ChemComm COMMUNICATION
Cite this: Chem. Commun., 2013, 49, 9932 Received 29th July 2013, Accepted 28th August 2013
Coiled-coil peptide motifs as thermoresponsive valves for mesoporous silica nanoparticles† ` via Capell and Alexander Kros* Giuliana Martelli, Harshal R. Zope, Mireia Bro
DOI: 10.1039/c3cc45790g www.rsc.org/chemcomm
Coiled-coil peptide motifs were used as thermo-responsive valves for mesoporous silica nanoparticles (MSNs). The controlled release of a model drug as a function of temperature was demonstrated.
Stimuli-responsive nanoparticles are attractive transport vehicles due to their ability to encapsulate and protect cargo molecules against enzymatic degradation or denaturation induced by the surrounding physiological environment.1 Mesoporous silica nanoparticles (MSNs) are particularly attractive as they can encapsulate large amounts of molecules inside their 2D-hexagonally arranged pores and are considered biocompatible. Therefore MSNs are promising nanocarriers which potentially enable targeted2 and controlled drug delivery.3 MSNs have been used to deliver a variety of compounds such as imaging agents, hydrophobic drugs4 or bio-active compounds like DNA,5 peptides6 and also proteins inducing bone tissue regeneration.7 Additionally the external surface of MSNs can be chemically modified enabling the construction of smart nanovalves,8 which are responsive to a specific external stimulus. These nanovalves cap the pores by steric hindrance and thereby prevent release of the cargo from the MSNs. Removal of the steric hindrance (i.e. activation of the nanovalves) results in opening of the pores, and the concomitant controlled release of the encapsulated drug. In recent years smart mesoporous silicabased delivery systems have been developed in which the delivery of molecules from the pores can be controlled by a variety of external stimuli such as pH, temperature, magnetic fields, redox reactions, enzymes, and even antibodies.9 As building blocks for these valves, many different types of molecules have been used including pH-switchable rotaxanes,10 organic polymers11 and biomolecules like carbohydrates or peptides. Recently MSNs have been modified with TAT peptides to ensure efficient and targeted cellular uptake.12 At the same time, the increased knowledge about de novo peptide design,
Leiden Institute of Chemistry, Leiden University, P.O. Box 2300, RA Leiden, The Netherlands. E-mail:
[email protected] † Electronic supplementary information (ESI) available: Experimental details of synthesis, characterization and release study. See DOI: 10.1039/c3cc45790g
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structure and folding has raised interest in the use of peptide motifs as building blocks for novel bio-active materials13 and to develop new functions. For example, Fletcher14 and coworkers showed that careful design of short complementary peptides enables their assembly into new supramolecular biomaterials offering control over chemistry, self-assembly, reversibility and size. Furthermore, peptides designed to assemble through noncovalent interactions can be programmed to be responsive to a variety of stimuli, including temperature and pH.15 Previous studies demonstrated that the temperature-dependency of coiled coils can be utilized to control the structure of supramolecular assemblies.16 In this contribution, we used a heterodimeric coiled coil motif as a thermoresponsive valve for MSNs. Formation of coiled-coil motifs at the MSN surface effectively blocks the pores, preventing the release of its cargo. At high temperature, coiled-coil motifs disassemble (i.e. opening of the MSN), resulting in the release of its cargo. For this, the MSN surface was covalently modified via a small spacer with the well-characterized heterodimeric peptide pair E/K acting as the nanovalves (Scheme 1).17
Scheme 1 Schematic representation of the thermoresponsive MSN valve system. MSNs are modified with spacers and coiled-coils forming peptides to produce MSNA-E and MSNB-E. Fluorescein is loaded and nanovalves are closed with the complementary peptide K or pegylated peptide K (K-PEG). As the temperature increases the coiled coil valves open and the cargo is released.
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Communication For the formation of coiled coil valves at the surface of MSNs, peptide E was covalently bound and the complementary peptide K or pegylated peptide K (K-PEG) was used to close the pores by coiled coil formation. K-PEG was employed to study the effect of increased valve size on the kinetics of cargo release and also to enhance the colloidal stability of the silica nanoparticles in the body due to the ‘stealth’ like properties of the PEG-shell.18 Here we present for the first time that specific supramolecular peptide–peptide interactions can be exploited to control the release of a cargo from MSNs, thereby creating a new function for coiled-coils as thermoresponsive valves. Mesoporous silica nanoparticles (MSNs) were synthesized using a base-catalyzed sol–gel process at high temperature,19 tetraethyl orthosilicate (TEOS) as the silica source and hexadecyltrimethyl-ammonium bromide (CTAB) as the surfactant template. The surfactant was subsequently removed from the pores of the MSNs by refluxing with acidic methanol. CTAB removal was confirmed using Fourier transform infrared spectroscopy (FTIR, see ESI,† Fig. S3). Scanning electron microscopy (SEM) showed that the particles were spherical with a diameter of 136 nm 38 (see ESI,† Fig. S4) while the 2Dhexagonal porosity was shown using transmission electron microscopy (TEM). The pore size was 2.9 nm as confirmed by XRD. In order to construct the nanovalves at the MSN surface, the surface was modified with either (3-aminopropyl) triethoxysilane (APTES) (see ESI,† Scheme S1, 2) or 3-mercaptopropyltriethoxysilane (ESI,† Scheme S1, 3), followed by addition of an activated thiol, which was introduced by coupling N-succinimidyl 3-[2-pyridyldithio]-propionate (ESI,† Scheme S1, 4) or aldrithiol (ESI,† Scheme S1, 5). Finally, the MSNs were incubated overnight with cysteine modified peptide ‘E’ [amino acid sequence C(EIAALEK)3] (ESI,† Scheme S1, 6 and 7). The thiol of the cysteine was exchanged with the activated disulfide bond of the silica nanoparticles, resulting in peptide attachment. The concomitant release of thiopyridone was used to quantify the amount of peptide bound to the MSN surface using UV-spectroscopy and typically 0.33 mmol of peptide was bound per 10 mg of MSNs. FTIR spectroscopy (ESI,† Fig. S8) confirmed the presence of amide bonds of peptide E at the MSN surface. The MSNs were subsequently loaded with fluorescein (2.5 mM), as a model compound for the release studies. After 24 hours the valves were closed by addition of the complementary peptide ‘‘K’’ [(KIAALKE)3] to the solution. The loading capacity for fluorescein was 7 nmol mg 1 as determined using UV-VIS spectroscopy. The prevention of premature and uncontrolled drug release is an important criterion for any potential drug delivery system.12c Therefore the spacer length was varied (MSNA and MSNB) in order to minimize leakage, which was evaluated by suspending 5 mg of each MSN system in PBS at room temperature and at 10 minute intervals the amount of fluorescein released into the buffer solution was measured. Peptide ‘‘E’’ modified MSNA showed significant leakage (Fig. 1). As expected closing the pores by the addition of peptide K or K-PEG resulted in a lower amount of fluorescein leakage. However, the coiled coils did not fully close the pores. Therefore we redesigned our MSNs by shortening the spacer length between the silica surface and peptide E (MSNB). In this case This journal is
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Fig. 1 Fluorescein leakage assay from peptide and coiled coil motif modified MSNs at room temperature after 240 minutes.
the leakage of fluorescein from silica nanoparticles at room temperature was significantly reduced even after 4 hours (Fig. 1). This showed that coiled coils can be used to effectively cap the pores of MSNs and that the spacer length is highly important. Next we investigated whether the coiled coil motif could be disassembled by increasing the temperature resulting in the release of the dye. Robson Marsden16b showed that the melting temperature of coiled coils increases significantly when the peptides are confined to an interface. When the temperature reaches 80 1C, the ellipticity ratio as measured by circular dichroism spectroscopy indicated that the peptides were no longer forming a coiled coil complex. Therefore, the release profile was evaluated by suspending the MSNs in PBS (pH 7.4) at 20 1C and after 60 minutes the temperature was raised to 80 1C, which was the optimal temperature for the release study (see ESI,† Fig. S9). In the absence of a temperature increase, a negligible leakage of the dye was observed from MSNs even after 240 min (Fig. 2). In contrast, when the temperature of the suspension was raised to 80 1C, significant release of fluorescein was observed. The MSNB-E/K system released 60% and the MSNB-E/K-PEG system released 75% of the dye. The difference between the two systems is not yet fully understood and further studies are required. The release of fluorescein as a function of time and
Fig. 2
Fluorescein release at t = 240 minutes at room temperature and at 80 1C.
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Fig. 3 Fluorescein release of MSNB-E/K-PEG at room temperature and raise of temperature after 60 minutes to 80 1C.
temperature confirmed that the dye was only released upon increasing the temperature, showing that the release is controlled by the coiled coil disassembly (Fig. 3). In summary, we have demonstrated a potential new drug delivery system based on the coiled coil ‘nanovalve’ concept. MSNs were synthesized and conjugated to the ‘‘E’’ peptide that forms a coiled-coil motif when mixed with the complementary peptide K or K-PEG. Increasing the temperature above the melting point of the coiled coil results in the release of the cargo. This demonstrates that we developed a new function for coiled coils, namely to act as a thermoresponsive nanovalve controlling the release of its cargo. In future studies we will use the coiled coil valves on iron-oxide core MSNs.20 Applying an oscillating magnetic field to these particles results in a local temperature increase and this should initiate the unfolding of the coiled coil valves resulting in the controlled release of drugs, which could be used in the field of targeted in vivo drug delivery. GM, HZ and AK acknowledge the support from the European Research Council (Project 240394). Wim Jesse, Fabiola Porta and Aimee Boyle are acknowledged for experimental assistance and fruitful discussions.
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