A Protein Capsid-Based System for Cell Delivery of ... - ACS Publications

Subscriber access provided by Hong Kong University of Science and Technology Library

Article

A Protein Capsid-Based System for Cell Delivery of Selenocysteine Shuxin Wang, Aneesa T. Al-Soodani, Geoffrey C. Thomas, Bethany Buck-Koehntop, and Kenneth J. Woycechowsky Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00302 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

A Protein Capsid-Based System for Cell Delivery of Selenocysteine Shuxin Wang†, Aneesa T. Al-Soodani‡, Geoffrey C. Thomas‡,§, Bethany A. Buck-Koehntop‡, and Kenneth J. Woycechowsky†,‡,#,* †

School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin

Road, Nankai District, Tianjin, China 300072 ‡

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City,

USA 84112 §

Present address: College of Medicine, The Ohio State University, 370 W 9th Ave,

Columbus, OH 43210 #

Present address: School of Pharmaceutical Science and Technology, Tianjin

University, 92 Weijin Road, Nankai District, Tianjin, China 300072 *

e-mail: [email protected]

1

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

ABSTRACT: Selenocysteine (Sec) has received a lot of attention as a potential anti-cancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery. Here, we demonstrate for the first time that an engineered version of the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for delivery of Sec to cells. Specifically, a previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with the diselenide dimer of Sec (Sec2) to generate a selenenylsulfide conjugate between the capsid and Sec (AaLS-IC-Sec). Importantly, it was determined that the structural context of the reactive cysteine was important for efficient capsid loading. Further, the encapsulated Sec could be quantitatively released from AaLS-IC-Sec by reducing agents such as glutathione or dithiothreitol. To assess cellular penetrance capabilities of AaLS-IC-Sec and subsequent cytotoxic response, six different cells line models were examined. Across the cell lines analyzed, cytotoxic sensitivity correlated with cellular uptake and intracellular trafficking patterns. Together these findings suggest that the engineered AaLS-IC capsid is a promising vehicle for targeted cell delivery of Sec.

2


Page 3 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Selenocysteine (Sec) is a redox-active amino acid with promising anti-cancer properties. In air, Sec will rapidly oxidize to selenocystine (Sec2), the diselenide dimer of Sec. When added to culture media, Sec2 displays cytotoxicity to a broad range of mammalian cells, as well as yeast and bacteria, with IC50 values typically in the low-µM range1-7. The mechanism of Sec-induced cytotoxicity remains unclear, but may involve disruption of redox homeostasis in the cell, leading to overoxidation and apoptosis3, 8. Additional factors may also contribute to its cytotoxic effects, including misincorporation of Sec into proteins via charging of an inappropriate tRNA and metabolism of free Sec by a dedicated β-lyase enzyme9-11. Regardless of the mechanism(s), the wide-ranging cytotoxicity of Sec limits its potential utility as an anti-cancer drug. To overcome this limitation, targeted delivery systems for Sec are needed. A carrier for Sec2 based on mesoporous silica nanoparticles decorated with transferrin and a cell-penetrating peptide has been reported to enter cells via receptor-mediated endocytosis and slowly release its cargo at low pH12. This system was shown to sensitize cancer cells to X-ray radiation, which could be useful for a combined radio-/chemo-therapy treatment strategy. However, some potential disadvantages with this kind of Sec2 delivery system include the complex procedure for the preparation of the Sec2-loaded and surface-modified nanoparticles, the use of organic solvents in this preparation procedure, the release of some Sec2 prior to cell uptake (due to the non-covalent nature of the interaction between the nanoparticle and Sec2), the ability to induce certain undesirable metabolic changes, and the propensity of silanols on the 3



Page 4 of 37

nanoparticle surface to interact with the membranes of some non-cancerous cells, causing undesired off-target effects13. Protein capsids represent potentially highly attractive alternative nanocarriers for Sec delivery that could conceivably avoid these issues. Indeed, the native biological functions of protein capsids generally involve acting as nanoscale containers for storage and delivery of various molecular cargoes. These closed-shell supramolecular structures have a high degree of symmetry, are monodisperse, and are readily modified by genetic and chemical methods14. The hollow interior of a protein capsid provides ample space for housing guest molecules, shielding them from the bulk environment. Indeed, a variety of protein capsids have been used to deliver drugs and imaging agents into cells15-23. The protein capsid formed by lumazine synthase from Aquifex aeolicus (AaLS) is a particularly attractive scaffold for developing a Sec delivery system. AaLS assembles into a 60-subunit dodecahedron with a diameter of 160 Å and a hollow interior that is roughly 90 Å across. The capsid has 12 pores located at the C5 symmetry axes24. The narrowest point of these pores is 8.9 Å across, which allows small molecules to diffuse freely across the capsid shell25. AaLS also possesses remarkable thermal stability, with a reported Tm value of 120 °C24. Variants of AaLS have previously been engineered to encapsulate RNA26, 27 and protein cargoes28-31. Small-molecule drugs have also been conjugated to the exterior surface of AaLS for delivery into cells32, 33. Previously, we reported the encapsulation of a thiol-bearing analog of curcumin 4


Page 5 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(cur-SH) by a variant of AaLS, called AaLS-IC25. As a consequence of two point mutations (C37A/E122C), AaLS-IC possesses one cysteine residue per subunit, located at the interior surface of the capsid. Cur-SH was loaded into AaLS-IC via a two-stage thiol-disulfide exchange process. Encapsulated cur-SH had a dramatically higher water solubility than free cur-SH and could be released from the capsid by treatment with the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Here, we utilize AaLS-IC to encapsulate Sec via direct, spontaneous selenenylsulfide bond formation and examine the ability of the encapsulated Sec to exert a cytotoxic response in six different mammalian cell lines. Notably, the engineered AaLS-IC-Sec capsid shows a significant degree of cell-type selectivity in Sec delivery that directly correlated with cellular uptake and trafficking. As such, AaLS-IC represents a promising new vehicle for targeted cell delivery of Sec.

RESULTS Loading of Sec. The engineered cysteine of AaLS-IC (C122) is capable of performing thiol-disulfide exchange reactions with small, exogenously added disulfides25. In principle, a small molecule containing a diselenide bond, such as Sec2, could similarly react with the thiol of C122 inside the capsid to give a selenenylsulfide conjugate (AaLS-IC-Sec) and free selenol (Sec). Continuous rapid recycling of the selenol by-product back to the diselenide34, 35 would then drive the accumulation of AaLS-IC-Sec (Figure 1). Thus, we measured the extent of reaction between AaLS-IC and Sec2 at 37 °C for 5



Page 6 of 37

Figure 1. Design of AaLS-IC and AaLS-IC-Sec. A: The AaLS capsid, with individual pentamers differentially colored (pdb ID: 1HQK). B: A model of the AaLS-IC capsid, showing the interior surface along the back half of the capsid. The cysteine residues introduced at sequence position 122 in each subunit are shown in yellow. A close-up view of the three-fold symmetry axis, with C122 shown in a stick representation (sulfur in yellow, carbon in green). The E122C mutation model was prepared using PyMOL, starting from the structure of wild-type AaLS (pdb ID: 1HQK). C: Reaction scheme for generating AaLS-IC-Sec. Sec can be loaded into AaLS-IC by thiol-diselenide exchange with Sec2 to produce a selenenylsulfide adduct in the capsid. In the presence of atmospheric O2, the byproduct of this reaction (Sec) rapidly recycles back to regenerate Sec2 starting material, which likely helps to boost the loading efficiency. different reactant stoichiometries and reaction times, using Ellman’s assay36 to detect any free thiol groups left inside of the protein capsid. The product yield after 4 h was found to increase with increasing Sec2 concentration, reaching a plateau at Sec2:protein ratios above 4:1 (Figure 2A). Using a 6-fold excess of Sec2, the reaction was already >50% complete within 30 min, and after 3 h no thiol groups that can react with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) remained in the protein (Figure 2B). 6


Page 7 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2: Effect of stoichiometry and reaction time on Sec encapsulation yield. A: The disappearance of protein thiols was measured at different molar ratios of Sec2 to AaLS-IC monomers after 4 h for each reaction. B: The disappearance of protein thiols was measured at different times following the addition of six-fold molar excess Sec2 to AaLS-IC. For each data point, the number of thiol groups (SH) per capsid was derived from a combination of DTNB assay measurements and Bradford assay measurements of total protein concentration. The percent values of SH per capsid are reported relative to a theoretical maximum of 60 SH groups per capsid. For fully reduced AaLS-IC, the percent value of SH per capsid is 79%. C: Size exclusion chromatography analysis of AaLS-IC (black) and AaLS-IC-Sec (red) using a HiPrep 16/60 Sephacryl S-400 HR column (running buffer: 50 mM sodium phosphate, 200 mM NaCl, pH 8.0). Thus, the yield of AaLS-IC-Sec was ≥75%, since only 75% of the cysteines in the AaLS-IC starting material reacted with DTNB. To further examine the loading yield, we

used

the

2-nitro-5-thiosulfobenzoate

(NTSB)

assay37

to measure

the

selenenylsulfide bond content in AaLS-IC-Sec that was produced using the optimized reaction conditions and then treated with iodoacetamide (IAA) to block any unreacted thiol groups. The yield of AaLS-IC-Sec determined in this way was found to be 81%, which is similar to the yield found using the DTNB assay (Table S1). Since AaLS-IC can react with NTSB more completely than with DTNB (for reasons that remain unclear), the yield determined with NTSB should be more accurate. Thus, one AaLS-IC capsid can carry about 48 Sec molecules. The presence of an adduct between AaLS-IC and Sec was confirmed by mass 7



Page 8 of 37

spectrometry (Figure S1). Further, size-exclusion chromatography (Figure 2C) and transmission electron microscopy (TEM) (Figure S2) analyses showed that the capsid assembly state was unaffected by the reaction. Thus, Sec2 efficiently forms selenenylsulfide-linked conjugates with AaLS-IC, but does not otherwise induce any major structural changes in the capsid.

Impact of capsid architecture and cysteine location in the reaction with Sec2. Diselenides can catalyze the oxidation of thiols by atmospheric O2 to form disulfide bonds in proteins35. As discussed above, the reaction of AaLS-IC with Sec2 gives a selenenylsulfide adduct rather than disulfide-crosslinked protein. This observation prompted us to questions why AaLS-IC is not prone to disulfide cross-linking during treatment with Sec2. We hypothesized that the combined features of the protein capsid assembly and the location of the cysteine on the capsid may play a critical role in determining the outcome of this reaction. To test this hypothesis, we studied the reaction of Sec2 with both a different capsid-forming AaLS variant (AaLS-EC), which has a cysteine on the exterior surface instead of the interior surface, and a pentameric variant of AaLS-IC (AaLS-IC-pent), which has a similar engineered cysteine as AaLS-IC along with additional mutations that prevent capsid assembly38. In both cases, the cysteine residue should be exposed to the bulk solvent. To generate AaLS-EC, a unique reactive cysteine was installed at an exposed surface loop on the outside of the AaLS capsid by making two point mutations, C37A and E70C relative to wild-type AaLS (Figure S3A). These mutations did not significantly alter the capsid assembly of AaLS-EC, relative to AaLS-IC, as assessed 8


Page 9 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by size-exclusion chromatography with a Sephacryl S-400 HR column. Reaction of AaLS-EC with Sec2, under conditions identical to those used with AaLS-IC, substantially altered its size-exclusion chromatography elution profile (Figure 3A). The peak at 70 mL, corresponding to the 60-subunit capsid, was greatly diminished, while a new peak emerged in the void volume around 40 mL, indicating disulfide bond cross-linking between capsids to give much larger particles (Figure S3B). The negative control lacking Sec2 also showed the accumulation of earlier-eluting species, although to a lesser extent (Figure 3A), indicating that Sec2 increases the rate of disulfide cross-linking between AaLS-EC capsids. In contrast, the product of the reaction between AaLS-IC and Sec2 eluted identically to the unreacted capsid (Figure 2C). The formation of disulfide bonds in AaLS-EC was confirmed by SDS-PAGE (Figure 3B). After exposure to Sec2, AaLS-EC gave a band under non-reducing conditions whose mobility corresponded to that expected for a dimer, which disappeared under reducing conditions. In contrast, the reaction product of AaLS-IC and Sec2 did not show a significant dimer band under non-reducing conditions. Thus, the location of the reactive cysteine on the capsid has important consequences for the reaction with Sec2. To examine the importance of capsid assembly for the reaction with Sec2, we installed a unique cysteine at position 122 of a previously reported pentameric AaLS variant (containing the mutations R40E/H41E/I125H)38 by adding two additional point mutations, C37A and E122C, to give AaLS-IC-pent (Figure S3C). After incubation in buffer for 3 h, the size-exclusion chromatograpy profile of 9



Page 10 of 37

Figure 3. Structural effect of Sec2 treatment on AaLS variants with alternative cysteine placement and assembly state. A: Analytical size-exclusion chromatography of AaLS-EC (1 mg/mL) after incubation with either 330 µM Sec2 (red) or buffer only (black). Samples were incubated for 3 h at 37 °C in buffer (50 mM sodium phosphate, 200 mM NaCl, pH 8.0) prior to injection onto a Hiprep 16/60 Sephacryl S-400HR column. B: Reducing and non-reducing SDS-PAGE analysis of AaLS-EC-Sec, AaLS-EC, AaLS-IC-Sec, and AaLS-IC. Each sample was mixed with loading buffer, either with or without DTT, and incubated at 99 °C for 5 min prior to gel loading. C: Analytical size-exclusion chromatography of AaLS-IC-pent (1 mg/mL) after incubation with either 330 µM Sec2 (red) or buffer only (black). Samples were injected onto a Hiprep 16/60 Sephacryl S-300HR column, but otherwise handled as described for panel A. D: Reducing and non-reducing SDS-PAGE analysis of AaLS-IC-pent-Sec, AaLS-IC-pent, AaLS-IC-Sec, and AaLS-IC. Each sample was treated and analyzed as described for panel B. AaLS-IC-pent elution from a Sephacryl S-300 HR column (Figure 3C) showed a major peak at 70 mL, corresponding to a pentameric assembly, and a minor peak around 50 mL, presumably disulfide cross-linked dimers-of-pentamers resulting from partial air oxidation of the protein. In comparison, after incubation of AaLS-IC-pent in 10


Page 11 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


buffer containing Sec2 for 3 h, the 70 mL peak diminished, the 50 mL peak grew, and a shoulder appeared on the left side of the 50 mL peak (Figure 3C). This shift to earlier eluting species is consistent with the notion that Sec2 catalyzes disulfide bond formation between pentamers of AaLS-IC-pent (Figure S3D). Non-reducing SDS-PAGE analysis of the Sec2 reaction product also showed a band corresponding to a dimer of AaLS-IC-pent (Figure 3D). It therefore seems likely that the capsid assembly is necessary to prevent disulfide cross-linking in AaLS-IC. Taken together, these results demonstrate that the conjugation of Sec to a protein requires careful design in order to avoid heterogeneous product distribution. If the cysteine is exposed to the bulk environment, Sec2 will catalyze disulfide bond formation, giving cross-linked proteins. In AaLS-IC, the location of the cysteine inside the capsid prevents cross-linking between capsids, and the protein structure holds the interior cysteines apart from each other to prevent cross-linking within the capsid. The high yield of selenenylsulfide adduct depends on both a stable capsid structure and sequestration of the reactive cysteine within the capsid lumen.

Release of Sec. For AaLS-IC-Sec to be a viable delivery vehicle, it must be able to release Sec once it reaches its target location. In principle, encapsulated Sec could be released from AaLS-IC upon reduction of the selenenylsulfide bond. Glutathione is an abundant reducing agent in cells, especially in cancer cells where the concentration of reduced glutathione (GSH) in the cytoplasm is often in the range of 1 to 10 mM39-41. Therefore we examined the ability of GSH to release encapsulated Sec in vitro, using 1 mM GSH to simulate the condition of cancer cells at the low end 11



Page 12 of 37

of the cellular concentration range. After reaction of GSH and AaLS-IC-Sec, the small molecules were labeled with a coumarin dye (Figure S4), separated by HPLC, and the amount of Sec released was estimated by measuring the area under the peak corresponding to dye-labeled Sec (Figure S5). The resulting time course (Figure 4) showed that 50% of the Sec was released within 3 h and 90% was released within 10 h. Adding a second dose of GSH at 24 h after the initial addition of GSH did not stimulate any further release of Sec, confirming full release of Sec from the capsid. As an alternative to GSH, the ability of dithiothreitol (DTT) to release the encapsulated Sec was also tested, since DTT is a more powerful reductant. The reaction of DTT with AaLS-IC-Sec reached completion in about 10 h, slightly faster than GSH-triggered release of Sec. The observed release yields were consistent with the loading yields reported above. In contrast, a negative control with no added reducing agent resulted in no observable Sec release after 24 h. Thus, common reducing agents can trigger the complete release of encapsulated Sec in vitro with a half-time of a few hours.

Cytotoxic effect of AaLS-IC-Sec on various cell line models. Sec2 has been reported to exhibit high toxicity towards a broad range of cancer cells. As such, there is an interest in developing methodologies for targeted delivery of Sec. Thus, we investigated the potential of our engineered AaLS-IC-Sec capsid to deliver Sec and induce a cytotoxic response in several cell line models. After 72 h of exposure to either free Sec2 or the AaLS-IC-Sec capsid, cell viability was measured using the MTT assay for one non-cancerous cell line (mouse embryonic fibroblasts (MEFs)) 12


Page 13 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Timecourse of Sec release from AaLS-IC-Sec. The amount of free Sec in solution was measured at different times after mixing AaLS-IC-Sec (90 µM) in pH 8.0 buffer with either 1 mM GSH (black) or 0.5 mM DTT (red). A negative control lacking any reducing agent is shown in blue. The percent of Sec released from the capsid is reported based on the ratio of the mol free Sec at each time point to the total mol of Sec loaded into the capsid. and five cancerous cell lines of varying phenotype: HeLa (cervical cancer), PC3 (a model for aggressive prostate cancer), LNCaP (a model for indolent prostate cancer), HCT116 (colon cancer), and MCF7 (breast cancer). Free Sec2 was observed to be toxic to all six cell lines, confirming the general cellular cytotoxicity of this molecule. However, the sensitivity appeared to vary between cellular phenotypes as the IC50 values ranged from 2 - 20 µM, on a per selenium basis (Figure 5, Table S2). Intriguingly, delivery of encapsulated Sec resulted in a wider range of cellular sensitivity across the tested cell lines. HCT116 and HeLa cells were the most sensitized to AaLS-IC-Sec (Figure 5), yielding IC50 values similar to free Sec2 (2.9±1.1 µM and 4.1±1.2 µM, respectively). MCF7 and PC3 cells showed IC50 values of 38.8±1.3 µM and 20.8±1.2 µM, respectively, indicating that these cell lines were more moderately sensitized to encapsulated Sec than HCT116 and HeLa cells and 13



Page 14 of 37

Figure 5. MTT assay shows differing levels of cytotoxicity for Sec2, AaLS-IC-Sec, and AaLS-IC in six cell lines.

14


Page 15 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. MTT assay shows differing levels of cytotoxicity for Sec2, AaLS-IC-Sec, and AaLS-IC in six cell lines (continued from previous page). Cell viability is plotted against the concentration of the tested compound (free Sec2 (black), AaLS-IC-Sec (red), or unmodified AaLS- IC (blue)) for HCT116 (A), HeLa S3 (B), MCF7 (C), PC3 (D), LNCaP (E), and MEF (F) cell line models. The concentrations of tested compound ranged from 5.49 nM to 90 µM in HCT116, HeLa, LNCaP, and MEF cells, and from 5.49 nM to 147 µM in MCF7 and PC3 cells. Cell viability is reported as a percent value, relative to a control in which buffer only was added to the cells. G: Comparison of calculated IC50 values for Sec2 and AaLS-IC-Sec treatments of each cell line. exhibited an ~2-fold decrease in sensitivity relative to free Sec2 (Figure 5 and Table S2). In contrast, neither LNCaP nor MEFs showed a significant response to AaLS-IC-Sec, even though both cell lines demonstrated a cytotoxic response for free Sec2 (Figure 5). The lack of cytotoxic response in these two cells lines after AaLS-IC-Sec exposure may indicate that the capsid was unable to penetrate these cells, or possibly that the encapsulated Sec was degraded in these cellular environments. Notably, no significant toxicity for the AaLS-IC capsid alone was observed for any of the cell lines, indicating that the capsid itself was not toxic and, where encountered, the cytotoxic response to AaLS-IC-Sec was directly related to targeted Sec delivery. Combined, these findings suggest that the engineered AaLS-IC capsid may be an effective tool for selective delivery of Sec to a number of cancerous cell types. The additional observation that cytotoxic response is cell phenotype dependent, may also afford tunability in capsid drug delivery design.

Cytotoxic response to AaLS-IC-Sec correlates with cellular penetrance and localization. To further investigate the observed differences in cytotoxic response to AaLS-IC-Sec across the six cell lines, confocal fluorescence microscopy was utilized

15



Page 16 of 37

to simultaneously evaluate the level of cell penetrance as well as discern any potential variances in cellular localization (Figure 6). For these studies, each cell line was independently treated with either buffer alone or buffer containing AaLS-IC capsids labeled with 5-iodoacetamidofluorescein (AaLS-IC-5-IAF) to facilitate direct capsid monitoring. AaLS-IC-5-IAF encapsulates the dye via a stable thioether linkage with the engineered cysteine. Dye-labeling does not change the size of the capsid (Figure S6A), and the dye-labeled capsid remains stable in cell culture media for 72 h (Figure 6B and 6C). Thus, the toxic effects observed for AaLS-IC-Sec likely result from the uptake of intact capsids by the cells. The buffer-treated controls for all cell lines exhibited little to no green fluorescent signal, with any residual signal most likely representing cellular autofluorescence. The ability of AaLS-IC-5-IAF to penetrate the cells and differentially localize to the cytoplasm alone or cytoplasm and nucleus correlated with the cytotoxic response of each cell line (Figure 5 and Table S2). Intriguingly, HCT116 and HeLa cells, which exhibited the highest sensitivity to AaLS-IC-Sec, showed significant nuclear presence relative to the cytoplasm after treatment with AaLS-IC-5-IAF. MCF7 and PC3 cells treated with AaLS-IC-5-IAF appeared to have a notable cytoplasmic presence, which appears to coincide with their decreased sensitivity to AaLS-IC-Sec. Relative to MCF7, more nuclear localization of the fluorescent signal was observed in PC3 cells, which again is consistent with the moderately increased sensitivity to AaLS-IC-Sec for these cells. Finally, very little green fluorescence was observed for both LNCaP and MEF cells, correlating with the lack of response from these cells to AaLS-IC-Sec 16


Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Cell penetrance and localization of AaLS-IC-5-IAF differs between cellular phenotype in a manner that correlates with cytotoxic response to AaLS-IC-Sec. Representative confocal fluorescence microscopy images for the five cancerous cell lines (HCT116, HeLa, MCF7, PC3, LNCaP) and noncancerous MEFs after incubation with either S400 buffer (control) or AaLS-IC-5-IAF (treatment). CellTracker Orange CMRA dye (red) was used to stain the cell body. Each image represents one of several z-stacks that was selected for presentation based on the clarity of cellular structures and AaLS-IC-5-IAF localization (viewed using the NIS-Elements Viewer from Nikon).

treatment, and suggesting that the capsid does not enter these cells efficiently. Together, these data suggest that overall cytotoxic sensitivity to AaLS-IC-Sec is dependent on the ability of the capsid to penetrate the cell and have an increased localization within the nucleus. DISCUSSION Here we report the reversible loading of a versatile protein capsid with Sec, as well as demonstrate delivery of this potential cancer therapy agent to a number of cancer cell line models. The reactivity provided by selenium allows simple, one-step 17



Page 18 of 37

loading of Sec2 into the AaLS-IC capsid. The previously reported process for loading thiol-bearing molecules, such as cur-SH, into AaLS-IC required activation of the cysteine in the capsid with DTNB prior to conjugation of the guest thiol25. Elimination of the DTNB activation step allowed the loading yield to increase somewhat (~48 Sec per capsid vs. ~42 cur-SH per capsid), as the yield for thiol guests is limited by the yield of the reaction between DTNB and AaLS-IC. The basis for this difference in encapsulation yield is not clear, but could perhaps stem from different amounts of steric hindrance in the late stages of the reaction of AaLS-IC with Sec2 vs. DTNB. Additionally, the rapid air oxidation of the Sec (selenol) by-product to regenerate Sec2 (diselenide) is a key aspect of the capsid loading strategy reported here (Figure 1C), as it provides a means to achieve a high yield of conjugation, despite the lower thermodynamic stability of the selenenylsulfide product relative to the diselenide starting material. While it is difficult to compare the release kinetics quantitatively, given the different reducing agents and solution conditions used, release proceeds fairly quickly for both Sec and Cur-SH guests. Half of the encapsulated cur-SH was released by 5 mM TCEP in less than one hour25, and 1 mM GSH can release 50% of the encapsulated Sec in about three hours. The rate of release may benefit from the relatively low pKa value of 5.2 for the selenol42, which should make Sec a good leaving group. Thermodynamically, it should be more difficult to release Sec than cur-SH, given that selenenylsulfide bonds are more stable than disulfide bonds43-45. However, Sec release by GSH is nearly quantitative (Figure 4), whereas GSH was 18


Page 19 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


unable to release cur-SH from AaLS-IC. As with loading into the capsid, air oxidation of released Sec likely provides a strong energetic driving force for release from AaLS-IC. Additionally, released Sec accumulates to a higher extent in bulk solution than released cur-SH25, since it is not prone to degradation by the reducing agent. While the reactivity of selenium can benefit loading and release, it needs to be appropriately harnessed

to avoid additional, undesired consequences. The

selenenylsulfide bond that results from attachment of Sec to a protein is kinetically labile. Displacement of Sec by a free Cys on a second protein molecule will cause a disulfide bond to form (Figure S3). The liberated Sec will rapidly oxidize to form a thermodynamically stable diselenide, further favoring disulfide crosslinking. Therefore the site for Sec attachment must be carefully chosen. Indeed, Sec2 will induce the formation of large aggregates with AaLS-EC, in which the reactive Cys is placed on the exterior of the AaLS capsid, or with AaLS-IC-pent, which assembles as pentamers rather than a capsid (Figure 3). Reaction of AaLS-IC with Sec2 avoids such cross-linking because the placement of the reactive cysteine on the interior surface precludes reaction with a second capsid and the stable folded structure prevents disulfide bond formation within the capsid. Thus, the capsid architecture and the position of the reactive cysteine within the capsid are both crucial for the ability of AaLS-IC to function as a nanocarrier for this guest. The usefulness of AaLS-IC as a nanocarrier for drug delivery also depends on the ability of the capsid to enter cells and release the guest in an active form. Since free Sec2 is generally toxic, cell viability represents a useful reporter for Sec delivery. 19



Page 20 of 37

While all cell lines investigated showed cytotoxic sensitivity to free Sec2, exposure to AaLS-IC-Sec showed a surprising degree of cell-type specificity in its toxic effects. In general, the six cell lines can be roughly classified as being highly sensitive, moderately sensitive, or insensitive to AaLS-IC-Sec. Differences in the efficiency of cellular uptake and intracellular trafficking of AaLS-IC-Sec may be the primary contributing factors for the observed differences in cytotoxic response across cell types. It is notable that capsid uptake was very limited in normal cells, but efficient in the majority of cancer cell line models evaluated, suggesting that specific disease cell targeting may be achievable. Based on the observation that the cell lines with the highest sensitivity to AaLS-IC also display greater fluorescence in the nucleus upon treatment with AaLS-IC-5-IAF, it is tempting to speculate that nuclear localization of the capsid makes AaLS-IC-Sec more toxic. In principle, the AaLS capsid, with a diameter of 16 nm, should be able to gain access to the nucleus via the nuclear pore complex, which can transport nanoparticles with diameters up to 39 nm46, although it may be too wide to enter the nucleus by passive diffusion through the nuclear pore47. Alternatively, disassembly or partial degradation of the capsid prior to the entry of AaLS-IC-5-IAF into the nucleus cannot be ruled out. Further, the extent to which Sec release precedes the entry of AaLS-IC-Sec into the nucleus is not known. Understanding the basis for the observed variance in cell-type specific sensitivity to AaLS-IC-Sec will require further investigation into the mechanism of capsid entry into the cell, the intracellular trafficking pathway, and the timing and location of intracellular Sec release. 20


Page 21 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


While the mechanism of Sec toxicity remains unclear and is likely complex, recent studies provide further hints that nuclear accumulation of AaLS-IC-Sec could boost its toxicity. For instance, the nuclear concentration of GSH can range up to four times higher than that of the cytoplasm, depending on cell cycle phase48-50, which might afford more efficient Sec release within the nucleus. Sec released in the nucleus could then directly deplete GSH by catalyzing thiol oxidation51, and depletion of nuclear GSH is known to interfere with cellular proliferation50. Five selenoproteins (selenoprotein H, methionine-R-sulfoxide reductase 1, glutathione peroxidase-4, thioredoxin reductase-1, and thioredoxin glutathione reductase) are known to reside in the nucleus52. Thioredoxin reductase-1 is an essential enzyme that is overexpressed in many tumor cells, and Sec2 has been variously reported to be a substrate53 or inhibitor4 of this enzyme. Either way, interaction of Sec with thioredoxin reductase-1 could potentially contribute to the mechanism of toxicity by disrupting the intracellular redox status. Interaction of Sec with some (or all) of the nuclear selenoproteins, possibly via diselenide bond formation, could help to throw off the redox balance in the nucleus and thereby induce lethal damage. In line with this notion, selenoprotein H is known to be overexpressed in HCT116 cells52, which in our studies were shown to be highly sensitive to AaLS-IC-Sec and exhibit an increased localization of AaLS-IC-5-IAF in the nucleus. It is further possible that inhibition of the cytoplasmic versions of these selenoproteins by localized Sec release might lead to a less potent response. Alternatively, the mechanism of toxicity by AaLS-IC-Sec in cell lines where cytoplasmic localization predominates might involve diffusion of 21



Page 22 of 37

released Sec into the nucleus or another subcellular compartment, resulting in an effective molecular dilution. A better understanding of the differential cellular toxicity mechanism(s) will be essential for informing additional efforts to further engineer the AaLS capsid for targeted Sec delivery.

CONCLUSIONS Here we report the development of a protein capsid nanocarrier for efficient cellular delivery of Sec. The design of the AaLS-IC capsid affords facile loading by simply mixing the reduced protein with Sec2 in buffer under ambient conditions. Efficient capsid loading depends on the location of the reactive cysteine along the interior surface of the capsid to prevent catalysis of disulfide crosslinking between proteins by Sec2. GSH can release the encapsulated Sec on a time-scale of several hours. This property should be useful for the triggered release of Sec in cancer cells, which tend to have higher levels of GSH, compared to healthy cells. Indeed, AaLS-IC-Sec was observably cytotoxic to four out of five human cancer cell lines tested and was not toxic to a normal mouse cell line. Intriguingly, while all cell lines tested were sensitive to µM levels of free Sec2, their response to AaLS-IC-Sec was highly variable. The findings reported here suggest that the variance in cytotoxic response across cell lines is correlated with the efficiency of cellular uptake of AaLS-IC-Sec and increased trafficking to the nucleus. In sum, we demonstrate the first engineered protein capsid capable of selective and efficient cellular delivery of toxic Sec to a number of cancerous cell line models.

22


Page 23 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL PROCEDURES Materials. L-Selenocystine (Sec2, 98% pure) was purchased from Acros Organics.

5,5'-Dithiobis-(2-nitrobenzoic

acid)

(DTNB,

99%

pure),

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98% pure) were from Sigma-Aldrich (St. Louis, MO, USA). Dithiothreitol (DTT, 99.5% pure) and reduced glutathione (GSH, >98% pure) were from Solarbio (Beijing, China). Tris(2-carboxyethyl)phosphine hydrochloride was from Adamas-beta (Shanghai, China).

Iodoacetamide

(IAA)

was

from

Biosharp

(Beijing,

China).

4-Bromomethyl-7-methoxycoumarin (Br-Mmc) was from TCI (Tokyo, Japan). 5-(Iodoacetamido)fluorescein

(5-IAF)

was

purchased

from

Marker

Gene

Technologies (Eugene, OR, USA). E. coli strains BL21 (DE3) and DH5α were purchased from Baibei (Tianjin, China). LNCaP, PC3, HCT116 and MCF7 cells were from ATCC; HeLa S3 and MEFs were a generous gift from the laboratory of Dr. Cynthia Burrows (University of Utah). DMEM, MEM, F-12K, RPMI-1640 media were from VWR (USA). Fetal bovine serum (FBS), 4',6-diamidino-2-phenylindole (DAPI), and CellTracker Orange CMRA Dye were purchased from Thermo Fisher Scientific (USA). All other chemicals and reagents were from BBI Life Sciences (Shanghai, China), Concord Technology (Tianjin, China), Dingguo (Beijing, China), Genview (Florida, USA), OXOID (Hampshire, UK), Sigma-Aldrich (St. Louis, MO, USA), Solarbio (Beijing, China), TIANGEN (Tianjin, China), Tianjin Da Mao (Tianjin, China), or Yuanli Huagong (Tianjin, China). LC/MS analyses were performed using a 1260 infinity 6420 triple quad LC/MS 23



Page 24 of 37

from Agilent Technologies (Santa Clara, USA). Protein purifications were carried out using either an ÄKTA pure or ÄKTA Prime Plus fast protein liquid chromatography (FPLC) instrument. UV-Vis spectroscopy was performed with either a TECAN Infinite M200 Pro plate reader (TECAN, Männedorf, Switzerland), Eppendorf BioPhotometer

(Eppendorf,

Hamburg,

Germany),

or

a

Hitachi

U-3900

spectrophotometer (Hitachi High-Tech Science Corporation, Tokyo, Japan). Fluorescence images were obtained from Nikon A1 Confocal Microscope (Nikon, Tokyo, Japan). Site-Directed Mutagenesis. A plasmid encoding AaLS-EC was generated by site-directed mutagenesis, using the plasmid pMG-AaLS-C37A25 as a template. The primers

GT_E70CF

and

GT_E70CR

(5'-GGGTGAACTGGCGCGTAAATGCGACATTGATGCTGTTATCG-3'

and

5'-CGATAACAGCATCAATGTCGCATTTACGCGCCAGTTCACCC-3', respectively, mutations indicated in bold) were used to introduced the E70C mutation, yielding the plasmid pMG-AaLS-EC. The mutagenesis procedure was carried out as previously

described.

A

plasmid

encoding

C37A/R40E/H41E/E122C/I125H

(AaLS-IC-pent) was generated by a similar procedure, using the plasmid pMG-AaLS-R40E/H41E/I125H38 as a template. The primers GT_C37A2F and GT_C37A2R (5'-GTGGAGGGTGCAATTGATGCCATAGTCGAGGAGGGCGGC-3' 5'-GCCGCCCTCCTCGACTATGGCATCAATTGCACCCTCCAC-3',

and respectively)

were used to introduce the C37A mutation; the primers GT_E122C2F and 24


Page 25 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GT_E122C2R (5'-GTTATTACAGCTGACACCTTGTGCCAGGCTCATGAGCGCGCCGGC-3' and 5'-GCCGGCGCGCTCATGAGCCTGGCACAAGGTGTCAGCTGTAATAAC-3', respectively) were used to introduced the E122C mutation, yielding the plasmid pMG-AaLS-IC-pent. The coding portions of plasmids pMG-AaLS-EC and pMG-AaLS-IC-pent were confirmed by DNA sequencing. Protein Production and Purification of AaLS variants. AaLS-IC and AaLS-EC were produced and purified using a previous published procedure25. AaLS-IC-pent was also produced and purified as previously described with the exception that the final purification step utilized a HiPrep 16/60 Sephacryl S-300 HR size-exclusion column instead of a HiPrep 16/60 Sephacryl S-400 HR column. Protein concentrations were determined using either the BCA assay or the Bradford assay, essentially as described54. Molar concentrations of protein solutions are reported on a monomer basis, unless otherwise specified. Reaction of Sec2 with AaLS variants. Sec was loaded into capsids by incubating reduced AaLS-IC with Sec2. A stock solution of Sec2 (1 mM) was prepared by dissolving the powder (10 mg) in 30 mL of S400 buffer (50 mM sodium phosphate, 200 mM NaCl, pH 8.0). The concentration of Sec2 was confirmed by NTSB assay37. AaLS-IC-Sec was then prepared by the room-temperature addition of Sec2 (710 µL of a 1 mM stock solution) dropwise with stirring to AaLS-IC (1 mL of a 117 µM solution in S400 buffer). The reaction mixture (1.71 mL) was then incubated at 37 °C for 3 h. Unreacted Sec2 was removed by four rounds of dilution with S400 buffer and 25



Page 26 of 37

concentration in Vivaspin 20 (30,000 MWCO) ultrafiltration units (Sartorius Stedim Biotech GmbH, Goettingen, Germany), by size-exclusion chromatography, or dialysis at 4 °C. Both unreacted AaLS-IC and the product were compared by electrospray mass spectrometry using a Waters LCT XE Premier TOF mass spectrometer. The mass ([M+H]+) calculated for unreacted AaLS-IC was 16890 and the observed mass was 16891. The mass ([M+H]+) calculated for AaLS-IC-Sec was 17056 and the observed mass was 17055. The standard loading procedure described above was based on investigations into how reactant stoichiometry and reaction time influence the yield of AaLS-IC-Sec. Reactant stoichiometry was varied by adding different amounts of Sec2 (63 nmol, 126 nmol, 251 nmol, 377 nmol, 502 nmol, or 628 nmol) to AaLS-IC (59 nmol monomers) in S400 buffer. The mixtures were incubated at 37 °C for 4 h. The reaction timecourse was analyzed by a similar procedure at final concentrations of 59 µM and 377 µM for AaLS-IC and Sec2, respectively. At various times after initiation (5 min, 30 min, 60 min, 120 min, 180 min, and 240 min), the reaction was diluted 15-fold with S-400 buffer, and the free Sec2 was removed by four rounds of ultrafiltration and re-dilution. Determination of Sec content in AaLS-IC-Sec. The extent of reaction between AaLS-IC and Sec was determined using two different colorimetric assays, Ellman’s assay36 and the NTSB assay37. Prior to both assays, unreacted small molecules were removed from the protein by ultrafiltration. In Ellman’s assay, the amount of free protein thiol groups was quantitated by measuring the amount of 5-nitrothiobenzoate (NTB, ε412

nm

= 14,150 M-1cm-1)55 26


Page 27 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


formed upon addition of DTNB. Briefly, either AaLS-IC or AaLS-IC-Sec (0.5 mL of a ~1.8 mg/mL stock solution in S400 buffer) was combined with DTNB (1 mL of a 0.5 mM solution in buffer containing 200 mM Tris-HCl and 3 mM EDTA at pH 9.5). Following a 20 min incubation at room temperature, the absorbance at 412 nm was recorded to determine the concentration of reactive thiol groups. The number of Sec per capsid was estimated from the difference in (mol DTNB-reactive thiol groups)/(mol capsid) between AaLS-IC and AaLS-IC-Sec. The NTSB assay was employed to more directly measure the number of selenenylsulfide bonds in AaLS-IC-Sec. Each selenenylsulfide bond should generate one molecule of free NTB in the NTSB assay. NTSB was prepared by reacting DTNB and sodium sulfite under an oxygen balloon for ~8 h, according to a published procedure37. Any free thiol groups in AaLS-IC-Sec were capped using IAA by mixing 1 mg/mL AaLS-IC-Sec (1 mL) and 500 mM IAA (1 mL). After incubation for 3 h at room temperature, the solution volume was decreased to 0.5 mL by ultrafiltration and the IAA-treated AaLS-IC-Sec was added to 1 mL NTSB assay buffer (0.5 mM NTSB, 0.1 M sodium sulfite, 0.2 M Tris-HCl, 3 mM EDTA, pH 9.5). Following a 20 min incubation on ice, the absorbance at 412 nm was measured to determine the concentration of free NTB produced. The number of Sec per capsid was estimated from [NTB]/[capsid]. As controls, the NTSB assay was also performed for AaLS-IC, with and without IAA treatment. SDS-PAGE analysis. To check for the presence of disulfide bonds between subunits of AaLS-IC, AaLS-IC-Sec, AaLS-EC, AaLS-EC-Sec, AaLS-IC-pent, or 27



Page 28 of 37

AaLS-IC-pent-Sec, the mobility of the protein (20 µg) during SDS-PAGE was examined under non-reducing and reducing conditions (0 mM and 20 mM DTT, respectively). Samples were incubated at 99 °C for 5 min prior to SDS-PAGE analysis with a 15% polyacrylamide resolving gel. The gel was run for 1 h at 250 V, and then visualized after Coomassie Blue R-250 staining. Release of Sec. To examine the ability of a small thiol to trigger release of Sec from the capsid, AaLS-IC-Sec was incubated with GSH. Briefly, GSH (13 µL of a 80 mM stock solution in S400 buffer) was added to AaLS-IC-Sec (1 mL of a 1.6 mg/mL stock solution in S400 buffer) and incubated at room temperature. The final concentrations of AaLS-IC-Sec and GSH were 92 µM (protein monomers) and 1 mM, respectively. At different times (10 min, 60 min, 120 min, 180 min, 240 min, 360 min, 480 min, 600 min, and 1440 min), an aliquot (0.1 mL) was removed from the reaction and the small molecules were separated from the protein by ultrafiltration at 13,622 g for 5 min using a Sartorius Vivaspin 500 spin concentrator (30,000 MWCO). To reduce the sample, TCEP (2 µL of a 100 mM stock solution in S400 buffer) was combined with a portion (50 µL) of the flow-through from the spin concentrator. Exactly 10 min after the addition of TCEP, Br-Mmc (150 µL of a 2.67 mM stock solution dissolved in acetone) was added and the sample was incubated for an additional 20 min. After filtration through a 0.22 µm nylon membrane, each time point was injected onto a reverse-phase HPLC-MS system equipped with a 4.6 mm x 150 mm BioBasic-18 column with a 5 µm particle size and a 300 Å pore size (Thermo Scientific, USA). The HPLC running conditions were 40 °C column 28


Page 29 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temperature, 1 mL/min flowrate, and 328 nm detection wavelength. The elution procedure involved a gradient from 5:95:0.1 (acetonitrile:water:formic acid) to 30:70:0.1 (acetonitrile:water:formic acid) from 0 min to 32 min followed by 100:0:0.1 (acetonitrile:water:formic acid) for an additional 8 min. The concentration of free Sec was calculated based on peak area and a calibration curve constructed via LC-MS analysis of Sec2/TCEP/Br-Mmc reactions carried out using various concentrations of Sec2. The Sec release reaction was also performed using AaLS-IC-Sec (88 µM monomers) and DTT (0.5 mM) in place of GSH. As a negative control, AaLS-IC-Sec (88 µM) in the absence of DTT or GSH was subjected to the same treatment as described above. The GSH- and DTT-triggered Sec release reactions were each performed in triplicate. MTT assay. Cell lines were cultured at 37 °C under 5% CO2 in DMEM (HeLa and MEF), EMEM (HCT116 and MCF7), F-12K (PC3) or RPMI (LNCaP) media; each of which was supplemented with 10% FBS. Cell counting was performed on a Countess Automated Cell Counter (Thermo Fisher Scientific). The cytotoxicity of AaLS-IC, AaLS-IC-Sec and free Sec2 for all six cell lines was examined by MTT assay. Upon reaching 80-90% confluency, the cells were diluted with fresh media, seeded into a 96-well plate (4000 cells/well for HeLa, MCF7, PC3, LNCaP, and MEFs, 2000 cells/well for HCT116) and incubated for 24 h. Varying amounts of AaLS-IC-Sec, AaLS-IC, or free Sec2 was then added to cells, with the final drug concentrations ranging from 5 nM to 90 µM for all cells except MCF7 and PC3, 29



Page 30 of 37

where the maximum final AaLS-IC-Sec concentration was 147 µM. After 72 h, the media was replaced with fresh media containing 0.5 mg/mL MTT, except for LNCaP cells where PBS containing MTT was used in place of media, and incubated for 4 h. The MTT solution was then replaced with 100 µL DMSO to dissolve formazan crystals formed by metabolically active living cells. Absorbance values at 490 nm were measured using a plate reader. The percentage of viable cells was calculated from the ratio of ∆A490 (between the sample and a blank with no cells and no drug) to ∆A490,

reference

(between a control with no drug and the blank). At each drug

concentration, the viability was measured 6 to 18 times, using 1 to 3 independently prepared cell cultures. To determine IC50 values, the average percentage of viable cells was plotted against the corresponding concentration of AaLS-IC-Sec or Sec2 (on a per selenium basis) and the data fit to a sigmoidal equation using GraphPad Prism 6.0. Cell imaging. For confocal fluorescent microscopy, ~16,000 cells were cultured on chambered cover-glass overnight prior to treatment with either S400 buffer, 90 µM AaLS-IC-5-IAF (MCF7, PC3, LNCaP, and MEFs), or 70 µM AaLS-IC-5-IAF (HeLa and HCT116). AaLS-IC-5-IAF was prepared by adding AaLS-IC (1 mL of a 249 µM stock solution) into 2.5 mM 5-IAF (996 µL) and incubating the mixture for 30 min in the dark at room temperature. Fluorescein-labeled protein was obtained by removing unreacted 5-IAF with a 30,000 MWCO spin concentrator until the flow-through lacked detectable fluorescence. Cells were treated with AaLS-IC-5-IAF for 8 h, washed with 1x PBS, and fixed with 4% 30


Page 31 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


formaldehyde in PBS for 4 min. After fixation, cells were washed with 1x PBS, and stained with DAPI (nuclear stain) and 10 µM CellTracker Orange CMRA dye in serum-free media for 30 min prior to coating with mounting medium. Z-stack images were captured using a Nikon A1 confocal microscope and analyzed with the NIS Elements Viewer software (Nikon). Stability of AaLS-IC-5-IAF in cell culture media. To examine whether the cell culture media conditions cause the capsid to disassemble or degrade, AaLS-IC-5-IAF was incubated in cell culture media and then analyzed by size-exclusion chromatography and SDS-PAGE. Briefly, DMEM containing 10% FBS was conditioned using HeLa cells, 1% penicillin, and 1% streptomycin for 24 h at 37 °C. The conditioned media (1.9 mL) was then removed from the cells and transferred to a clean 2 mL vial. AaLS-IC-5-IAF (100 µL of a 1900 µM stock solution) was added to the DMEM and the mixture (containing AaLS-IC-5-IAF at a final monomer concentration of 94 µM) was incubated in the dark at 37 °C for 76 h. The resulting solution was then dialyzed against 2 L of S400 buffer for 1 day, filtered, and injected onto the HiPrep 16/60 Sephacryl S-400 HR column. The fluorescence of each fractions was measured using a TECAN Infinite M200 Pro plate reader. As a control, the stock solution of AaLS-IC-5-IAF was also analyzed using the same size-exclusion chromatography procedure. Interesting fractions from the size-exclusion column were analyzed by SDS-PAGE, along with samples of conditioned DMEM containing 10% FBS, before and after incubation with AaLS-IC-5-IAF.

31



Page 32 of 37

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Determination of loading yield for AaLS-IC-Sec; Mass spectra of AaLS-IC and AaLS-IC-Sec; TEM images of AaLS-IC and AaLS-IC-Sec; Reactions schemes for AaLS-EC and AaLS-IC-pent; Reaction scheme for dye labeling of Sec released from AaLS-IC-Sec; LC-MS data for quantitation of Sec released from AaLS-IC-Sec; Table of IC50 values for Sec2 and AaLS-IC-Sec.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China grant # 31670764 (KJW and SW) and the Department of Chemistry, University of Utah (BAB-K and AA-S). We thank Dr. Hsiao-Nung Chen for assistance with mass spectrometry (University of Utah, Department of Chemistry Mass Spectrometry Core Facility). Sequencing was performed at the DNA Sequencing Core Facility, University of Utah. Imaging was performed at the Fluorescence Microscopy Core Facility, a part of the Health Sciences Cores at the University of Utah. Microscopy equipment was obtained using a NCRR Shared Equipment Grant #1S10RR024761-01. Electron microscopy was performed with the help of Dr. David Belnap at the University of Utah Electron Microscopy Core Laboratory.

NOTES 32


Page 33 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The authors declare no competing financial interest.

ABBREVIATIONS AaLS, Aquifex aeolicus Lumazine Synthase; AaLS-EC, C37A/E70C-AaLS; AaLS-IC, C37A/E122C-AaLS;

AaLS-IC-5-IAF,

thioether

conjugate

of

AaLS-IC

and

5-iodoacetamidofluorescein; AaLS-IC-pent, C37A/R40S/H41S/E122C/I125H-AaLS; AaLS-IC-Sec,

selenenylsulfide

conjugate

of

AaLS-IC

and

Sec;

DTNB,

5,5′-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, reduced glutathione; IAA, iodoacetamide; NTSB, 2-nitro-5-thiosulfobenzoate; Sec, selenocysteine; Sec2, selenocystine.

33



Page 34 of 37

References (1)

Batist, G., Katki, A. G., Klecker, J., R.W., and Myers, C. E. (1986) Selenium-induced cytotoxicity of human leukemia cells: interactions with reduced glutathione. Cancer Res. 46, 5482-5485.

(2)

Beld, J., Woycechowsky, K. J., and Hilvert, D. (2010) Small-molecule diselenides catalyze oxidative protein folding in vivo. ACS Chem. Biol. 5, 177-182.

(3)

Chen, T., and Wong, Y.-S. (2009) Selenocystine induces reactive oxygen species-mediated

(4)

Fan, C.-d., Fu, X.-y., Zhang, Z.-y., Cao, M.-z., Sun, J.-y., Yang, M.-f., Fu, X.-t., Zhao, S.-j., Shao,

apoptosis in human cancer cells. Biomed. Pharmacother. 63, 105-113. L.-r., Zhang, H.-f., et al. (2017) Selenocysteine induces apoptosis in human glioma cells: evidence for TrxR1-targeted inhibition and signaling crosstalk. Sci. Rep. 7, 6465. (5)

Hoefig, C. S., Renko, K., Köhrle, J., Birringer, M., and Schomburg, L. (2011) Comparison of different selenocompounds with respect to nutritional value vs. toxicity using liver cells in culture. J. Nutr. Biochem. 22, 945-955.

(6)

Misra, S., Boylan, M., Selvam, A., Spallholz, J. E., and Björnstedt, M. (2015) Redox-active selenium compounds - from toxicity and cell death to cancer treatment. Nutrients 7, 3536-3556.

(7)

Short, M. D., Xie, Y., Li, L., Cassidy, P. B., and Roberts, J. C. (2003) Characteristics of selenazolidine prodrugs of selenocysteine: toxicity and glutathione peroxidase induction in V79 cells. J. Med. Chem. 46, 3308-3313.

(8)

Chen, T., and Wong, Y. S. (2008) Selenocystine induces apoptosis of A375 human melanoma cells by activating ROS-mediated mitochondrial pathway and p53 phosphorylation. Cell. Mol. Life Sci. 65, 2763-2775.

(9)

Zorn, M., Ihling, C. H., Golbik, R., Sawers, R. G., and Sinz, A. (2013) Selective selC-independent selenocysteine incorporation into formate dehydrogenases. PLoS ONE 8, e61913.

(10)

Esaki, N., Nakamura, T., Tanaka, H., and Soda, K. (1982) Selenocysteine lyase, a novel enzyme that specifically acts on selenocysteine. J. Biol. Chem. 257, 4386-4391.

(11)

Lazard, M., Dauplais, M., Blanquet, S., and Plateau, P. (2017) Recent advances in the

(12)

He, L., Lai, H., and Chen, T. (2015) Dual-function nanosystem for synergistic cancer

mechanism of selenoamino acids toxicity in eukaryotic cells. BioMol Concepts 8, 93-104. chem-/radiotherapy through ROS-mediated signaling pathways. Biomaterials 51, 30-42. (13)

Bharti, C., Nagaich, U., Pal, A. K., and Gulati, N. (2015) Mesoporous silica nanoparticles in target drug delivery system: A review. Int. J. Pharm. Investig. 5, 124-133.

(14)

Schoonen, L., and Van Hest, J. C. M. (2014) Functionalization of protein-based nanocages for

(15)

Ahn, B., Lee, S.-G., Yoon, H. R., Lee, J. M., Oh, H. J., Kim, H. M., and Jung, Y. (2018) Four-fold

drug delivery applications. Nanoscale 6, 7124-7141. channel-nicked human ferritin nanocages for active drug loading and pH-responsive drug release. Angew. Chem. Int. Ed. 10.1002/anie.201800516. (16)

ElSohly, A. M., Netirojjanakul, C., Aanei, I. L., Jager, A., Bendall, S. C., Farkas, M. E., Nolan, G. P., and Francis, M. B. (2015) Synthetically modified viral capsids as versatile carriers for use in antibody-based cell targeting. Bioconjug. Chem. 26, 1590-1596.

(17)

Flenniken, M. L., Liepold, L. O., Crowley, B. E., Willits, D. A., Young, M. J., and Douglas, T. (2005) Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture. Chem. Commun., 447-449.

34


Page 35 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18)

Fu, Y., and Li, J. (2016) A novel delivery platform based on Bacteriophage MS2 virus-like

(19)

Galaway, F. A., and Stockley, P. G. (2013) MS2 viruslike particles: a robust, semisynthetic

particles. Virus Res. 211, 9-16. targeted drug delivery platform. Mol. Pharm. 10, 59-68. (20)

Ma, Y., Nolte, R. J., and Cornelissen, J. J. (2012) Virus-based nanocarriers for drug delivery. Adv. Drug. Deliv. Rev. 64, 811-825.

(21)

Masarapu, H., Patel, B. K., Chariou, P. L., Hu, H., Gulati, N. M., Carpenter, B. L., Ghiladi, R. A., Shukla, S., and Steinmetz, N. F. (2017) Physalis mottle virus-like particles as nanocarriers for imaging reagents and drugs. Biomacromol. 18, 4141-4153.

(22)

Pan, Y., Zhang, Y., Jia, T., Zhang, K., Li, J., and Wang, L. (2012) Development of a microRNA

(23)

Rhee, J.-K., Baksh, M., Nycholat, C., Paulson, J. C., Kitagishi, H., and Finn, M. G. (2012)

delivery system based on bacteriophage MS2 virus-like particles. FEBS J. 279, 1198-1208. Glycan-targeted virus-like nanoparticles for photodynamic therapy. Biomacromol. 13, 2333-2338. (24)

Zhang, X., Meining, W., Fischer, M., Bacher, A., and Ladenstein, R. (2001) X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 Å resolution: determinants of thermostability revealed from structural comparisons. J. Mol. Biol. 306, 1099-1114.

(25)

Guo, Q., Thomas, G. C., and Woycechowsky, K. J. (2017) Reversible loading of thiol-modified

(26)

Azuma, Y., Edwardson, T. G. W., Terasaka, N., and Hilvert, D. (2018) Modular protein cages for

curcumin in an engineered protein capsid. RSC Advances 7, 34676-34686. size-selective RNA packaging in vivo. J. Am. Chem. Soc. 140, 566-569. (27)

Lilavivat, S., Sardar, D., Jana, S., Thomas, G. C., and Woycechowsky, K. J. (2012) In vivo encapsulation of nucleic acids using an engineered non-viral protein capsid. J. Am. Chem. Soc. 134, 13152-13155.

(28)

Azuma, Y., Zschoche, R., and Hilvert, D. (2017) The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase. J. Biol. Chem. 292, 10321-10327.

(29)

Azuma, Y., Zschoche, R., Tinzl, M., and Hilvert, D. (2016) Quantitative packaging of active enzymes into a protein cage. Angew. Chem. Int. Ed. 55, 1531-1534.

(30)

Seebeck, F. P., Woycechowsky, K. J., Zhuang, W., Rabe, J. P., and Hilvert, D. (2006) A simple

(31)

Wörsdörfer, B., Woycechowsky, K. J., and Hilvert, D. (2011) Directed evolution of a protein

tagging system for protein encapsulation. J. Am. Chem. Soc. 128, 4516-4517. container. Science 331, 589-592. (32)

Min, J., Kim, S., Lee, J., and Kang, S. (2014) Lumazine synthase protein cage nanoparticles as modular delivery platforms for targeted drug delivery. RSC Adv. 4, 48596-48600.

(33)

Kim, H., Kang, Y. J., Min, J., Choi, H., and Kang, S. (2016) Development of a antibody-binding modular nanoplatform for antibody-guided targeted cell imaging and delivery. RSC Adv. 6, 19208-19213.

(34)

Singh, R., and Whitesides, G. M. (1991) Selenols catalyze interchange reactions of dithiols and

(35)

Beld, J., Woycechowsky, K. J., and Hilvert, D. (2008) Catalysis of oxidative protein folding by

disulfides in water. J. Org. Chem. 56, 6931-6933. small-molecule diselenides. Biochemistry 47, 6985-6987. (36)

Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77.

35



Page 36 of 37

(37)

Thannhauser, T. W., Konishi, Y., and Scheraga, H. A. (1983) Sensitive quantitative analysis of

(38)

Chen, H.-N., and Woycechowsky, K. J. (2012) Conversion of a dodecahedral protein capsid

disulfide bonds in polypeptides and proteins. Anal. Biochem. 138, 181-188. into pentamers via minimal point mutations. Biochemistry 51, 4704-4712. (39)

Meister, A. (1995) Glutathione metabolism. Methods Enzymol. 251, 3-7.

(40)

Balendiran, G. K., Dabur, R., and Fraser, D. (2004) The role of glutathione in cancer. Cell Biochem. Funct. 22, 343-352.

(41)

Hider, R. C., and Kong, X. L. (2011) Glutathione: a key component of the cytoplasmic labile iron pool. Biometals 24, 1179-1187.

(42)

Huber, R. E., and Criddle, R. S. (1967) Comparison of chemical properties of selenocysteine

(43)

Li, F., Lutz, P. B., Pepelyayeva, Y., Arnér, E. S. J., Bayse, C. A., and Rozovsky, S. (2014) Redox

and selenocystine with their sulfur analogs. Arch. Biochem. Biophys. 122, 164-173. active motifs in selenoproteins. Proc. Nat. Acad. Sci. U.S.A. 111, 6976-6981. (44)

Metanis, N., Keinan, E., and Dawson, P. E. (2006) Synthetic seleno-glutaredoxin 3 analogues are highly reducing oxidoreductases with enhanced catalytic efficiency. J. Am. Chem. Soc. 128, 16684-16691.

(45)

Steinmann, D., Nauser, T., and Koppenol, W. H. (2010) Selenium and sulfur in exchange reactions: a comparative study. J. Org. Chem. 75, 6696-6699.

(46)

Panté, N., and Kann, M. (2002) Nuclear pore complex is able to transport macromolecules

(47)

Paine, P. L., Moore, L. C., and Horowitz, S. B. (1975) Nuclear envelope permeability. Nature

with diameters of ~39 nm. Mol. Biol. Cell 13, 425-434. 254, 109-114. (48)

Bellomo, G., Vairetti, M., Stivala, L., Mirabelli, F., Richelmi, P., and Orrenius, S. (1992) Demonstration of nuclear compartmentalization of glutathione in hepatocytes. Proc. Nat. Acad. Sci. U.S.A. 89, 4412-4416.

(49)

Markovic, J., Borrás, C., Ortega, A., Sastre, J., Viña, J., and Pallardó, F. V. (2007) Glutathione is recruited into the nucleus in early phases of cell proliferation. J. Biol. Chem. 282, 20416-20424.

(50)

Markovic, J., García-Gimenez, J. L., Gimeno, A., Viña, J., and Pallardó, F. V. (2010) Role of glutathione in cell nucleus. Free Radic. Res. 44, 721-733.

(51)

Dickson, R. C., and Tappel, A. L. (1969) Reduction of selenocystine by cysteine or glutathione. Arch. Biochem. Biophys. 130, 547-550.

(52)

Zhang, X., Zhang, L., Zhu, J.-H., and Cheng, W.-H. (2016) Nuclear selenoproteins and genome maintenance. IUBMB Life 68, 5-12.

(53)

Björnstedt, M., Hamberg, M., Kumar, S., Xue, J., and Holmgren, A. (1995) Human thioredoxin reductase directly reduces lipid hydroperoxides by NADPH and selenocystine strongly stimulates the reaction via catalytically generated selenols. J. Biol. Chem. 270, 11761-11764.

(54)

Han, X., and Woycechowsky, K. J. (2017) Encapsulation and controlled release of protein guests by the Bacillus subtilis lumazine synthase capsid. Biochemistry 56, 6211-6220.

(55)

Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman's reagent. Methods Enzymol. 91, 49-60.

36


Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic

37


A Protein Capsid-Based System for Cell Delivery of ... - ACS Publications

Recommend Documents