-sensitive noncovalent interactions between the PLL moieties and the QD ligands (see below) can be used as a mode of release. Another desirable trait of the QD-polypeptide assemblies is their biocompatibility conferred by the multiple diethylene glycol “coating” (analogous to PEG-coating1,11,12,22-25) that is expected to render the structures resistant to nonspecific protein adsorption and aggregation and reduce the cytotoxicity of the QDs (Figure 1). Our current work has demonstrated that, by changing the relative dimensions of the “amphiphilic” molecular unit, the shape and size of the QD-polypeptide structure can be altered in highly predictable manners. Therefore, this QD-polypeptide system has the potential of being a tunable system for generating effective and biocompatible simultaneous drug-delivery and imaging agents. This work investigates the self-assembly properties of a quantum dot (QD)-diblock copolypeptide hybrid system that forms either shell-like structures or dense aggregates and the effect of the self-assembly on the secondary structure of the polypeptide. Much of the previous work on polyelectrolytedriven nanoparticle assembly has focused primarily on characterizing the structures formed.26-30 Our work extends the previous studies by establishing correlations between the amphiphilic lipid and QD-polypeptide systems; specifically, this report describes the analogy of the self-assembly properties between the QD-polypeptide and lipid systems. To our knowledge, such an analogy has not been reported. Evidence of this analogy comes from the observation that there is a transition in the shape of the QD-polypeptide assembly from shell-like structure to dense aggregate that is very much analogous to the vesicle-to-micelle transition that has been observed extensively in lipid systems. Furthermore, we will show that this shape transition can be explained by a change in the ratio of the “hydrophobic” area to the “headgroup” area (a parameter that is known as the surfactant number (N) in lipid systems) of the QD-polypeptide “amphiphilic” molecular unit (Figure 1). In addition, the size of the QD-polypeptide shell-like structure changes as a function of the dimensions of the individual “amphiphilic” molecular unit in a manner that is similar for lipid vesicles. Our findings here not only reinforce the understanding of the molecular interactions that are responsible for the self-assembly, but they also provide (19) Pathak, S.; Choi, S. K.; Arnheim, N.; Thompson, M. E. J. Am. Chem. Soc. 2001, 123, 4103–4104. (20) Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47–51. (21) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016–2018. (22) Photos, P. J.; Bacakova, L.; Discher, B.; Bates, F. S.; Discher, D. E. J. Controlled Release 2003, 90, 323–334. (23) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Muller, R. H. Colloids Surf., B 2000, 18, 301–313. (24) Ballou, B.; Lagerholm, B. C.; Ernst, L. A.; Bruchez, M. P.; Waggoner, A. S. Bioconjugate Chem. 2004, 15, 79–86. (25) Akerman, M. E.; Chan, W. C. W.; Laakkonen, P.; Bhatia, S. N.; Ruoslahti, E Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12617–12621. (26) Murthy, V. S.; Cha, J. N.; Stucky, G. D.; Wong, M. S. J. Am. Chem. Soc. 2004, 126, 5292–5299. (27) Rana, R. K.; Murthy, V. S.; Yu, J.; Wong, M. S. AdV. Mater. 2005, 17, 1145. (28) Euliss, L. E.; Grancharov, S. G.; O’Brien, S.; Deming, T. J.; Stucky, G. D.; Murray, C. B.; Held, G. A. Nano Lett. 2003, 3, 1489–1493. (29) Wong, M. S.; Cha, J. N.; Choi, K. S.; Deming, T. J.; Stucky, G. D. Nano Lett. 2002, 2, 583–587. (30) Cha, J. N.; Bartl, M. H.; Wong, M. S.; Popitsch, A.; Deming, T. J.; Stucky, G. D. Nano Lett. 2003, 3, 907–911.

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Table 1. Sizes and Shapes of Structures Formed from the Self-Assembly of the PEGLL-PLL Diblock Copolypeptide Molecules and Citrate-Functionalized CdSe/CdS QDs diblock copolypeptide PEGLL11-PLL129 PEGLL11-PLL90 PEGLL113-PLL30

charge ratio, Ra 1.0 1.0 1.1

fb 11.7 8.2 0.3

size (nm) variance c

830 533d 31e

0.144 0.027 0.017

Table 2. Effect of Dimensions of QD-Polypeptide “Amphiphilic” Molecular Unit on Size of the Shell-like Structuresa

shape of structure

diblock copolypeptide

shell-like shell-like Dense aggregate

a See Materials and Methods. b f ) ratio of number of PLL residues that are neutralized by the citrate charges to remaining number of PEGLL and PLL residues that are not neutralized. c Both intensity- and number-weighted particle-size distributions are bimodal; both show maxima at ∼170 nm (weak) and 900 nm (strong). The latter peak corresponds to the shell-like QD-polypeptide structures, while the former may correspond to relatively small QD agglomerates formed through their inherent aggregation (see text). d Both intensity- and number-weighted particle-size distributions show single peaks. e The number-weighted particle-size distribution shows a single peak. The intensity-weighted distribution is bimodal with maxima at ∼32 nm (strong) and 170 nm (weak); the former peak corresponds to the QD-polypeptide dense aggregates, while the latter peak may correspond to small QD agglomerates present in the solution.

ways to tailor the shape and size of the assembly according to the intended application. In addition to investigating the selfassembly properties of the hybrid system, the use of a diblock copolypeptide in which one of the blocks has a well-defined secondary structure, R-helix (Figure 2), allows us to determine the effect of self-assembly on the secondary structure. This will in turn help confirm the types of molecular interactions that are responsible for the formation of the shell-like structures and dense aggregates.

Materials and Methods Polypeptide and QD Syntheses. Please refer to the Supporting Information (SI I-III) for details on the syntheses of the polypeptides and CdSe/CdS core-shell QDs as well as the characterization of the former by NMR and GPC. The size of the CdSe/CdS core-shell QD is determined to be between 3 and 4 nm by transmission electron microscopy. Formation of QD-Polypeptide Shell-like Structures or Dense Aggregates. Polypeptides, regardless of molecular weight, are dissolved in Milli-Q water to give concentrations ranging between 1 and 2 mg/mL. The QD-polypeptide assembly is spontaneously formed upon mixing the polypeptide solution (50-500 µL) with the appropriate amount of the CdSe/CdS QD solution (concentration of citrate ∼ 4-6 mM) by repeated manual pipetting. The amount of CdSe/CdS QD solution mixed with the polypeptide solution is determined based on the desired charge ratio, R, which is defined as the molar ratio of citrate in the QD solution (total amount of citrate used to synthesize the QDs) to the amine residues on the PLL blocks. In other words, R is proportional to the percentage of the amine residues on the PLL block that are neutralized by the citrate molecules present in the QD-polypeptide mixture. SI IV in the Supporting Information provides details on the conditions that we have explored in the formation of various QD-polypeptide assemblies. For the investigation on the effect of the dimensions of the “amphiphilic” molecular unit, that is, R value, on the size of the shell-like structure (Discussion, Table 2) assembled from a particular diblock copolypeptide architecture, the concentration of the polypeptide solution is kept constant for all R values. Specifically, for each R value, the polypeptide solution is diluted with the appropriate amount of water (Milli-Q) before adding the corresponding amount of the QD solution such that the final concentration of the polypeptide in the QD-polypeptide mixture is constant. This way, any effect of the concentration on structure size is eliminated31 (see Discussion, section II.B), and the change in the structure size as R is increased (31) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1991.

R

f

Lb

structure size (nm)

PEGLL11-PLL90c

0.7 0.8 1.0

1.7 2.5 8.2

63 72 90

188 334 533

0.018 0.037 0.027

PEGLL11-PLL129d

0.6 0.8 1.0

1.2 2.8 11.7

77 103 129

283 595e 830f

0.052 0.087 0.144

variance

a See Materials and Methods for experimental details. b L is the number of PLL residues neutralized by the QDs. c Both number- and intensityweighted particle-size distributions show single peaks and agree closely with each other for all values of R. d For R g 0.8, both number- and intensityweighted distributions are bimodal. e The intensity-weighted particle-size distribution has maxima at ∼170 and 620 nm. f The intensity-weighted particle-size distribution has maxima at ∼170 and 930 nm. In each system, the ∼170 nm peak may correspond to small QD aggregates.

is solely attributed to changes in the dimensions of the molecular unit. Circular Dichroism (CD). CD measurements were obtained using CD spectrometer Aviv model 202-01. Far-UV CD spectra were taken in a 0.1 cm quartz cell at 20 °C in Milli-Q water. For all samples, pH values of the solutions were left unadjusted. Concentrations of the polypeptides in the samples varied between ∼2 and 8 µM. For the sample containing the QD-polypeptide assemblies, a small amount of the QD-polypeptide mixture was first prepared and then diluted with Milli-Q water to achieve the desired concentration of polypeptide in the mixture. CD measurements were taken at 1.0 nm intervals, and they are reported in terms of mean residue ellipticities (m-r ellipticities), [θ], in units of degrees cm2 dmol-1 at the specified wavelengths. The fraction of R-helicity, fH, is calculated using the following equation:32-34

fH )

[θ]obs - [θ]C [θ]H - [θ]C

where [θ] is the m-r ellipticity measured at 222 nm, [θ]obs is the m-r ellipticity of the polypeptide sample, [θ]C is the reference m-r ellipticity for a 100% random coil, and [θ]H is the reference m-r ellipticity for a 100% R-helix. Here, we take [θ]C to be the measured m-r ellipticity of polylysine homopolymer (PLL153) at neutral pH in water, which is known to adopt a pure random coil conformation ([θ]C ) 5757), and [θ]H to be the average of m-r ellipticities for five 100% R-helical proteins ([θ]H ) -30 000).32 The measured m-r ellipticity of PLL153 at neutral pH, [θ]222 ) 5757, is roughly consistent with the published value for PLL115 at pH 5.7, [θ]222 ) 3900,33 which is known to adopt a pure random coil conformation. The average of m-r ellipticities for five 100% R-helical proteins, [θ]222 ) -30 000, is also roughly consistent with that of PLL115 at pH 11.1 ([θ]222 ∼ -33 000)33 and the measured m-r ellipticity of PEGLL48 homopolymer in water ([θ]222 ∼ -28 000); both polymers have been established as 100% R-helical. Slight discrepancies in the m-r ellipticity values for each conformation may be attributed to differences in the chain length and solvent condition. Accordingly, fH calculated in this way is only an approximation, as the value is certainly dependent upon the chosen reference values ([θ]H and [θ]C). Dynamic Light Scattering (DLS). All QD-polypeptide assemblies were prepared immediately before DLS measurements, (32) Chen, Y. H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350–3359. (33) Greenfield, N; Fasman, G. D. Biochemistry 1969, 8, 4108–4116. (34) Park, S. H.; Shalongo, W.; Stellwagen, E. Protein Sci. 1997, 6, 1694– 1700.

710 Langmuir, Vol. 25, No. 2, 2009 which were made using a Brookhaven Instrument BI-200SM with a laser source emitting at 633 nm. Each sample was placed in a 10 mL cylindrical vial and diluted with Milli-Q water to achieve a count rate that ranged between 100 and 400 kcps. All measurements were done at a fixed angle of 90° and room temperature. Data were collected over an analysis time of 6 min, with a first delay time of 5 µs and last delay time of 100 ms, and using a dust filter with dust cutoff of 30.0. We ensured that the baseline difference was kept within 2%. Data analyses of particle-size distributions were carried out using the BIC Dynamic Research software. Measured autocorrelation functions were analyzed by means of the method of cumulants and the CONTIN algorithm.35,36 The CONTIN algorithm was used to determine whether the particle-size distribution was multimodal; if so, the mean particle size was reported based on this method. In order to be consistent, the mean particle size for the sample for which the distribution showed a single peak was also reported based on the CONTIN algorithm. In this case, we observed that the mean diameter obtained using the method of cumulants agreed closely with that obtained using the CONTIN algorithm. For the sample containing the shell-like structures, the particle-size distribution was intensity-weighted, while for that containing the dense aggregates the distribution was number-weighted. Since the shell-like structures are relatively large and large particles contribute more significantly to the scattered intensity, the intensity-weighted distribution will minimize the effect of any small QD agglomerates (see Results, section I) present in the solution on the average structure size. On the other hand, since the QD-polypeptide dense aggregates are likely to be smaller than the random QD agglomerates, the numberweighted distribution will be affected less by the presence of the latter. Polydispersity of the assemblies in solution is reported in terms of the variance of the particle-size distribution obtained using the CONTIN algorithm. The exact refractive indices of both the shell-like structures and dense aggregates are unknown; a value of 1.59 (refractive index of polystyrene particles) is nonetheless used. The hydrodynamic diameter of the structure is ultimately calculated as an average of three to four samples, and the data for each sample were collected twice. Fluorescence Optical Microscopy. Imaging of the shell-like structures was done using a Nikon Eclipse E 800 microscope. A small amount (∼30 µL) of the QD-polypeptide mixture containing the structures was sandwiched between a glass slide and a coverslip, and the imaging was performed immediately. Laser Scanning Confocal Microscopy. Laser scanning confocal microscopy was performed using a Leica SP2 microscope with a laser excitation source emitting at 405 nm, and observations were made in the range of 550-620 nm. A droplet (∼30 µL) of the QD-polypeptide mixture was sandwiched between a glass slide and a coverslip and imaged immediately. Deconvolution Microscopy. A QD-polypeptide mixture was diluted 1:10 in Milli-Q purified water and placed in a #1.5 LaboratoryTek cambered coverglass (Nunc, Inc.). Samples were imaged using wide-field, deconvolution microscopy (DeltaVision RT, Applied Precision) utilizing an inverted Olympus IX-70 microscope and a 60×, NA 1.2, water immersion lens. Excitation was achieved using a 100 W Hg bulb and a 490/20 (FITC) band pass filter, and sample fluorescence was passed through a 617/73 band pass filter and collected by a CoolSnap HQ, cooled CCD camera (Princeton Instruments). Axial sampling was at 200 nm/step. Image restoration was performed using SoftWoRx software (Applied Precision), and the images were uniformly adjusted for contrast and brightness over the whole image. Transmission Electron Microscopy (TEM). TEM images were acquired using an FEI CM20 microscope equipped with a fieldemission gun (FEG) at 200 kV electron beam acceleration voltage. Five µL of the QD-polypeptide mixture was loaded onto a TEM grid (Ted Pella, Carbon type-B, 300 Mesh Copper Grid) using a pipet. The TEM grid was then allowed to air-dry for 10 h prior to imaging. (35) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213–227. (36) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 229–242.

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Results I. Aqueous Formation of Diblock Copolypeptide-QD Supramolecular Assemblies. The mixing of citrate-functionalized CdSe/CdS QDs with poly(diethylene glycol-Llysine)-poly(L-lysine) (PEGL-PLL) at the appropriate charge ratio, R (final pH ∼ 8.2), leads to the spontaneous formation of either shell-like structures or dense aggregates in water (Table 1). It is important to note that these QD-polypeptide dense aggregates have morphology and shape that are distinct from those of random QD agglomerates that are formed through their inherent aggregation (see Results, section I.B). For each system, the size of the assembly in solution is determined using dynamic light scattering (DLS), while the shape is determined using either a combination of deconvolution and laser scanning confocal microscopies, or TEM. As the control for the DLS experiments of each of the PEGLLx-PLLy systems (Table 1), we used a stock QD solution that is diluted with water (no polypeptide added) such that the concentration of the QD in the control is the same as that in the sample. We find that the control yields a highly erratic and distorted autocorrelation function that prevents a meaningful analysis by the software for size determination. This observation may be attributed to the fact that the size of an individual QD (∼3 nm by TEM) is at the lower limit of our DLS instrument. I.A. EVidence of QD-Polypeptide Shell-like Structure Formation. The formation of the QD-polypeptide shell-like structures using PEGLL11-PLL90 and PEGLL11-PLL129 are characterized by means of confocal and deconvolution microscopies (Figures 3 and 4). In order to resolve the interior morphology of the assembly, we have acquired cross-sectional images of the individual structures using the two microscopy techniques (Figure 4 and Supporting Information SI V). Considering the resolution of these optical microscopy techniques, we have deliberately chosen to image structures that are g1.5 µm in size. From Figure 4, we can see the distinct contrast in luminescence intensity between the periphery and core of the structure, which confirms its shell-like nature. The bright “ring”/ ”shell” around the periphery corresponds to the cluster of excited QDs that are interacting closely with the PLL blocks; that is, the bright ring corresponds to the “membrane” portion of the shelllike structure (Figure 1 and Supporting Information SI IV). It is also evident that the core of the shell-like structure is absent of fluorescence, that is, it is filled with water, and the inner surface of the “membrane” appears to be rough and undulating. Interestingly, as the structure size reaches g2.5 µm, we start to see small “QD islands” that are found in close vicinity of and associated with the inner surface of the “membrane” (see Supporting Information SI V). The average sizes of the structures formed using PEGLL11-PLL90 and PEGLL11-PLL129 as revealed by fluorescence optical and confocal microscopies are roughly consistent with those measured by DLS (Table 1), which are 533 and 830 nm, respectively. From the variance of the particle-size distribution (DLS) and confocal microscopy image (Figure 3b), the structures formed using the PEGLL11-PLL129 system are relatively polydisperse. This polydispersity is likely to be attributed to the effect of the large hydrophobic section (significant hydrophobic repulsion) on the intrinsic thermodynamics of the self-assembly.31 I.B. EVidence of QD-Polypeptide Dense-Aggregate Formation. Transmission electron microscopy (TEM) is used to show the shape of the assembly formed from the PEGLL113-PLL30 system (Figure 5). TEM reveals the widespread presence of what appears to be QD-polypeptide dense aggregates that have sizes ranging between 12 and 15 nm; the polypeptide molecules are not detected

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Figure 3. Laser scanning confocal microscopy images of QD-polypeptide shell-like structures. (a) PEGLL11-PLL90; charge ratio ∼ 1.0; scale bar ) 4 µm. (b) PEGLL11-PLL129; charge ratio ∼ 1.0; scale bar ) 4 µm. Excitation of the QDs by the light source resulted in the fluorescence detected on the shell-like structures.

Figure 4. Images of single shell-like structures formed using PEGLL11-PLL129 with charge ratio ∼ 1.0. (a) Laser scanning confocal microscopy image of a single structure ∼2 µm in size. (b) Deconvolution microscopy image of three single structures (shown in each frame); the structure in the middle has a size ∼ 2.2 µm. In (a) and (b), cross sections of the structure(s) were taken from roughly the top to bottom of the structure(s) (left to right); they clearly show the shell-like nature and the hollow interior of the structure.

that have completely random shapes and sizes, and their morphology is different from that of the QD-polypeptide dense aggregates (Figure 5b,c). The sizes of the individual dense aggregates as seen by TEM (∼12-15 nm) are roughly consistent with the average size obtained from DLS (∼30 nm) considering the following factors. The DLS measures the hydrodynamic size of the QD-polypeptide dense aggregate, while only the QD particles are detected by TEM. In addition, the TEM sample has undergone processing, which includes drying and transfer onto a surface (TEM grid) prior to imaging. The high vacuum conditions of electron microscopy may also have altered the size of the dense aggregates. From the TEM data, although some of these aggregates tend to form relatively small clusters, most of them are individually dispersed. The minimal aggregation on the surface may be attributed to the substantial steric repulsion imposed by the relatively long PEGLL segments of the PEGLL113-PLL30 molecules. This phenomenon is analogous to the steric repulsion that exists between liposomes and micelles that bear PEG molecules on their surfaces.37-39 The large number of diethylene glycol side chains along the long R-helical PEGLL block (Figure 2) impose a significant steric repulsion against other PEGLL blocks, which in turn stabilizes the dense aggregates formed from PEGLL113-PLL30. II. Effect of Self-Assembly of QDs and Diblock Copolypeptides on Their Secondary Structures. Before we can compare the secondary structures of the PEGLLx-PLLy diblock copolypeptide molecules before and after their self-assembly with the QDs, their secondary structures in solution (without the QDs) first need to be determined (see Supporting Information SI VI). It is known that the PEGLL and PLL homopolymers adopt 100% R-helical and random coil conformations,15,33 respectively, in water at neutral pH and pH ∼ 8.2 (pH of selfassembly) (Supporting Information Figure S2). Intuitively, the PEGLLx-PLLy diblock copolypeptide molecules adopt an average secondary structure which has both random coil and R-helical characters (Supporting Information Figure S2), with the fraction of R-helicity, fH (see Materials and Methods) increasing as the ratio of x:y increases (Supporting Information

in these images, as they were not negatively stained (Figure 5a). For this hybrid system, a dense aggregate is envisioned to constitute a few QDs that are densely clustered together with the associating polypeptide molecules (Figure 1a). The QD control shows the widespread presence of random QD agglomerates

(37) Johnsson, M.; Hansson, P.; Edwards, K. J. Phys. Chem. B 2001, 105, 8420–8430. (38) Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171– 199. (39) Woodle, M. C. Chem. Phys. Lipids 1993, 64, 249–262.

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Figure 5. TEM images of QD-polypeptide dense aggregates (a) and QD control (b,c). (a) Dense aggregates formed using PEGLL113-PLL30 with charge ratio ∼ 1.1. Amount of QDs loaded onto TEM grid ∼ 0.7 µL. Scale bars: 60 nm (left) and 35 nm (right). At these magnifications, each QD-polypeptide aggregate appears as a dark and dense “spot”. The high vacuum condition of TEM might have led to the partial collapse of the aggregates, which may explain why some aggregates are irregular in shape. (Right) Magnified image of the same sample as that shown (left) for easy comparison with the QD control sample. Because the polypeptide molecules were not stained, they do not appear in these TEM images. (b) Control sample: CdSe/CdS QD particles (no polypeptide); amount of QD solution loaded onto the grid is the same as that in (a). Scale bar: 170 nm. The control shows widespread presence of large QD agglomerates that are not seen in the QD-polypeptide dense-aggregate sample together with smaller QD agglomerates that are less widespread. (c) Zooming into the small QD agglomerates found in the same control sample as in (b). Scale bar: 80 nm.

Table S1). In addition, the fraction of R-helicity of each diblock copolypeptide corresponds roughly to the fraction of the PEGLL residues in each molecule, which indicates that the incorporation of the random-coil PLL block into the copolypeptide does not alter the R-helical conformation of the PEGLL block. In other words, the diblock copolypeptide molecule can be envisioned to comprise two segments each with a distinct secondary structure, which are covalently linked together: an R-helical section that constitutes the PEGLL residues and a random coil section that constitutes the PLL residues. We have further shown that the secondary structure of the diblock copolypeptide is virtually unchanged upon interaction with the QDs to form the supramolecular assemblies (Figure 6). Figure 6a shows that, for the PEGLL113-PLL30 system, the CD spectra of the polypeptide molecules remain relatively unchanged before and after their self-assembly with the QDs. However, the CD signal of the PEGLL42-PLL47 molecules after their interaction with the QDs can be observed to be reduced

slightly relative to that of the copolypeptide molecules alone in the limited region between 190 and 220 nm, and such reduction is exacerbated in the PEGLL11-PLL90 system (Figure 6b,c). Considering that the average size of the assembly formed using the PEGLL113-PLL30 system is ∼30 nm, while those formed using the PEGLL42-PLL47 and PEGLL11-PLL90 systems are ∼99 nm and ∼200 nm (by DLS), respectively, we can also observe a trend in which the deviation in the CD spectra becomes more pronounced as the structure size increases. Based on these findings, it is unlikely that the reduction in the CD signal is attributed to a change in the secondary structure of the polypeptide; rather, this deviation is presumably caused by differential absorption flattening,40-44 which is commonly observed in the CD of (40) Wallace, B. A.; Mao, D. Anal. Biochem. 1984, 142, 317–328. (41) Gordon, D. J.; Holzwart., G Arch. Biochem. Biophys. 1971, 142, 481–& (42) Phillips, C. L.; Mickols, W. E.; Maestre, M. F.; Tinoco, I. Biochemistry 1986, 25, 7803–7811. (43) Wallace, B. A.; Teeters, C. L. Biochemistry 1987, 26, 65–70.

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membrane proteins (see Supporting Information SI VII for further discussion).

Discussion I. Formation of Supramolecular Assemblies: Conditions, Molecular Arrangements, and Interactions. The primary interactions responsible for the self-assembly are likely to be localized between the PLL residues and citrate ligands of the QDs, since the PEGLL residue is uncharged and the diethylene glycol side chain is relatively “inert”. This hypothesis is supported by our CD data, which show that the R-helical structure of the PEGLL block is maintained during the selfassembly (Figure 6); one would expect that its well-defined and ordered secondary structure would be destroyed if there were any significant interactions between the PEGLL residues and QDs. Electrostatic and hydrogen bonding (H-bonding) interactions between the protonated PLL residues (pKa ) 10.5) and the carboxylate moieties of the citrate ligands (pKa ) 6.4)45 seem to lead to the partial or complete charge neutralization of the PLL block, which in turn creates a pseudo “amphiphilic” molecular unit. It is believed that only one of the three carboxylate moieties of the citrate ligand is involved in these interactions, as the other two carboxylate moieties with the lower pKa’s (3.1 and 4.8) are bound by the Cd2+ during the initial CdSe QD synthesis.45 In this “amphiphilic” molecular unit, the “hydrophobic” section comprises the charge-neutralized PLL residues together with the associating QD, while the hydrophilic section (“headgroup”) comprises the R-helical PEGLL block and any remaining PLL residues of which the charges are not neutralized. The charge ratio, R, controls whether the charge neutralization of the PLL block is complete (R g 1) or partial (R < 1) (see Materials and Methods). Please refer to the Supporting Information section SI IV for discussion on the conformations of the PEGLL-PLL molecules as well as the arrangements of the polypeptide molecules and QDs in the QD-polypeptide assemblies. II. Analogy between QD-Polypeptide Hybrid and Lipid Systems. II.A. Applicability of the Concept of Surfactant Number, N, to Predict Shapes of Assemblies. From the data presented above (Figures 3, 4, and 5, and Table 1), we can see that the shape of the QD-polypeptide assembly undergoes a transition from a shell-like structure to dense aggregate that is very much analogous to the vesicle-to-micelle transition known to occur in lipid supramolecular assemblies.31 Furthermore, we will show that this shape transition occurs due to a change in a parameter that is analogous to the surfactant number, N (eq 1), which has been applied successfully to predict the shapes of lipid supramolecular structures:11,13,31,46

Figure 6. CD data of PEGLL-PLL diblock copolypeptides as they self-assemble with the QDs in water at 20 °C. (a) PEGLL113-PLL30 solution at 2.5 µM (×); PEGLL113-PLL30 mixed with CdSe/CdS QD solution at R ) 1.1 (+). (b) PEGLL42-PLL47 solution at 8 µM ([); PEGLL42-PLL47 mixed with CdSe/CdS QD solution at R ) 1.0 (×) (see Supporting Information SI VIII for details on this system). There is no apparent CD signal in the long-wavelength region (data not shown). (c) PEGLL11-PLL90 solution at 5.5 µM (2); PEGLL11-PLL90 mixed with CdSe/CdS QD solution at R ) 0.7 (9). (Top) Range ) 190-250 nm. (Bottom) Range ) 250-360 nm; there is no apparent differential absorption of circularly polarized light in the long-wavelength region, which indicates that the differential scattering effect is probably minimal. In each QD-polypeptide sample, we assume that all of the polypeptide molecules have interacted with the QDs such that there are no excess “unreacted” polypeptides in the QD-polypeptide mixture. The CD spectrum of the control sample (QD solution) shows a flat signal over the same range of wavelengths: 190-250 nm (data not shown).

N)

V ahglc

∼

ahc ahg

(1)

where V is the volume of the hydrocarbon chain, lc is the critical chain length, ahg is the optimal headgroup area, and ahc is the hydrocarbon or “hydrophobic” area. It has been shown that a lipid molecule with a given set of ahg, V, and lc and, hence, a particular N, will pack into a unique structure that is consistent with these geometric constraints. As N decreases from 1, the shape of the lipid assemblies will change from a vesicle to a micelle.31,46,47 (44) Woody, R. W. Biochem. Spectrosc. 1995, 246, 34–71. (45) Cha, J. N.; Birkedal, H.; Euliss, L. E.; Bartl, M. H.; Wong, M. S.; Deming, T. J.; Stucky, G. D. J. Am. Chem. Soc. 2003, 125, 8285–8289. (46) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1976, 72, 1525–1568.

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Evidently, the transition in the shape of the QD-polypeptide assembly occurs as the parameter f, which is defined as the ratio of the number of PLL residues neutralized by QDs to the number of PEGLL residues and remaining PLL residues that are not charge neutralized, changes (Table 1). Noting the structure of the QD-polypeptide amphiphilic molecular unit (see Discussion, section I), it is then intuitive that f is proportional to the ratio of the “hydrophobic” area (ahc) to the “headgroup” area (ahg), which then implies that f is completely analogous to the surfactant number (N) (eq 1). Therefore, we have shown that the shape transition (shell-like to dense aggregate) that occurs in the QD-polypeptide hybrid system can be explained by a concept that is completely analogous to the surfactant number. II.B. Change in Assembly Size as Dimensions of Molecular Unit are Altered. Here, we will show that the size of the QD-polypeptide shell-like structure changes as a function of the dimensions of its amphiphilic molecular unit in a manner that is known to occur for lipid vesicles, thus reinforcing the analogous self-assembly properties between the two systems. Based on simple geometric considerations, for 1/2 < N < 1, the radius, Rc, of the smallest vesicle formed while preventing the headgroup area of the lipid molecule from exceeding ahg is given by31,48

Rc ∼

lc (1 - N)

(2)

where N is the surfactant number and lc is the critical hydrocarbon chain length of the lipid molecule. It would be entropically unfavorable for the radius of the vesicle to increase beyond Rc and energetically unfavorable for the radius to decrease below Rc.31,46 Although we have established above that N is analogous to the parameter f, we have no direct way of quantifying the “hydrophobic chain length”, lc, for the QD-polypeptide system. Nonetheless, we shall assume that lc is proportional to the size of the “hydrophobic” section or the number of residues of the PLL block that are neutralized by the QD, L. According to eq 2, as both lc and N (N < 1) of the lipid molecule increase, the critical vesicle size increases. We have shown that the QD-polypeptide shell-like structure exhibits the same trend: as f and L increase, which are analogous to increases in N and lc, respectively, the structure size increases (Table 2). For the shelllike structures formed using PEGLL11-PLL90 and PEGLL11PLL129, the average size increases from ∼200 to 500 nm and ∼300 to 800 nm, respectively, as determined from DLS. For each polypeptide system, the increase in structure size has also been confirmed visually using fluorescence optical microscopy (data not shown). Additionally, we have found that the size of the shell-like structures assembled using a particular diblock copolypeptide architecture can be reduced by decreasing the concentration of the polypeptide molecules before mixing with the QD solution (see Supporting Information SI IX). Since it is known that the size of lipid vesicles decreases when the total concentration of the surfactants is decreased,31 this finding can therefore be taken to further highlight the analogy in the self-assembly properties between the QD-polypeptide and lipid systems.

Conclusions We have demonstrated the aqueous self-assembly of poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) (47) Stokes, R. J.; Evans, D. F. Fundamentals of Interfacial Engineering; Wiley-VCH: Canada, 1996. (48) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. ReV. Biophys. 1980, 13, 121–200.

diblock copolypeptides with citrate-stabilized CdSe/CdS quantum dots (QDs) to form either the shell-like structures or dense aggregates. Electrostatic and H-bonding interactions between the positively charged PLL residues and the negatively charged carboxylate moieties of the citrate ligands (one carboxylate moiety per citrate ligand) are believed to be responsible for the selfassembly. The resulting partial or complete charge-neutralization of the PLL block yields a “hydrophobic” section, while the PEGLL residues and the remaining PLL residues that are not neutralized by the QD ligands constitute the hydrophilic section (“headgroup”). Thus, a pseudo “amphiphilic” molecular unit is created, which in turn drives the formation of the QD-polypeptide assemblies. One aspect of this report focuses on the analogy of the self-assembly properties between our QD-polypeptide hybrid and amphiphilic lipid systems. Evidence of this analogy comes from the observed transition in the shape of the assembly (shelllike structure to dense aggregate) that is very much analogous to the vesicle-to-micelle transition seen in lipid systems. Furthermore, we have shown that this shape transition occurs due to a decrease in the ratio of the “hydrophobic” area to the “headgroup” area (f) of the QD-polypeptide amphiphilic molecular unit. The applicability of the parameter f, which is conceptually analogous to the surfactant number, N (extensively applied in lipid systems), to explain the shape transition substantiates the analogy between the two systems. In addition, we have found that the size of the QD-polypeptide shell-like structure increases with the length of the hydrophobic section (L) and f. Since the size of a lipid vesicle is known to increase with the hydrophobic chain length (lc) and N, this finding further highlights the analogous self-assembly properties between the lipid and QD-polypeptide hybrid systems. The presence of a well-defined secondary structure (R-helix) within the diblock copolypeptide allows us to investigate the effect of the self-assembly on the secondary structure. CD measurements have shown that the secondary structure of the PEGLL-PLL molecules remains unchanged upon their selfassembly with the QDs. The reduction of the CD signal in the wavelengthregionbetween190-210nmfromtheQD-polypeptide mixture relative to the polypeptide solution (without the QDs) is likely to be an artifact of the differential absorption flattening effect rather than a real change in the secondary structure. The fact that the secondary structure of the PEGLL-PLL molecule is unaltered as a result of the self-assembly with the QD supports the hypothesis that the main interactions (electrostatic and H-bonding) responsible for the self-assembly occur between the PLL residues and the citrate ligands. Otherwise, it would be likely that the ordered R-helical conformation of the PEGLL block would be destroyed. Acknowledgment. We thank Krystyna Brzezinska of the Materials Research Laboratory at U.C.S.B for performing the GPC experiments on our polypeptide samples; Kitty Lee of the Cell Sciences Imaging Facility (CSIF) at Stanford University for her valuable guidance in using laser scanning confocal microscopy; and Jon Mulholland of the CSIF for acquiring the deconvolution microscopy images. And we thank the Pehr. A. B. Harbury and Alex. T. Brunger groups at Stanford University for the access to their CD instruments. We are also very grateful to Victor Lee, Russell Pratt, Andrew Mason, and Bob Miller of the IBM Almaden Research Center for the valuable advice on and assistance in the syntheses of the polypeptides. And we thank Yacop Halim for his tremendous help in creating and rendering the schematics of the self-assembled structures. Finally, we thank Bryan Parrish for his help in the synthesis and analyses of the

Self-Assembly of Copolypeptides and Nanocrystals

diblock copolypeptides. This work is supported by the Center on Polymer Interfaces and Macromolecular Assemblies. Supporting Information Available: Syntheses and characterization of polypeptides; synthesis of citrate-stabilized CdSe/CdS core-shell QDs; QD-polypeptide supramolecular assembly conditions, molecular arrangements, and conformation; QD-polypeptide shell-like

Langmuir, Vol. 25, No. 2, 2009 715 structures via confocal and deconvolution microscopy; secondary structuresofpolypeptideswithoutQDs;characterizationofQD-polypeptide assembly formed from PEGLL42-PLL47; effect of concentration on the size of QD-polypeptide shell-like structures; and deviation of polypeptide CD spectra due to differential absorption flattening. This material is available free of charge via the Internet at http://pubs.acs.org. LA801848D