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Mechanosensation at the molecular levelYilmaz, Duygu

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Chapter 5

Bioorthogonal Chemistry as a Tool for

Triggered Release from Liposomes: Click

To Free

Duygu Yilmaz, Martin Walko, Marc Robillard, Armağan Koçer

Abstract

Due to their rapid and highly selective nature, bioorthogonal chemical reactions

attracted a significant amount of interest in recent years. An exciting application of

these reactions is in the field of ‘pre-targeting’, for both possible treatment and imaging technologies. Here we adapt a click reaction to establish controlled release

from liposomes comprising MscL, as a valve. We incorporated a trans-cyclooctene

(TCO) to an engineered cysteine at the 22nd position of MscL. After reconstitution of

MscL into liposomes, tetrazine is added to the liposome mixture. The conjugation

between TCO and tetrazine results in the introduction of charge (from the amine or

the acid on the tetrazine) in the pore of the channel, leading to its opening. By this

way, controlled release of the liposomal content is achieved.

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Introduction

Protein labeling in vivo with synthetic probes offers a powerful tool to study protein structure, dynamics and function in cells and living organisms. However, the study of biomolecules in their native environments is a challenging task because of the structural complexity and functional reactivity of the biological systems. Researchers are trying to find new tools for the selective manipulation of molecules, cells, particles and surfaces, and the tagging and tracking of biomolecules in vitro and in vivo. In this respect, bioorthogonal chemistry has emerged as a very useful toolbox in the last years, demonstrating utility for labeling in complex environments.

The term bioorthogonal chemistry refers to selective chemical transformations among abiotic reactants that can proceed in living systems without interfering with surrounding biological entities (Boyce and Bertozzi, 2011). Bioorthogonal reactions involve two-steps and need a pair of functional groups. First, the bioorthogonal functional moiety (chemical reporter) of a compound is incorporated into a substrate. Second, the reporter is covalently linked to an exogenous probe through a reaction, which allows for detection and isolation of the target (Figure 1).

Figure 1. A common experimental platform for biomolecule probing using

bioorthogonal chemistry. First, a non-native functional roup, a “chemical reporter”, is installed in a biomolecule of interest. The modified biomolecule is subsequently labeled using a bioorthogonal chemical reaction. (adapted from Sletten and Bertozzi, 2011, ACS)

To be maximally useful in biological research, bioorthogonal reactions must proceed smoothly in aqueous environment at physiological pH, temperature and pressure, provide good yield and reasonable kinetics at low reagent concentrations, and produce only nontoxic (or no) side products (Boyce and Bertozzi, 2011). Furthermore, the chemical reporter should be inert in vivo, have no reaction with the biological environment and small enough to modify the target substrate without any functional and spatial interference. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems. These reactions include the

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Staudinger ligation, the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), and the inverse-electron-demand Diels–Alder (inv-DA) reaction (Blackman et al., 2008; Debets et al., 2013) (Figure 2).

Figure 2. Overview of the most common bioorthogonal reactions. 1) The Cu(I)- catalyzed Azide - Alkyne Click Chemistry reaction (CuAAC) relies on the presence of Cu(I) ions whereas 2) the Copper-free Strain-Promoted Alkyne - Azide - Click Chemistry reaction (SPAAC) and 3) Tetrazine – trans-Cyclooctene (TCO) Ligation efficiently proceed without metal catalysis.

The Staudinger reaction involves a reaction between an azide and a phosphine functional group to form an aza-ylide (Staudinger and Meyer, 1919). After modification by Saxon et al., it is recognized as a highly chemoselective ligation method for the preparation of bioconjugates (Saxon, 2000). It has been used successfully for the modification of proteins (Kiick et al., 2001), and the engineering of cell surfaces both in vitro (Saxon, 2000; Agard et al., 2006) and in living animals (Prescher et al., 2004). It has also been used in combination with fluorophores in in

vitro studies targeting azide groups present on the surface of live cells (Chang et al., 2007; Hangauer and Bertozzi, 2008). A study conducted by Vugts et al. explored the possibility of using the Staudinger ligation to facilitate a pretargeted imaging strategy (Vugts et al., 2011). In this case, an anti-CD44v6 chimeric monoclonal antibody that had been modified with multiple azide functionalities was employed as a targeting vector. A series of phosphine-containing small molecules

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incorporating radionuclides for imaging (67/68Ga, 89Zr, 123I, and 177Lu) and

therapeutic applications (177Lu) were then evaluated as secondary agents.

Following the administration of a 67Ga-DFO-phosphine agent in non-tumor bearing mice, the presence of Staudinger products in the blood pool was monitored, however, no evidence of any ligation was observed. Experiments in serum revealed that Staudinger ligation efficiency was primarily hampered by the formation of a side product. Furthermore, the fact that phosphine species is prone to oxidation makes it unable to undergo reaction with azide groups. Therefore, the rate of the Staudinger ligation was found to be sub-optimal for in vivo bioorthogonal reactions, particularly considering the rapid clearance and elimination of the secondary phosphine agent. CuAAC is a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole. Being the most used click reaction, many applications of CuAAC were demonstrated. One of the applications of CuAAC is in the development of drug delivery vehicles for therapeutic, imaging, or diagnostic purposes. This includes surface modification of lipid vesicles to alter their zeta potentials, hydrophobicities, and targeting capabilities. Surface modifications of these carriers can have significant impacts on their physical-chemical properties and therapeutic efficacy. To this extent, Hassane et al. developed a strategy for conjugating mannose ligands to the surfaces of preformed liposomes using CuAAC (Said Hassane et al., 2006). The resulting mannosylated liposomes were claimed to serve as vehicles to target human dendritic cells (Copland et al., 2003). In another study, Cavalli et al. used the copper- mediated [3 + 2] azide–alkyne cycloaddition as a chemical tool for the facile in-situ surface modification of liposomes. They used fluorescence resonance energy transfer (FRET) to demonstrate that the reaction takes place at the surface of the liposomes (Cavalli et al., 2006). In a recent study, Johnston et al. developed a method to couple azide-functionalized antibodies to alkyne-modified capsules using chelated Cu(I) catalyst and demonstrated specific targeting to cancer cells (Johnston et al., 2012).

Despite the ease and common use of CuAAC, it has been suggested that copper catalyst used in the reaction could have some adverse effects related to its toxicity (Link and Tirrell, 2003). For biomedical applications the use of Cu in the reaction and its retention post-synthesis, poses potential toxicity risks, and thus could limit the use of this method for products intended for biology. To overcome this drawback, chelating ligands such as ethylenediaminetetraacetic acid (EDTA) could be used to remove the copper metal after the reaction. However since EDTA is highly membrane impermeable, it would not capture the internal copper. Furthermore, considering the potential adverse effects, even at picomolar levels, development of Cu-free click strategies are preferred recently which reduce the risk of transition metal related toxicity.

For instance, the Diels−Alder reaction, as an alternative to CuAAC, avoids potential metal ion contamination and is a clean and simple click reaction that is effective in

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aqueous conditions. In 2008, Fox and co-workers reported the very fast reaction kinetics (rate constant, k2 = . × 1 3 M−1 s−1 in MeOH/H2O (9:1)) and in vitro bioorthogonality of the inverse-electron-demand Diels−Alder (inv-DA) reaction between trans-cyclooctene (TCO) and electron-deficient tetrazines (Blackman et al., 2008), which has led to several very promising bioconjugation applications (Devaraj et al., 2009; Li et al., 2010; Devaraj and Weissleder, 2011; Zeglis et al., 2011; Lang et al., 2012). These applications include its use in multistep labeling of live cell surface antigens, intracellular imaging of small molecules and in vivo

imaging applications as well as modification of cells with nanomaterials for clinical diagnostics (Devaraj and Weissleder, 2011). In a tumor pretargeting study for molecular imaging, it has been shown that the inv-DA reaction between antibody-conjugated trans-cyclooctene (TCO) and a radiolabeled tetrazine occurs effectively in mice at low equimolar concentrations (Rossin et al., 2010; Devaraj et al., 2012; Rossin et al., 2013; Zeglis et al., 2013).

Bearing this in mind, we envision that adapting such a click reaction to establish controlled release of liposomal content could have a high potential in the development of drug delivery vehicles for therapeutic purposes. To this extent, here we use inverse electron-demand Diels-Alder reaction as a chemical tool for controlled release of cargo from liposomes comprising a channel protein, the Mechanosensitive Channel of Large Conductance, MscL, as a sensory release valve.

MscL is a bacterial channel protein that opens to form a large non-selective pore of about 3 nm in diameter in the membrane, allowing the passage of ions, small molecules, peptides and small proteins. In nature, MscL gates in response to the tension in the membrane. It has been shown that the hydrophilicity of the 22nd amino acid position of MscL affects the mechanosensitivity of the channel up to a point where it starts to open even in the absence of tension (Yoshimura et al., 1999). On the basis of this principle, MscL protein was re-engineered to incorporate small modulators at Cys-22 in MscL. In this way the ability to control the release of liposome content with reversible channel opening and closing was demonstrated, using photoswitchable or pH-sensitive probes (Koçer et al., 2005). Here, we adopt a similar principle, taking advantage of a bioorthogonal reaction. The use of a biocompatible chemical reaction that does not rely on endogenous activation mechanisms for selective liposome activation could be valuable in the development of drug delivery systems. Selective activation of liposomes at the required site and time allows control over many processes within the body, including cancer. Therapies may be made more specific and effective, providing an increased therapeutic contrast between normal cells and tumor to reduce unwanted side effects.

Results and Discussion

In our system, we incorporate a trans-cyclooctene (TCO) to an engineered cysteine at the 22nd position of MscL. We anticipate that upon addition of tetrazine, the conjugation between TCO and tetrazine will result in the introduction of charge

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(from the amine or the acid on the tetrazine) in the pore of the channel, leading its opening. By this way, release of the liposomal content will be achieved. To this extent we firstly design the following inv-DA reaction (Figure 3).

Figure 3. Labeling of MscL with TCO 1 and inv-DA reaction between TCO and

tetrazines 3 and 4

G22C MscL is expressed in E. coli and purified as described previously (Koçer et al., 2007). G22C MscL is chemically modified with TCO maleimide 1 by attaching TCO 1 to the cysteine. Afterwards the presence of TCO 1 was confirmed with ESI-MS. As shown in Figure 4A and 4B, the unmodified protein has a mass of 15695 Da, and TCO 1-labeled protein has a mass of 16003 Da.

Then, proteins modified with TCO 1 are reconstituted into synthetic liposomes with a self-quenching dye, calcein, accordin to Koçer et al. (Koçer et al., 2007). After removal of the external dye, the fluorescence increase upon release of calcein from MscL-reconstituted liposomes was monitored. After the initial fluorescence reading, tetrazines 3 and 4 are added to the solution. The fluorescence is measured continously and an increase in fluorescence is expected if the inv-DA reaction leads

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to calcein release. However, no release was observed, suggesting that the reaction between TCO and tetrazine may not have taken place. ESI-MS analysis showed that tetrazine 3 reacts only partially with TCO, while tetrazine 4 is reacting with TCO (Figure 4C and 4D).

We reason that this may be due to the position of the incorporated TCO label, which may not be accessible to tetrazine 3. We shifted the position of TCO 1 towards the upper residues in TM1 of the channel. We mutated residues 23-29 of MscL individually to cysteine and labeled these residues with TCO 1. When we reconstituted these mutant proteins into the liposomes and incubated with tetrazines, we detected about 10 % of calcein release upon addition of tetrazines.

Figure 4. ESI-MS of (A) G22C MscL (B) G22C MscL labeled with TCO 1 (C) G22C MscL

labeled with TCO 1 reacted with tetrazine 3 (D) G22C MscL labeled with TCO 1 reacted

with tetrazine 4

In order to increase the release efficiency, we designed another TCO-tetrazine reaction (Figure 5). This reaction, a cleavage, is different from the previous conjugation, which adds bulky groups to the protein, resulting in the positioning of the charge away from the pore of the channel. As a result of the cleavage reaction, here, the charge ends up close to the pore of the channel. Furthermore in this system, the use of a smaller size tetrazine facilitates its accessibility to the TCO, attached in the narrow pore region of the channel.

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Figure 5. Labeling of MscL with TCO 7 and inv-DA reaction between TCO and tetrazine

9

The modification of MscL cysteine with TCO 7 and the subsequent generation of a primary amine in the reaction with tetrazine 9 are shown in the scheme above. TCO 7 labeled MscL carries no charge. Upon reaction with tetrazine 9, a primary amine is formed, and protonation at a pH value below its pKa (7.75) in turn leads to opening of the channel and liposomal release.

Figure 6 shows the liposomal release in TCO 7-modified liposomes and in control liposomes at different pH values. At both pH 8 and pH 6, there is a significant difference in release between the TCO 7- modified liposomes and control liposomes (P<0.05). This, together with the ESI-MS data (Figure 7) indicates that the anticipated inv-DA reaction between the TCO 7 and tetrazine 9 is taking place. However, the presence of the intermediate product 12 in the ESI-MS spectrum shows that this reaction does not result in 100 % product formation. Further modification to improve the dissociation of the intermediate product to final product would be useful in terms of increasing the calcein release.

Furthermore, the observed release in liposomes without TCO 7 modification suggests that there is also a release due to non-specific interaction of tetrazine 9 with the lipid bilayer. We reason that this non-specific interaction is due to the hydrophobic nature of the tetrazine 9.

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Figure 6. Calcein release from TCO 7-modified liposomes at pH 8 and pH 6. Each bar reflects the average value obtained from three independent experiments. Asterisk indicates statistical significance (one sample t-test, P<0.05).

Figure 7. ESI-MS of (A) G22C-labeled with TCO 7 (B) G22C-labeled with TCO 7 reacted

with tetrazine 9

In conclusion, here, we show that the inv-DA reaction can be used as a chemical tool in the activation of MscL embedded liposomes. With further fine-tuning, such a reaction could become useful in the development of systems for the targeted delivery and/or controlled release of therapeutic agents.

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Materials and Methods

Synthesis of compounds

Tetrazine 3 is synthesized according to Rossin et al. (Rossin et al., 2010). Tetrazine 4 is synthesized according to Devaraj et al. (Devaraj et al., 2009). The tetrazine 9 is synthesized according to Versteegen et al. (Versteegen et al., 2013).

Plasmid and Strain

As previously described (Birkner et al., 2012) E. coli MscL was encoded in p1BAD vector and protein expression was performed in an MscL null E. coli PB104 strain. The p1BAD vector provides ampicillin resistance, a 6His-tag for purification, and arabinose control over protein expression. Site-directed mutagenesis was accomplished by polymerase chain reaction, using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Briefly, wild-type MscL in p1BAD was used as a template and oligonucleotide primers were designed that incorporated the desired codon change accompanied by two 12- to 18-base pair flanking sequences on each side. Mutants were verified by sequencing and enzymatic digestion.

Protein expression and purification

Mutant proteins were expressed as described previously (Koçer et al., 2007). In short, mutant constructs were transformed into CaCl2-competent E. coli PB104 cells and were rown in LB medium in the presence of 1 μ /mL chloramphenicol and 1 μ /mL ampicillin. Cells were rown in a bioreactor with pH 7.5, temperature 7 °C, and oxy en control (dissolved oxy en >7 %), usin a complex medium [1 g/L Bacto-Tryptone (BD), 24 g/L yeast extract (BD), potassium phosphate (17 mM KH2PO4 and 72 mM K2HPO4) (pH 7), supplemented with chloramphenicol and ampicillin]. 40% (v/v) glycerol/L medium was used as additional carbon source and 0.1% (w/v) L-arabinose to induce the protein expression. Cells were harvested after 120 minutes.

Membrane vesicles were prepared as described elsewhere (Koçer et al., 2007). Briefly, cells were broken using a cell disrupter (Type TS/40; Constant Systems) at 1.7 kbar and 5 °C. After two subsequent centrifu ation steps, membrane vesicles were resuspended and homogenized in ice-cold 25 mM Tris-HCl (pH 8.0) to 7 g (wet weight)/mL, and frozen in liquid nitro en and stored at −8 °C.

Protein modification and Isolation

Protein was isolated as described by Kocer et. al. (Koçer et al., 2007) with some modifications. Briefly, membrane vesicles were solubilized by solubilization buffer (10 mM NaPi pH:8.0, 300 mM NaCl, 1%(v/v) Triton X-100 and 35 mM imidazole), and unsolubilized material was removed by ultracentrifugation. The solubilized fraction was then applied to Ni-NTA agarose resin (Qiagen), which was equilibrated

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with solubilization buffer. After 30 minutes incubation, the unbound material was let washed through and the column was washed with 15 CV of wash buffer (10 mM sodium phosphate (pH 8.0), 300 mM NaCl, 0.2% (v/v) Triton X-100, 35 mM imidazole). After this washing step, the column was further washed with a second wash buffer that contains all the components of the first wash buffer except for imidazole. Then the column matrix was incubated with TCO label dissolved in DMSO (2 mg/ml). After 45 minutes at room temperature the column was washed with the second wash buffer and 50 mM histidine buffer (wash buffer containing 50 mM Histidine). Finally, the protein was eluted with 235 mM histidine buffer (wash buffer containing 235 mM Histidine) and the fractions were analyzed for protein content by the Bradford assay.

Protein Reconstitution into Liposomes

Proteins were reconstituted into synthetic liposomes according to Koçer et al. (Koçer et al., 2007). Briefly, lipid suspension (asolectin) was homogenized by extrusion 11 times through a 400-nm filter. Liposomes were destabilized by the addition of Triton X-100. Protein and lipids were mixed at 1:50 weight ratio and incubated for min at 5 °C. Subsequently, the mixture was supplemented with 6 mg (wet weight) Biobeads (SM-2 Absorbents; Bio-Rad) per microliter of detergent (10% Triton X-100) used in the sample and lipid preparation. For detergent

removal, the sample was incubated overnight (∼16 h) at °C under mild a itation.

Before fluorescence assay, the mixture was applied to a size-exclusion column (Sephadex G50 Pharmacia) in order to collect the liposomes.

Fluorescence Assay

Elution fractions were assayed in a Varian Cary Eclipse Fluorometer at an excitation wavelength of 495 nm and recording the emission at 515 nm. In a standard assay, 5 μL calcein-filled proteoliposomes were diluted into 2.2 ml efflux buffer. At t = 1 min, tetrazine was added. The fluorescence was measured continuously, and the total fluorescence of the sample was determined by dissolving the proteoliposomes by the addition of 0.5% (v/v) Triton X-100 at t = 100 min. As a control, empty liposomes were recorded in the presence of tetrazine. The data sets were normalized by using the initial fluorescence of each sample as 0 % and the signal after the Triton X-100 addition as 100 %.

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