Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a

Nov 30, 2017 - In order to both impart a greater degree of specificity to our design and address the need to develop a platform which would be modular...
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Communication Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release Joshua J. Santiana,† Binglin Sui,† Nicole Gomez, and Jessica L. Rouge* Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States S Supporting Information *

ABSTRACT: Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme−substrate recognition as a triggered release mechanism. Using photocatalyzed thiol−yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

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chemical photo-cross-linking strategy for incorporating peptide sequences into our nanocapsule design. Using thiol−yne chemistry, and thiolated peptides and nucleic acids, we can achieve rapid peptide incorporation that enables highly specific, enzyme-triggered degradation and cargo release. A major goal of embedding peptides into our nucleic acid nanocapsules (NANs) was to be able to utilize enzyme expression in a cell as a determining factor as to whether or not a cargo (i.e., cytotoxic drug or sensing dye) would be released. Many sensing applications attempt to determine the presence of enzymes expressed, which can vary dramatically during the course of a cell’s life, and differ depending on different disease states. With this in mind, we tested cathepsin B and MMP9, as they are well-studied and their peptide substrates are known.15,16 They are also both found to be upregulated in several cancer types,17,18 and therefore are involved in a variety of sensing applications,4,5 serving as the intended trigger for cargo or drug release. Importantly, MMP9 is located in the extracellular matrix (ECM) of cells, and functions optimally at physiological pH (pH 7),19 whereas cathepsin B is an endosomal protease that functions optimally at pH 5.16 As a control we compared the localization of these enzymeresponsive peptide cross-linked-nucleic acid nanocapsules (pep-NANs) to the activity of the previously studied estercross-linked NAN using electron microscopy. In doing so, the new hybrid peptide-NANs were found to have important advantages in controlling the release of cargo in cell specific

ontrolling the timing of cargo release from nanoparticle systems is of great importance when considering such materials as potential drug carriers,1−3 or for sensitive biosensing applications.4,5 Many research groups have focused on various chemical6,7 and biochemical8 stimuli to trigger the degradation of a nanoscale material to release small molecules such as drugs and dyes for therapeutic and diagnostic applications, respectively. Peptides in particular have been shown to be useful building blocks for self-assembling nanoscale delivery vehicles under biologically relevant conditions.9−11 Yet, although individual peptide sequences have shown efficacy in controlling degradation of nanomaterials,12,13 there currently exist few chemical strategies for rapidly integrating different peptide sequences into nanomaterials to screen against variable enzyme targets, thus limiting one’s ability to rapidly tailor materials for diverse enzymatic applications. Recently, we have shown that an esterified cross-linker works well for gating the release of small molecule drugs from a nucleic acid functionalized nanomaterial by the endosomal enzyme, esterase.14 Although the ester-embedded cross-linker effectively gated the release of cargo from our nanocapsules by esterases, the ester-cross-linker lacked the ability to be tuned to open in one specific cell type or cellular environment over another, limiting its broad application as a nucleic acid delivery vehicle. This specificity limitation is due to the ubiquitous nature of esterases as enzymes, found in the endosomes of numerous cell types. In order to both impart a greater degree of specificity to our design and address the need to develop a platform which would be modular in nature and amenable to rapid integration of diverse peptide sequences, we developed a straightforward © XXXX American Chemical Society

Received: October 17, 2017 Revised: November 22, 2017

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DOI: 10.1021/acs.bioconjchem.7b00629 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry locations. This added level of control and specificity over degradation and release of internalized cargo, coupled with the rapid and modular nature of the assembly approach, offers significant advantages in the design of future hybrid peptidebased biomaterials for various therapeutic and diagnostic applications. In order to synthesize a pep-NAN, we first modified the synthetic route for making our original nucleic acid nanocapsules to accommodate thiol−yne cross-linking between alkyne-functionalized surfactants rather than azide−alkyne cross-linking.14 This was enabled by introducing a peptide substrate whose sequence was modified at both the N and C terminus with cysteine residues to allow for chemical attachment points (thiols) for reacting with the alkynes presented at the surface of the NAN’s micellular core (Figure 1). Characterization of the successful assembly of the pep-

Figure 2. Nanoscale characterization of peptide cross-linked NANs. (A) Representative DLS measurement of peptide-cross-linked NANs (complete summarized DLS and zeta potential values for all pepNANs tabulated in Table S2). (B) Representative transmission electron micrographs showing peptide cross-linked NANs stained with 0.5% uranyl acetate for both CathB and MMP9 peptide substrates. Dotted outline indicates region of micrograph expanded for CathB NANs, additional TEM for pep-NANs found in Figures S2−S3. (C) 1% agarose gel showing the change in mobility of individual pep-NANs (CathB-NAN and MMP9-NAN) post attachment of a thiolated polyT20 DNA ligand using UV light-driven thiol− yne chemistry.

Figure 1. Assembly and programmed degradation of peptide crosslinked nucleic acid nanocapsules (pep-NANs). Peptide sequences of interest are modified with terminal cysteines to provide thiolated attachment points for covalently photo-cross-linking peptides to the alkyne-modified nanocapsule surface. The cross-linkers are incorporated using thiol−yne chemistry in the presence of a photoinitiator and UV light.14

NANs was achieved using a combination of dynamic light scattering (DLS), transmission electron microscopy (TEM), zeta potential measurements, and gel electrophoresis (Figure 2). Collectively these results indicated nanocapsule populations approximately 35 and 24 nm in size for cathepsin B peptide substrate linked NANs (CathB-NANs) and matrix metalloproteinase 9 peptide substrate linked NANs (MMP9-NANs), respectively (Figure 2B, Table S2). To test individual enzyme specificity for the peptide substrates within each pep-NAN (summarized in Table S1), a fluorescence assay14 was used to monitor the rate and extent of dye release (Figure 3). By monitoring the relative fluorescence change, the specificity of individual enzymes for the surface of different NAN formulations could be evaluated. MMP9-NANs and CathB-NANs released their dye when treated with their intended enzyme targets, MMP9 and cathepsin B, respectively (Figure 3A,B). We also evaluated cathepsin B activity on MMP9-NANs, in which no fluorescence change was observed (Figure 3C). This indicates that proteases from different families of enzymes are less likely to recognize the substrates of other enzymes within the pep-NAN formulations. The results of these experiments also indicated that the pH of the solution can control the release or lack of release of dyes

Figure 3. Fluorescence monitoring of pep-NAN degradation. (A) Representative raw fluorescence intensity plots showing the release of dye from MMP9-NANs cross-linked with MMP9 peptide in the presence of MMP9 enzyme. One μM MMP9-NANs were treated with 0.5 μg of MMP9 and monitored for 3 h. (B) Dye-loaded CathB-NANs in the presence of 0.5 μg of cathepsin B monitored for 3 h. (C) Fluorescence monitoring of cathepsin B activity on MMP9-NANs and the effect of varying pH on MMP9 cleavage rates. Exposure of MMP9NANs to cathepsin B and MMP9 at pH 5 resulted in no observable change in fluorescence. (D) Fluorescence plots of MMP9-NANs treated with MMP1 and MMP2 lacked the specificity to cleave the MMP9-NANs at pH 7. All experiments were conducted in triplicate.

from a pep-NAN formulation despite the presence of the appropriate enzyme trigger in solution. However, it is B

DOI: 10.1021/acs.bioconjchem.7b00629 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

enzymes in specific cellular locations for regulating cargo release. In light of this, it became particularly important to develop a more precise way of tracking the pep-NANs intracellularly in order to be able to anticipate the biochemical environment that they would experience. Achieving this would allow us to correlate where a nanocapsule is located and whether its cross-linked surface is still intact (cargo retained) or degraded (cargo released). To visualize the nanocapsules intracellularly, we developed a protocol for encapsulating gold nanoparticles (AuNPs), into the interior of the pep-NANs. Starting with a 10 nm tetradecylamine-modified AuNP, the addition of surfactant in water could be used to build the NAN’s exterior shell around the alkane-functionalized AuNP (Figure 4B). The gold nanoparticle-embedded NANs, referred to here as pep-Au-NANs, were incubated with HeLa cells to reveal the intracellular location post uptake and to determine if the stability of the pep-NANs could be visualized (i.e., shell intact or degraded) using uranyl acetate staining of charged organic species. Using this approach, both pep-NANs and chemically crosslinked NANs could be successfully visualized (Figure 4C,D). By controlling the stoichiometric ratio of surfactant, AuNP and cross-linker, roughly 1 AuNP per NAN could be achieved per particle, regardless of cross-linker utilized, as observed by TEM (Figures S4−S9). After characterizing the pep-Au-NANs, they were incubated with HeLa cells and subsequently sectioned, stained, and imaged for evidence of pep-Au-NAN uptake into cells (Figure 5A−D). As shown in Figure 5, diverse formulations of the AuNANs could be found within endosomes with varying degrees of stability, within the cross-sectioned HeLa cells. In Figure 5A,B the negative and positive controls for NAN disassembly are shown (negative control is a diazido-diol cross-linker, compound 1, SI, unable to be cleaved by cellular enzymes, and the positive control is the ester cross-linker, compound 2, SI14). As anticipated, the diazido-diol cross-linked NANs could not release their cargo in the endosomes, and the ester-cross-linked NANs could, giving us an image of what an intact and degraded NAN shell looks like intracellularly, respectively. This interpretation is based on the fact that an intact gray halo, similar to those seen in Figure 4C,D, is clearly seen surrounding each of the AuNPs in the endosomal compartments in Figure 5A. In addition, the lack of organic stained halo seen in Figure 5B along with the aggregated nature of the AuNPs suggests the loss of surface functionalization. The results of these studies indicate that the NAN’s outer structure could indeed dictate the release of the cargo from the nanocapsule. This was seen in the context of both the chemically cross-linked as well as peptide-cross-linked NANs. Importantly, the MMP9-NANs did not open despite being endocytosed, which correlates well with the fact that MMP9 enzyme is not expressed intracellularly, nor secreted in significant levels by HeLa cells.22 Cathepsin B, although found in HeLa cells, did not open the CathB-NANs, potentially due to the incubation time within the HeLa cells might not have been long enough to result in the breakdown of the pepNANs. The later reasoning is thought to be the case based on the degradation rate observed in vitro in the fluorescence assay results (Figure S15). Our results show it takes ∼20× as long to see comparable fluorescence changes in solution when cathepsin B is added to a solution of CathB-NANs as compared to MMP9-NAN degradation by MMP9. Additionally, it is

important to note that these experiments do not take into account that there are several proteases at any given time that pep-NANs could encounter with similar proteolytic roles (i.e., there are many MMP enzymes available in the extracellular space) which could result in false signal detection or premature release of a cargo depending on the application. Therefore, to test the relative rate of cleavage of the MMP9-NANs in the presence of closely related MMP enzymes, we treated the MMP9-NANs with various MMP proteins, including MMP1 and MMP2 (a collagenase and a gelatinase, respectively), both of which function optimally at pH 7. The results of these experiments indicated that the MMP9-NAN is specific to the MMP9 enzyme (Figure 3D). Additional MMP enzymes were also tested, showing a similar inability to cleave the MMP9NANs (Figure S17). Dye-loaded pep-NANs were also incubated with HeLa cells and observed under confocal microscopy (Figure S14). Both the CathB and MMP9 NANs entered cells readily when incubated in serum free conditions (Figure 4A) indicating that,

Figure 4. Confocal and electron microscopy of pep-NANs. (A) Confocal microscopy of pep-NANs (MMP9 and CathB) indicating cellular uptake. (B) Schematic depicting the assembly of a pep-AuNAN where the cross-linker consists of either esters, diols, or peptides, cross-linked using either Cu(I) azide alkyne catalyzed click chemistry or thiol−yne chemistry, depending on the terminal modification presented by the cross-linker. (C) TEM of diazido-diol (compound 1, SI) cross-linked NANs, individual shells could be seen surrounding the Au NP by TEM when stained with 0.5% uranyl acetate. (D) TEM of MMP9-NANs formed in the presence of an alkane-modified AuNP. Similar to the diol-cross-linked NANs, individual particles can be seen containing AuNPs incorporated into their centers.

like other spherical nucleic acid structures,20,21 they do not require addition of external transfection agents. The pep-NANs were also examined in cells over a range of concentrations in order to assess their relative toxicity in cell culture. The resulting cell survival indicated little to no toxicity for both the CathB-NANs and the MMP9-NANs (Figure S12, A,B) indicating their potential use in both in vitro and cell culture settings. It is also important to note that the central hypothesis of our work relies on our ability to engage individual populations of C

DOI: 10.1021/acs.bioconjchem.7b00629 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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The results presented here describe a new hybrid peptidecross-linked biomaterial that can recruit specific enzymes to its surface for the programmed degradation and release of internalized material. Using electron microscopy, we directly monitored the pep-NANs as they journeyed through the cell, shedding light on the importance of location of uptake and anticipated degree of degradation of the nanocapsules. This biochemically responsive material enables the rapid integration of peptides for screening enzyme interactions and can be used for a variety of biosensing and delivery applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00629. All experimental details, along with all nucleic acid sequences, synthetic strategies, and assay conditions (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Binglin Sui: 0000-0003-0376-317X Jessica L. Rouge: 0000-0002-6051-3735 Author Contributions †

J.J.S. and B.S. contributed equally.

Notes

Figure 5. Cellular uptake of AuNP embedded NANs visualized through cell sectioning and staining using TEM. (A) TEM of a diazido-diol cross-linked NAN inside a sectioned HeLa cell. (B) EsterAu-NANs, used as a positive control, observed within endosomes by TEM. In TEM micrograph of (C) CathB-Au-NANs and (D) MMP9Au-NANs intact peptide shells can be observed within the cells. (E) HeLa cells treated with PMA (MMP9 inducer) for 21 h show no toxicity up to 150 ng/mL. (F) HeLa cells treated with PMA for 21 h followed by incubation with 40 μM MMP9-NANs loaded with 2.5% camptothecin. The results indicate that only after PMA induction of MMP9 is toxicity of the drug loaded pep-NANs observed.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the University of Connecticut for providing the financial support to carry out this work and Biosciences Electron Microscopy Laboratory (BEML) at UConn for providing the instrumentation and assistance for the TEM cell sectioning work. We particularly thank Dr. Martiza Abril, Dr. Marie Cantino, and Dr. Xuanhao Sun for TEM assistance and feedback necessary for carrying out the cellular uptake experiments detailed in this work.

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important to consider that enzyme concentrations rise and fall depending on the current level of mRNA expression, and can be dysregulated (often overexpressed) when cells become diseased. In order to test the correlation between enzyme expression level within a live cell and the ability of the pep-NANs to be degraded, an additional assay was run in which HeLa cells were treated with phorbol 12-myristate 13-acetate (PMA), a known inducer of MMP9 expression in HeLa cells.23,24 Cells were incubated in serum-free media and treated with MMP9-NANs carrying camptothecin, a known apoptotic cancer drug. The toxicity of the drug loaded MMP9-NANs was evaluated using an MTS assay post 21 h PMA treatment, allowing enough time for MMP9 expression. These studies resulted in a dose dependent increase in cellular toxicity, interpreted as a response to the PMA inducing MMP9 expression and secretion by the HeLa cells, ultimately catalyzing the release of the drug in response to the pep-NAN’s enzyme target (Figure 5E,F). Treatment of PMA and drug loaded MMP9-NANs without allowing time for MMP9 secretion resulted in no cell toxicity (Figure S13).

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DOI: 10.1021/acs.bioconjchem.7b00629 Bioconjugate Chem. XXXX, XXX, XXX−XXX