Reversible gold “locked” synthetic vesicles derived from stimuli-responsive diblock copolymers

Abstract

Polymersomes derived from a RAFT-generated, thermally responsive diblock copolymer, P(DMAEMA₁₆₅-b-NIPAM₄₃₅), were shell cross-linked by in situ gold nanoparticle formation. The cross-linking was subsequently reversed by the addition of the thiols capable of inducing a ligand exchange on the surface of the gold nanoparticle.

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The potential of nanostructured assemblies derived from block copolymers for drug delivery applications has been well established and has continued to be an area of intense research in recent years. However, certain limitations of self-assembled nanostructures preclude the realization of their use in practical applications. One major limitation is the dilution-induced dissociation as the concentration falls below the critical aggregation concentration after administration in vivo leading to burst release of the drug payload.¹ In an effort to address the stability of the self-assembled copolymer aggregates while maintaining high encapsulation efficiency, researchers have turned to various shell cross-linking methods. Since the seminal work by Wooley and coworkers² and the Armes research group,³ numerous shell cross-linking strategies have been developed, including our recent reports with in situ formation of gold nanoparticles (AuNPs) templated by amine-containing shells of micelles and polymeric vesicles, commonly called polymersomes.^4,5 These AuNP-decorated systems are of interest in combining therapeutic delivery and potential diagnostic imaging in a “theranostic” vehicle for potential applications in nanomedicine.

One disadvantage of using such shell cross-linked aggregates for drug delivery is that their large size prevents renal excretion.⁶ One method to circumvent both the effect of dilution and potential buildup of the aggregates in the kidneys is to use reversible cross-linking chemistries which allow the gradual breakdown of the cross-links after successful delivery. Recently, a major emphasis in our research has been the construction of stimuli-reversible cross-linked systems with cleavable disulfide bonds^7,8 or salt-^9–12 and pH-reversible⁹ interpolyelectrolyte complexes. Herein we describe the use of ligand exchange reactions in which the thiols cysteamine and poly(ethylene glycol) (PEG-SH) are utilized to reverse the AuNP cross-linking of micelles and vesicles self-assembled in aqueous solution from poly[(N,N-dimethylaminoethyl methacrylate)₁₆₅-b-(N-isopropylacrylamide)₄₃₅] (P(DMAEMA₁₆₅-b-NIPAM₄₃₅)).

Previously, we reported the synthesis of multiply responsive P(DMAEMA₁₆₅-b-NIPAM_x) copolymers capable of self-assembly into spherical micelles, worm-like micelles, and vesicles above the lower critical solution temperature (LCST) of PNIPAM.⁵ Utilizing that procedure and specifically targeting vesicle-forming diblocks, DMAEMA was first polymerized by RAFT in dioxane using CTP and V-501 as the chain transfer agent and initiator, respectively. After purification by dialysis and lyophilization, the PDMAEMA macroCTA (M_n = 26 200 g mol⁻¹, PDI = 1.04) was then used to mediate the polymerization of NIPAM to yield P(DMAEMA₁₆₅-b-NIPAM₄₃₅) (M_n = 75 400 g mol⁻¹, PDI = 1.17).

AuNP-“locking” or cross-linking of the resulting polymersome was achieved by adding a preheated aqueous solution of NaAuCl₄ to the P(DMAEMA₁₆₅-b-NIPAM₄₃₅) solution at 50 °C providing a DMAEMA repeat unit to Au ratio of 10 to 1 (Fig. 1).⁴ This reaction was allowed to proceed for 48 h prior to being cooled to room temperature. Fig. 2A shows the distribution of assembly sizes for thiolated AuNPs, vesicles and “locked” vesicles measured at a fixed angle of 173° using a Malvern Instruments Zetasizer Nano light scattering instrument. P(DMAEMA₁₆₅-b-NIPAM₄₃₅) (0.01 wt% in water at pH 7.0) when heated to 50 °C (1 °C min⁻¹) self-assembles into aggregates having a hydrodynamic diameter (D_h) of 178 nm (Fig. 2A, distribution a). This temperature-responsive self-assembly is induced by the sharp coil-to-globule transition above the LCST of the NIPAM block.^13–15 The in situ reduction of AuCl₄⁻ at 50 °C results in Au nanoparticles bound to the DMAEMA block, presumably through counterion exchange and subsequent chelation.^4,16 The cross-linked nanostructures remain intact upon lowering the temperature to 25 °C. A slight increase in the hydrodynamic size (Fig. 2A, b) is observed from the increased hydrophilicity of the PNIPAM segment.

Fig. 1 Reversible AuNP-“locking” of P(DMAEMA₁₆₅-b-NIPAM₄₃₅) accomplished by a ligand exchange of PDMAEMA for thiolated stabilizing agents.

Fig. 2 (A) DLS measurements showing the reversibility of the AuNP-“locking” of vesicles formed from P(DMAEMA₁₆₅-b-NIPAM₄₃₅). (a) 0.01 wt% P(DMAEMA₁₆₅-b-NIPAM₄₃₅) (pH 7.0, T = 50 °C), (b) AuNP cross-linked vesicles (T = 25 °C), AuNPs after ligand exchange with (c) cysteamine and (d) PEG-SH. (B) Angular dependent DLS and (C) SLS measurements for the AuNP cross-linked vesicles.

To provide greater insight into the morphology of the cross-linked structure, dynamic light scattering (DLS) and static light scattering (SLS) studies were performed at multiple angles using a Brookhaven BI-200SM goniometer with a TurboCorr correlator. A plot of the diffusion coefficient of the cross-linked aggregates versus the square of the scattering vector q reveals a slight angular dependence (Fig. 2B). The slight angular dependence suggests that the scattering comes from Brownian diffusion of particles with a heterodisperse distribution of sizes. Extrapolating to 0°, a D_h value of 201 nm is calculated using the Stokes–Einstein equation. Coincidentally, the D_h was also measured at 90° and found to be 177 nm, consistent with the Malvern instrument. Typically, the contributions of larger particles are suppressed at higher angles, and this is especially true for block copolymer vesicle formation. Static light scattering was also performed on the AuNP-“locked” solutions in order to determine the radius of gyration (R_g) from the angular dependence of the scattering intensity (Fig. 2C). A plot of the inverse of the measured scattering intensity (I_ex) versus the square of the scattering vector q provides a linear relationship leading to calculation of an R_g value of 98.2 nm. This value along with the radius of hydration extrapolated to 0° leads to an R_g/R_h ratio of 0.98 which is indicative of a vesicular structure.^17–19 TEM micrographs of the vesicles cross-linked by AuNP formation are shown in Fig. 3A.

Fig. 3 (A) AuNP cross-linked polymersomes formed from P(DMAEMA₁₆₅-b-NIPAM₄₃₅) and AuNP formed 48 h after addition of (B) cysteamine and (C) PEG-SH to the AuNP cross-linked polymersomes. (D) UV-vis absorption spectra of AuNP cross-linked vesicles (a) and nanoparticles formed after ligand exchange with cysteamine (b) and PEG-SH (c).

Previous studies have shown that thiolated ligands are capable of displacing amine-containing, polymeric stabilizing agents on the surface of AuNPs.^20,21 Since thiolated ligands should have a stronger affinity for the gold surface than the amine functionalities along the DMAEMA block,²² the addition of either a small molecule thiol, cysteamine, or a polymeric thiol, PEG-SH, should result in ligand exchange on the surface of the AuNPs leading to the disassembly of the cross-linked vesicles and subsequent binding of the AuNPs by the thiolated ligands.²³ After allowing the ligand exchange reaction with cysteamine and PEG-SH to proceed for 48 h, the free polymer was removed by centrifugation and DLS and TEM measurements were conducted to determine the size and morphology of the thiolated AuNPs. The hydrodynamic diameters measured from DLS after the ligand exchange reaction with cysteamine and PEG-SH are shown in Fig. 2A as curves c and d, respectively. For the reaction with cysteamine, the hydrodynamic diameter of the stabilized AuNPs is 16.0 nm, while the AuNPs stabilized by PEG-SH are slightly larger (20.4 nm), presumably due to the increased thickness of the PEG layer as compared to the bound cysteamine. Of note is the occurrence of a peak corresponding to residual AuNP cross-linked vesicles in both curves c and d, indicating that the ligand exchange was not quantitative within 48 h. Further experiments have shown that extending the reaction time to longer times, however, does indeed lead to complete disappearance of the residual cross-linked peak. TEM micrographs of both systems (Fig. 3B and C) show near identical sizes of the resulting AuNPs of ∼9 nm after the ligand exchange reactions. UV-vis spectroscopy was also used to follow the ligand exchange process by monitoring the absorbance before and after reaction. The AuNP-“locked” vesicles display the absorbance typically observed for AuNPs in aqueous solution with a λ_max of 522 nm (Fig. 3). Similarly, the UV-vis absorption spectra of the cysteamine and PEG-SH stabilized AuNPs show the characteristic absorbance attributed to the surface plasmon resonance of the AuNPs.

In summary, we have demonstrated a facile method for reversing the AuNP cross-linking of aggregates self-assembled from RAFT-generated polymers. Polymersomes self-assembled from the thermally responsive P(DMAEMA₁₆₅-b-NIPAM₄₃₅) were prepared and subsequently “locked”. Employing ligand exchange reactions, the DMAEMA units bound to the surface of the in situ formed AuNPs were displaced by the smaller, stronger binding thiols, reversing the cross-links formed in the shell of the vesicle. Although ongoing studies will be necessary to elucidate mechanistic details, this reversible cross-linking method may prove useful for the preparation and eventual degradation of AuNP-“locked” theranostic vehicles targeting cancerous tissue where thiol concentrations can be as high as 7 times those in surrounding tissue.^24,25

The Department of Energy (DE-FC26-01BC15317), MRSEC program of the National Science Foundation (NSF) (DMR-0213883), and the Robert M. Hearin Foundation are gratefully acknowledged for financial support. The authors also acknowledge the NSF Division of Materials Research/Major Research Instrumentation awards 0079450 and 0421406 for the purchase of the Varian Unity Inova 500 MHz NMR spectrometer and JEOL JEM-2100 electron microscope.

Notes and references

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Footnotes

† Paper number 146 in a series entitled “Water Soluble Polymers”.

‡ Electronic supplementary information (ESI) available: Materials, experimental, and instrumentation details. See DOI: 10.1039/c0py00071j

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