Sterically Bulky Caging of Transferrin for Photoactivatable Intracellular Delivery
Communication
Satoshi Yamaguchi,* Satoshi Takamori, Kazuho Yamamoto, Akira Ishiwatari, Kosuke Minamihata, Eriko Yamada, Akimitsu Okamoto, and Teruyuki Nagamune
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ABSTRACT: Photoactivatable ligand proteins are potentially useful for light-induced intracellular delivery of therapeutic and diagnostic cargos through receptor-mediated cellular uptake. Here, we report the simple and eff ective caging of transferrin (Tf), a representative ligand protein with cellular uptake ability, which has been used in the delivery of various cargos. Tf was modifi ed with several biotin molecules through a photocleavable linker, and then the biotinylated Tf (bTf) was conjugated with the biotin-binding protein, streptavidin (SA), to provide steric hindrance to block the interaction with the Tf receptor. Without exposure to light, the cellular uptake of the bTf-SA complex was eff ectively inhibited. In response to light exposure, the complex was degraded with the
release of Tf, leading to cellular uptake of Tf. Similarly, the cellular uptake of Tf-doxorubicin (Dox) conjugates could be suppressed by caging with biotinylation and SA binding, and the intracellular delivery of Dox could be triggered in a light-dependent manner. The intracellularly accumulated Dox decreased the cell viability to 25% because of the cell growth inhibitory eff ect of Dox. These results provided proof of principle that the caged Tf can be employed as a photoactivatable molecular device for the intracellular delivery of cargos.
igand protein-based carriers have recently been used as tools for the intracellular delivery of various cargo
molecules and particles for drug delivery, bioimaging, and tissue engineering because of their cellular uptake ability as
1-3
well as their biocompatibility and targeting ability. Therapeutic and imaging agents can be conjugated with ligand proteins via cleavable linkers, which are spontaneously cleaved in the intracellular environment, resulting in intracellular accumulation regardless of the cell permeability of the
4,5
agents. Moreover, to deliver biomolecules and genes,
Light stimulation has many advantages for cell manipulation, such as the ease of production, noninvasive nature, controllable intensity, and ability to spatially confine the irradiation.8,9 Therefore, a photoactivatable Tf complex might be a promising tool for light-guided intracellular delivery. However, no photoactivatable Tf has been reported yet. “Caging” has been widely reported as a tool for making biomolecules photoactivatable; the activities of the caged molecules are transiently blocked with photodegradable protecting groups, and restored using light irradiation with the desired location,
modification with ligand proteins has been employed for the
10,11
timing, and amount.
Caged proteins have been reported to
cellular uptake of various types of carriers, such as micelles,
3,6
liposomes, polymeric nanoparticles, and viruses. The iron binding protein, human transferrin (Tf), has been actively used as a ligand protein for delivery because of its relatively low cost,
6,7 high cellular uptake effi ciency, and tumor-cell specifi city. The receptor for Tf (TfR) is ubiquitously expressed in most normal human tissues. Additionally, the TfR is expressed on malignant tumor cells at levels several-fold higher than those on normal cells, making the TfR an attractive target for cancer
6,7
therapy. If Tf can be made stimuli-activatable, stimuli- responsive intracellular delivery could be achieved in a wide variety of cells and tissues. In addition, stimuli-activatable Tf- drug conjugates could potentially achieve even fewer side eff ects in cancer therapy by selectively applying external stimuli to tumor tissues.
12,13
exert photoactivatable activity both in vitro and in vivo. However, such proteins need elaborate genetic engineering technologies to incorporate the photocleavable protecting groups specifically at the active site of the proteins. Therefore, facile protein caging methods based on random chemical modifi cations have been developed with sterically bulky
14,15
protecting groups for effective protein caging. In this
Received: March 27, 2021 Revised: July 11, 2021
© XXXX American Chemical Society
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Figure 1. (a) Chemical structure of biotinylated caging reagent (BCR) 1 consisting of a biotinylated photocleavable protecting group (red) and an amino-reactive p-nitrophenyl carbonate group (black). (b) Schematic illustration of sterically bulky caged transferrin (Tf) with 1 and streptavidin. (c) Schematic illustration of light-induced intracellular uptake of Tf through photo uncaging, binding to the Tf receptor, and endocytic internalization.
study, we aimed to create a photoactivatable Tf by a simple sterically bulky caging method, followed by application of the complex to the light-induced intracellular delivery of cargo molecules.
A sterically bulky caged Tf was prepared by a two-step caging method using a biotinylated caging reagent (BCR) (Figure 1):15 in the fi rst step, Tf was modifi ed with 1 through an amide coupling reaction; and in the second step, streptavidin (SA) was conjugated with the biotin moiety on the BCR-modified Tf (biotinylated Tf: bTf) as a sterically bulky protecting group. The complex formed with the bulky SA was expected to drastically inhibit the binding of Tf to TfR without elaborate site-specifi c incorporation of a protecting group, resulting in facile suppression of intracellular uptake (Figure 1). Tf was modified with 1 by mixing in a buff er, and the average number of biotinylated protecting groups on each Tf molecule was 6.1 from the mass spectrum,16 where the peaks of the bTf with diff erent modifi cation numbers were overlapped due to the small molecular weight of BCR compared with that of Tf. A fluorescently labeled Tf molecule (transferrin-Alexa Fluor 647 conjugate) was employed for visualization in all subsequent experiments, except for the final drug conjugation experiment. Excess SA was added to the bTf solution for effi ciently caging one bTf molecule (the molar
ratio of SA to bTf was 12) to prevent aggregation of multiple bTf molecules through cross-linking with SA.16
To examine the conjugate formation, we employed SDS- PAGE analysis. It has been reported that the SA-biotin complex was stable in SDS-PAGE analysis.17 In fact, in our previous report, the bands of the complex of biotinylated protein and SA has been successfully detected in SDS-PAGE analysis.15 Diff erent from the SA-biotin complex, apo-SA that did not bind to biotin was reported to be mainly detected as the monomer (13 kDa) on the SDS-PAGE gel, because apo-SA tetramer was less stable against heating in the loading buffer than the complex.17 Therefore, in this study, most of the excess apo-SA in the conjugation sample migrated as the monomer in the SDS-PAGE gel, and subsequently, a slight band of the apo- SA tetramer was observed (Figure 2). It was confirmed by SDS-PAGE analysis that all the bTf was conjugated with SA: the band for bTf disappeared, and simultaneously, the bands for bTf-SA complexes appeared at the top of the resolving gel on the SDS-PAGE gel (Figure 2). From the molecular weights of Tf and SA, the complex of bTf and two SA molecules was calculated to be approximately 180 kDa. The bands of the bTf- SA complexes were clearly above the band of the largest marker protein (180 kDa); therefore, each bTf molecule was assumed to bind to more than three SA molecules. In the
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Figure 2. Bands of transferrin (Tf) and biotinylated Tf (bTf) with and without streptavidin (SA) on an SDS-PAGE gel. Samples of bTf with SA were exposed to light at the wavelength of 365 nm at doses of 0, 1, 5, and 10 J/cm2. The identifi cation of each band is assumed based on the band of size markers.
control experiment, the band for Tf was the same before and after addition of SA (Figure 2). From these results, Tf was confirmed to form complexes with SA through the specifi c biotin-SA interaction as designed.
Next, light-induced release of Tf from the bTf-SA complexes was examined by SDS-PAGE analysis. The photolabile moiety of the biotinylated protecting group, the methyl-6-nitro- veratryloxycarbonyl group, is cleaved by exposure to light at wavelengths from 350 to 420 nm.18 After exposure to light at 365 nm (2.5 mW/cm2) at room temperature, the ladder bands that presumably corresponded to the complexes of bTf with fewer SA molecules (from 0 to 3 SA on each Tf) appeared on the SDS-PAGE gel (Figure 2). In response to the light dose, the bands for the high-molecular-weight complexes disap- peared, and above 5 J/cm2, the band for unmodified Tf was observed (Figure 2). Simultaneously, the bands of the SA tetramer became thicker. These results indicate that the SA- biotin derivative complex was released from the bTf-SA complexes through photodegradation as designed. By image analysis of the bands, the percentage of the released Tf was determined to be 39% at 10 J/cm2 under experimental conditions where the protein concentration was relatively high (approximately 3 μM) so as to be detectable on the SDS- PAGE gel. Here, the Tf concentration used for the delivery of cargoes to cells are much lower than those for the SDS-PAGE analysis. For example, the half-maximal inhibitory concen- tration (IC50) of Tf-drug conjugates has been reported to be in the range 100-400 nM;19 therefore, the present Tf-releasing efficiency was considered to be suffi cient for the light-induced cellular uptake of Tf. The bTf-SA complexes were confirmed to be able to be photodegraded with the release of unmodified Tf.
The cellular uptake activity of uncaged Tf was examined in cultured cells. First, human colon cancer (DLD1) cells, which were reported to be used as target cells expressing the
Figure 3. (a) Confocal microscopic images of DLD1 cells treated with transferrin (Tf), biotinylated Tf (bTf), and bTf-streptavidin (SA) conjugates. Tf was fluorescently labeled with Alexa Fluor 647 (AF647). The bTf-SA conjugates were exposed to light at the wavelength of 365 nm at doses of 0 and 10 J/cm2. The AF647- fluorescent image and the diff erential interference contrast image were merged. The scale bars are 50 μm. (b) Normalized fluorescence intensities of the cells treated with Tf and bTf with and without SA. The bTf-SA conjugates were exposed to light at the wavelength of 365 nm at 0, 1, 5, and 10 J/cm2. Each bar represents the mean ± SE (n = 3).
(Figure S2). Compared with the fluorescence of the Tf-treated cells (normalized to 100%), the fluorescence of cells treated with the bTf and bTf-SA complexes was approximately 40% and <1%, respectively (Figure 3b). In addition, the presence of SA had a negligible infl uence on the intracellular uptake of Tf. Accordingly, these results clearly showed that the binding of SA to bTf blocked the cellular uptake of bTf because of the steric hindrance of SA. Next, the cells were treated with the bTf-SA complexes exposed to light (10 J/cm2). After treatment, fluorescence from Tf was observed in the cells, similar to the case for Tf only (Figure 3a). From this result, the uncaged Tf was clearly shown to regain the cellular uptake ability. By fl ow cytometer analysis, the normalized cellular fl uorescence increased according to the light dose and was saturated above 5 J/cm2 (Figure 3b). Thus, it was shown that sterically bulky caged Tf can achieve light-induced cellular uptake of Tf. However, in detail, the cellular uptake activity was only restored to nearly 80%. Here, it was also confirmed that light exposure causes no impact on the cellular uptake activity of Tf (Figure S3). Therefore, the present incomplete recovery of the activity is not due to light-induced denaturation. In the present method, less BCR modifi cation may lead to requirement of less light for activation, although it also may lead to inadequate inactivation before exposure to
transferrin receptor,7,20 were treated with the bTf-SA light, resulting in less clear-cut ON/OFF regulation. There is
complexes (bTf conc.: 60 nM) without light exposure (0 J/
cm2). Here, in a preliminary experiment, the cellular uptake of Tf was confirmed to saturate within 15 min under the present experimental condition (Figure S1). After incubation for 15 min and rinsing, no Tf was detected in the cells through microscopic observation of the fluorescent dye label on the Tf, whereas the fluorescence of Tf was clearly observed in the cells treated with Tf or bTf only (Figure 3a). The cellular fluorescence was quantitatively analyzed with a flow cytometer
still a possibility that further optimization of the modifi cation conditions will improve the photoresponsiveness of the present light-induced cellular uptake method.
Finally, to verify the usability of the caged Tf for the light- induced intracellular delivery of cargo molecules, a Tf-drug conjugate was caged with 1 and SA. Doxorubicin (Dox), a widely used anticancer drug, was selected as the cargo drug. Tf- Dox conjugates have been previously developed using various stimuli-sensitive linkers, which were reported to have enhanced
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Figure 4. (a) Schematic illustration of light-induced intracellular drug delivery with sterically bulky caged transferrin (Tf). (inset) The chemical structure of the doxorubicin (Dox) modification reagent 2 and the scheme of the Dox modifi cation at the side chain of a lysine residue of Tf. (b) Confocal microscopic images of DLD1 cells treated with Dox-modifi ed Tf (Tf-Dox) and biotinylated Tf-Dox (bTf-Dox)-streptavidin (SA) conjugates. The bTf-Dox-SA conjugates were exposed to light at doses of 0 and 5 J/cm2. The Dox-fluorescent image and the diff erential interference contrast image were merged. The scale bars are 50 μm. (c) Viabilities of nontreated DLD1 cells and DLD1 cells treated with bTf-Dox- SA conjugates. The cells were exposed to light at 0 and 6 J/cm2 and then cultured for 48 h. Each bar represents the mean ± SE (n = 3).
stability, cellular uptake ability, and tumor-targeting ability compared with free Dox.19,21 In this study, Dox was conjugated with thiolated Tf (without the fluorescent dye) via an acid- sensitive hydrazone linker according to previous reports.21 After cellular uptake of the Tf-Dox conjugates, Dox was designed to detach from Tf under the acidic conditions in endosomes and then transfer to the nucleus, leading to apoptosis of the cells (Figure 4a).21 The red fluorescence of Dox was clearly observed in cells treated with Tf-Dox for 15 min (Figure 4b, left). Moreover, treatment with Tf-Dox for 2 days was confirmed to decrease the cell viability to 24%,
compared with that of nontreated cells (Figure 4c). These results clearly showed that the Tf-Dox conjugate prepared in
19,21
this study functioned as previously reported.
Tf-Dox was modifi ed with 1, and then biotinylated Tf-Dox (bTf-Dox) was mixed with SA, leading to the sterically bulky caging of Tf-Dox. Similar to bTf without Dox, the formation of the complexes of bTf-Dox with more than four SA molecules, and the photodegradation of the complexes, were confirmed in vitro by SDS-PAGE analysis (Figure S5). Then, the light- induced intracellular delivery of Dox through the photo- degradation of the complex was evaluated in cell experiments.
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In the cells treated with the complex of bTf-Dox with SA (bTf- Dox-SA), the red fl uorescence of Dox was observed only at a negligible level (Figure 4b, center), whereas considerable red fluorescence was observed in the cells after treatment with light-exposed bTf-Dox-SA (Figure 4b, right). These results clearly showed that sterically bulky caging of Tf-Dox could achieve control of the intracellular accumulation of Dox. Next, light-induced cell growth inhibitory eff ects were investigated in a medium supplemented with bTf-Dox-SA. The cell culture wells of a 96-well plate were exposed to light (365 nm, 30 mW/cm2) for 200 s (6 J/cm2) and the cells were cultured for 2 days. Without exposure to light, the cell viability was 78% after culture with bTf-Dox-SA (Figure 4c). It can be seen that the growth inhibitory eff ect of Dox was suppressed for a long period by this caging method. After light exposure and culture, the cell viability of the wells with bTf-Dox-SA decreased to 25% (Figure 4c). This viability level was almost the same as that of cells cultured with noncaged Tf-Dox. These results indicated the successful induction of a cell growth inhibitory eff ect, comparable to that of noncaged Tf-Dox, by exposure to a low dose of light that did not have a significant infl uence on the cell growth. Furthermore, it was also confi rmed that this dose of light did not impact on the cell membrane barrier against direct transduction of the bTf-SA complex into the cells (Figure S4). From these results, the present light-induced eff ect was shown to be derived from uptake of the released Tf- Dox via binding to TfR as designed. Thus, the present sterically bulky caged Tf was confirmed to be applicable for the light-induced intracellular delivery of a drug in living cells.
Until now, the sterically bulky caging of proteins has only been demonstrated in vitro using the bacteriolytic enzyme,
into the intracellular region of living cells. Using this photoactivatable cellular uptake ability, Tf-Dox conjugates were introduced into cells in a light-dependent manner, leading to a light-induced cell growth inhibitory eff ect through the intracellular accumulation of Dox. Thus, this caged Tf method achieved the light-induced intracellular delivery of a cargo molecule without compromising the function of the molecule. Because of its simplicity, usability, and potential versatility, this caged Tf complex is a promising tool for the light-guided intracellular delivery of a wide variety of cargos, which will be useful in fundamental biological research, drug delivery, and tissue engineering.
■ ASSOCIATED CONTENT
sı* Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00159.
Protocols for caging transferrin, detailed experimental procedures, time-dependence of cellular uptake of transferrin, histogram of the flow cytometry, impact of light exposure on the transferrin activity, the cell membrane barrier and the Dox derivatives, SDS-PAGE analysis of bTf-Dox (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Satoshi Yamaguchi - Research Center for Advanced Science
14,15
lysozyme, as a model protein.
The present study shows for
and Technology (RCAST), The University of Tokyo, Meguro-
the first time that random modification-based sterically bulky caging is applicable to other proteins. In the present study, Tf was successfully caged, resulting in the light-induced delivery of a drug into cancer cells (Figure 4). The photoactivation of a variety of drugs has been actively studied because of the
8,9
therapeutic advantages of light. Among them, photo- sensitizers that generate reactive oxygen species in the extracellular region are used for the site-specific induction of
22,23
cell death in photodynamic therapy in the clinical setting.
The present caged Tf molecule can in principle transport various drugs into the intracellular region. Because the only requirement for the drug is the ability to be chemically conjugated with Tf via an appropriate linker, this technology can be applied to a wide variety of drugs, leading to the expansion of candidates for photoactivatable drugs. In addition, this method may be applied, not only to drugs, but also to various small molecule agents for bioimaging, nutritional supply, and immunotherapy. Moreover, by modifying Tf with orthogonally reactive tags before caging, the caged Tf can potentially be conjugated with larger cargos, such as micelles, liposomes, polymeric nanoparticles, and viruses, which may achieve the light-guided spatiotemporal delivery of therapeutic agents and genes.7 Thus, the present soluble caged Tf is expected to be a versatile molecular nanodevice applicable to a wide range of cargos for light- guided intracellular delivery.
In conclusion, the caging of Tf was performed by a two-step method using biotinylation via a photocleavable linker and biotin-mediated complex formation with sterically bulky SA. In response to the photodegradation of the complexes, unmodified Tf was confirmed to be released and transferred
ku, Tokyo 153-8904, Japan; orcid.org/0000-0003-4822- 7469; Phone: +81-3-5452-5202; Email: yamaguchi@ bioorg.rcast.u-tokyo.ac.jp; Fax: +81-3-5452-5209
Authors
Satoshi Takamori - Department of Chemistry &
Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Kazuho Yamamoto - Department of Chemistry &
Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Akira Ishiwatari - Department of Chemistry & Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Kosuke Minamihata - Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Nishi-ku, Fukuoka 819-0395, Japan
Eriko Yamada - Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Meguro-ku, Tokyo 153-8904, Japan
Akimitsu Okamoto - Research Center for Advanced Science and Technology (RCAST), The University of Tokyo, Meguro- ku, Tokyo 153-8904, Japan; Department of Chemistry &
Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan; orcid.org/
0000-0002-7418-6237
Teruyuki Nagamune - Department of Chemistry &
Biotechnology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.bioconjchem.1c00159
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Author Contributions
The manuscript was written through contributions of all authors.
Notes
The authors declare no competing fi nancial interest.
■ ACKNOWLEDGMENTS
This work was supported by a Ministry of Education, Culture, Sports, Science, and Technology Grant-in-Aid for Challenging Exploratory Research (19K22079), Scientific Research (C) (15K06575) and Scientific Research (B) (21H01723), and by the Japan Science and Technology Agency (JST) PRESTO (16815021) and MIRAI project (19217334).
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