Fluorescent Heterotelechelic Single-Chain Polymer Nanoparticles

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Cite This: ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

Fluorescent Heterotelechelic Single-Chain Polymer Nanoparticles: Synthesis, Spectroscopy, and Cellular Imaging Daniel N. F. Bajj, Michael V. Tran, Hsin-Yun Tsai, Hyungki Kim, Nathan R. Paisley, W. Russ Algar,* and Zachary M. Hudson* Department of Chemistry, The University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada

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S Supporting Information *

ABSTRACT: The folding and collapse of individual polymer chains into single-chain polymer nanoparticles (SCPNs) is a versatile and emerging platform for biological applications such as diagnostics, imaging, and therapy, where components for two or more of these functions can be combined onto a single polymer carrier. Here, we prepare heterotelechelic polymers with three sets of separately addressable chemical handles at their α-terminus and ω-terminus, and along their backbone. As a model system, the αand ω-termini are conjugated with a targeting ligand (folic acid or biotin) and therapeutic drug cargo (camptothecin), respectively, and the backbone is grafted with pendant fluorescent dye molecules, poly(ethylene glycol) oligomers, and benzene-1,3,5-tricarboxamide. These polymers fold in water to give fluorescent SCPNs, which are characterized with respect to their physical and photophysical properties. The latter reveals a relationship between polymer folding, quantum yield, and resistance to photobleaching. The SCPNs are then shown to be useful for immunolabeling of SK-BR-3 breast cancer cells and exhibit little or no acute cytotoxicity. This work demonstrates that SCPNs can be used as a viable platform for bioconjugation and cell labeling, helps establish a set of design criteria for optimizing future biological applications, and opens the door to the development of SCPNs for a broader range of theranostic applications. KEYWORDS: single-chain polymer nanoparticles, polymer synthesis, fluorescence spectroscopy, cellular imaging, heterotelechelic polymers

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INTRODUCTION Recent advances in polymer synthesis have made it possible to exercise an unprecedented level of control over the assembly of polymers into complex nanoscale objects. Sequence-defined polymers, hierarchical assembly strategies, and precise methods for end-group modification have all provided techniques for controlling polymer morphology on the nanometer scale. Furthermore, an improved fundamental understanding of polymer synthesis has been followed by the discovery of many polymer nanomaterials with applications in electronics, energy, and medicine.1−9 Among these discoveries, the folding of synthetic macromolecules into single-chain polymer nanoparticles (SCPNs) has attracted considerable research interest.10−13 These systems use supramolecular, covalent, or metal coordination-induced chain collapse to mimic the folding of linear polypeptides to yield globular proteins in biological systems.14−20 With the advantage of efficient and orthogonal chemistry for polymer functionalization, SCPNs have been successfully prepared for applications including sensing, light harvesting, and catalysis.21−25 Recent work by Albertazzi and Palmans demonstrated the preparation of SCPNs that were nontoxic to cells, and these materials were used for light-mediated singlet-oxygen generation to induce spatially controlled cell death.26 Like these authors, we reasoned that SCPNs also held promise for biological applications because functionality for targeting, © XXXX American Chemical Society

imaging, and therapy could potentially be combined in a single biocompatible polymer carrier. Recent advances in the synthesis of heterotelechelic polymers would enable the synthesis of SCPNs with two well-defined end groups suitable for bioconjugation, alongside multiple pendant side chains imparting luminescent, hydrogen bonding, and hydrophilic functionality to the particle. An SCPN-based platform would also have the advantage of maintaining precise control over the number of functional groups found on each particle. Other materials, including inorganic nanoparticles (e.g., metal, metal oxide, and semiconductor) and amphiphile-based systems (e.g., micelles, liposomes, polymersomes) are difficult to precisely functionalize because a majority of current methods for particle preparation rely on surface binding or encapsulation.27−31 The one-to-one stoichiometry of the α- and ω-termini of an SCPN offers a unique opportunity to generate nanoparticles bearing precisely two different functional units. If the end groups are prepared as orthogonal reactive handles, the final nanoparticle could be used to bind to any two appropriately functionalized biomolecules. The use of heterotelechelic polymers in bioconjugation experiments has been recently demonstrated Received: November 27, 2018 Accepted: January 18, 2019

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DOI: 10.1021/acsanm.8b02149 ACS Appl. Nano Mater. XXXX, XXX, XXX−XXX

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ACS Applied Nano Materials

Figure 1. Representation of the synthesis of heterotelechelic SCPNs bearing luminescent and H-bonding motifs and their self-assembly in aqueous media.

Scheme 1. Synthesis of Heterotelechelic SCPN-Forming Polyacrylamides Functionalized with (A) Biotin and (B) Camptothecin and Folic Acid Using a Combined Radical Cross-Coupling/CuAAC Strategy

constructs.32−36 Such a strategy could open the door to the eventual use of SCPNs in theranostics, which is an emerging

by Heredia, Boyer, and others, who have used functionalized polymer end groups to form a variety of polymer−protein B


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ACS Applied Nano Materials field of pharmaceutical development that aims to combine therapeutic and diagnostic agents onto a single carrier.3,37−45 SCPNs based on a benzene-1,3,5-tricarboxamide (BTA) hydrogen-bonding motif are a promising platform for the exploration of SCPNs for biological imaging. Recently developed by Palmans, Meijer, and co-workers, these SCPNs are constructed from a reactive ester prepolymer, allowing for postpolymerization functionalization with structurally diverse amines that could otherwise interfere with the synthesis of the polymer backbone.21 When functionalized with a combination of polyether and BTA side chains, these polymers spontaneously assemble into SCPNs in aqueous solution and have been used successfully in applications including singlet-oxygen generation, orthogonal self-assembly, and asymmetric catalysis.15,21,23,26 The recent demonstration of the biocompatibility of this platform offers an additional advantage, as the labeling of targeted cell types could potentially be achieved without nonspecific cytotoxicity. Herein, we describe a synthetic strategy for the preparation of fluorescent, heterotelechelic SCPNs and characterize these materials as labels for biological imaging (Figure 1). With the addition of the appropriate amines to a reactive precursor polymer, SCPN-forming polyamides are prepared containing distinct fluorescent, hydrophilic, and H-bonding motifs. Orthogonal chemistries are used to install biomolecules at the α- and ω-termini of the polymer, and heterotelechelic polymers are assembled into SCPNs in aqueous media. The photophysical properties of these SCPNs are studied in detail, revealing the relationship between polymer folding, particle quantum yield, and resistance to photobleaching. We then examine the suitability of end-functional SCPNs as a platform for selective cell labeling and investigate the cytotoxicity of SCPNs bearing a model therapeutic cargo. This work not only establishes proof-of-concept for termini-specific labeling but also elucidates design criteria for future SCPN materials that are better optimized for intended biological applications.

remained following the RAFT polymerization process. Conventionally used methods such as aminolysis or thermolysis were deemed unsuitable, as the former would result in competing substitution of the pentafluorophenyl ester backbone, and the latter would result in an unsaturated end group that would be difficult to functionalize with high fidelity in a fully substituted SCPN. Instead, radical-induced endgroup removal was used to both cleanly remove the trithiocarbonate and simultaneously impart the desired functionality to the polymer. 4,4′-Azobis(4-cyanovaleric acid) (ACVA), a derivative of the commonly used azobis(isobutyronitrile) (AIBN) radical initiator, presented a useful template for end-group radical cross-coupling as the terminal carboxylic acids can be easily conjugated to a variety of amines or alcohols. With preparation of an ACVA derivative covalently bonded to the biomolecule of interest, it is thus possible to functionalize the ω-terminus of a polymer synthesized via RAFT on heating, using the ACVA derivative as a radical trapping agent. Using this method, (S)-camptothecin (CPT) was installed at the pPFPA ω-terminus as a model therapeutic cargo. This commercially available alkaloid has been extensively studied as a cytotoxic agent49,50 and forms the structural basis of several anticancer drugs including topotecan and irinotecan. An immediate concern was that covalent bonding of camptothecin to the polymer via the C20 hydroxyl group could prohibit therapeutic activity, as the mode of binding of CPT to DNA topoisomerase I has been shown to involve the hydroxyl group in question.51−53 We postulated that, by formation of an ester linkage to the radical initiator and ultimately to the polymer backbone, esterases in the cellular environment could act to cleave this linkage and release free CPT. This strategy has been reported previously in the literature for drug delivery and would further allow the SCPN to remain in a dormant state until it is taken up by the appropriate cell.54,55 Initial trials of the radical cross-coupling with 1 equiv of the polymer chain to 20 equiv of bis(S)-camptothecin ACVA proved unsuccessful, producing only partial end-group substitution. A previous study by Chen et al. found that, while methacrylic and styrenic polymers could be substituted using this method, acrylate-based polymers required the addition of 2 equiv of lauroyl peroxide in addition to the AIBN for successful cross-coupling to occur.56 Employing these methods, it was possible to quantitatively remove the trithiocarbonate end group of P1 to give ω-CPT-pPFPA78-N3 (P3). The cross-coupling was first confirmed by 1H NMR, with the purified polymer showing peaks indicative of the CPT molecule, particularly the well-resolved peaks in the aromatic 7.3−8.5 ppm range (Figure S4). To further confirm our results, we examined the UV−vis absorbance spectra obtained from the GPC chromatogram of the polymer. In this manner, the absorbance of the sample was resolved according to elution volume, ensuring that any observed CPT absorbance occurred due to the polymer chain and not due to the presence of trace CPT impurities in the mixture. As shown in Figure S4, complete loss of the original trithiocarbonate absorption at 340 nm was observed following the radical cross-coupling, with the appearance of the characteristic dual absorbance band of CPT at 370 and 390 nm. Sequential Amine Addition and SCPN Formation. After functionalization of the ω-terminus, the reactive ester backbone of the polymer is easily functionalized upon stirring

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RESULTS AND DISCUSSION Synthesis of Polymer Backbone with Azide αTerminus. Poly(pentafluorophenyl) acrylate (pPFPA) has been shown by Palmans, Meijer, and co-workers to be a useful SCPN precursor, due to the ease with which the polymer backbone may be sequentially substituted with structurally diverse amines.21,46 Using a similar methodology, we prepared SCPN precursor polymers from PFPA using reversible addition−fragmentation chain-transfer (RAFT) polymerization, monitoring the reaction progress by 19F NMR and gel permeation chromatography (GPC). In order to introduce end-group functionality, 2-(dodecylthiocarbonothioylthio)-2methylpropionic acid 3-azido-1-propanol ester was used as the RAFT chain-transfer agent (CTA), bearing a terminal azide that could later be used for bioconjugation (Scheme 1). The kinetics of the polymerization process using a trithiocarbonate CTA were much faster compared to those of similar polymerizations using dithiobenzoate-based CTAs, as has been well-documented in the literature.47,48 By tuning polymerization time and the monomer/CTA ratio, two azidefunctionalized polymers pPFPA78-N3 (P1) and pPFPA150-N3 (P2) were prepared (Mn = 19 000 and 37 600, PDI = 1.34 and 1.38, respectively, Figure S8). Radical Cross-Coupling of ω-Terminus. To functionalize the ω-terminus of the polymer backbone, it was necessary to either remove or modify the trithiocarbonate moiety that C


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Figure 2. DLS data (A, B) and CD spectra (C) of P4 (blue) and P5 (green). Both CD and DLS measurements were done in water at room temperature with 0.07 and 0.2 mg/mL solutions, respectively. (D) AFM height image of P4 after self-assembly in water and spin-coating onto mica.

with a variety of amines in THF solution with mild heating. In agreement with the findings of Meijer and co-workers, we found that functionalization of 10% of the polymer backbone with amine-functionalized chiral benzene-tricarboxamide (BTA-NH2) yielded polymers that would form well-defined SCPNs in aqueous solution.21 Reaction of a further 10% of the available PFPA units with fluorescent dye was then undertaken to give SCPNs suitable for use in cell imaging. Amine-modified fluorescein isothiocyanate (FITC) was used here as a model dye due to its hydrophilic nature, low cost, and strong fluorescence in aqueous solution. Use of a hydrophilic dye was necessary to avoid incorporation into the core of the SCPN on the basis of hydrophobic encapsulation alone, and in principle many alternative amine-containing dyes could potentially be used in this way. Finally, the remaining 80% of the pPFPA backbone was substituted with an amine-terminated polyether (Jeffamine M-1000), to facilitate solubility in water and the eventual folding of the polymer into an SCPN. This sequential modification of the polymer can be readily monitored by 19F NMR, as the pentafluorophenyl acrylate repeat units are displaced by the added amines to give free pentafluorophenol (Figure S6). Performing this sequential amine substitution on P3 gave P4, bearing camptothecin and azide end groups with a backbone length of 78. An analogous reaction with P2 gave P5, having a backbone length of 150 and lacking the CPT moiety at the ω-terminus. The isolated polymers P4 and P5 were readily dispersible in water as well as a variety of common aqueous buffers (phosphate, borate, and acetate). Analysis by dynamic light scattering (DLS) indicated the formation of nanometer-scale particles in aqueous solution. The intensity-weighted DLS distribution indicated the formation of some particle

aggregates during the aqueous redispersion. The massweighted distribution, which provides a more accurate representation of the population of the particles, indicated a relatively narrow distribution of particle sizes, with P4 = 12 nm and P5 = 26 nm (Figure 2A,B and Figure S9). In agreement with Palmans and Meijer, both P4 and P5 show negative Cotton effects by circular dichroism (CD) spectroscopy at 223 nm (Figure 2C), consistent with the formation of left-handed BTA helices upon chain folding.21 Consistent with its longer chain length, the Cotton effect is larger for P5 (Δε = −39 M−1 cm−1) than for P4 (−30 M−1 cm−1). Spherical particles can also be observed by atomic force microscopy (AFM, Figure 2D) upon deposition of P4 onto mica, with average diameters in agreement with DLS data (12 ± 4 nm). Bioconjugation of the SCPN α-Terminus. The azide moiety at the SCPN α-terminus can be used to install alkynefunctionalized bioconjugates via copper(I) catalyzed azide− alkyne cycloaddition (CuAAC) at the micromolar scale for use in biological analyses. Assessment of the reactivity of the αterminus of the fully substituted SCPN is challenging, as the diverse functionality of the particles and small reaction scale preclude convincing quantification by many spectroscopic methods. To determine if the polymer α-terminus could be functionalized with high fidelity, we prepared SCPN-P6 by reaction of an azide-bearing P5 with a biotin-functionalized alkyne. Aqueous CuAAC from the in situ reduction of copper(II) sulfate with excess sodium ascorbate was used to functionalize the polymer, using a 2-fold excess of alkyne cargo to drive the reaction to completion. The polymer was then purified by spin filtration to give a 50 μM aqueous solution of biotin-conjugated SCPN-P6. D


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Figure 3. Fluorescence emission spectrum of P6 incubated with avidin-functionalized filter paper (green) and control (black). Photo (right) is taken under UV irradiation at 365 nm, showing fluorescence of functional paper (top) and control (bottom). Fluorescence measurements were made in air at room temperature.

Table 1. Photophysical Properties of SCPN-P7 and Reference Materials material

Φa

fluorescein FITC BSA-FITC

0.81 0.60 0.28

SCPN-P7

0.12

rc

kPB (min−1)d

kq (109 M−1 s−1)e

0.024 0.025 0.191

0.17 ± 0.02 0.10 ± 0.01 0.29 ± 0.04 (32%) 0.021 ± 0.002 (68%) 0.3 ± 0.2 (19%) 0.024 ± 0.004 (81%)

2.84 ± 0.46 2.68 ± 0.11 ∼0

τ (ns)b 4.0 3.9 1.0 3.8 1.8 4.3

(14%) (86%) (27%) (73%)

0.201

0.43 ± 0.27

Fluorescence quantum yield. All values ±5%. bFluorescence lifetime(s). All values ±0.1 ns. cFluorescence anisotropy. All values ±0.001. Photobleaching rate(s). eStern−Volmer quenching constant with iodide.

a

d

Figure 4. Photophysical characterization of SCPN-P7 and reference materials. (A) Spectra: (i) absorption; (ii) fluorescence excitation; and (iii) fluorescence emission. (B) Fluorescence decays. (C) Photobleaching curves. (D) Stern−Volmer plots for collisional quenching by iodide ion. All measurements were made in 1× PBS buffer at pH 7.2 (see Supporting Information for recipe) between 21 and 26 °C. Error bars represent the standard deviation of three replicate measurements. The concentrations for each sample can be found in the Supporting Information.

These fluorescent SCPNs were then used to label avidinfunctionalized cellulose substrates, providing evidence for the success of this bioconjugation strategy via the avidin−biotin binding interaction. A similar strategy was recently employed by Rowan, Palmans, and Meijer to immobilize SCPNs with catalytic activity to an avidin-functionalized surface.57 Avidin-

functionalized cellulose filter paper was first prepared by literature methods,58 generating a surface with an affinity for biotin due to the four binding sites present on each avidin protein. If the biotin was successfully conjugated to the SCPN by CuAAC, selective binding to the filter paper would be observed versus a negative control substrate bearing no avidin. E


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at shorter wavelengths than those of the dianion (∼470 and ∼450 nm, respectively, versus a peak at ∼490 nm for the dianion), and an emission peak and shoulder at longer wavelengths (∼510 and ∼550 nm, respectively, versus a peak at ∼515 nm for the dianion), which may account for the new spectral features († and ‡).62 An alternative interpretation is that the new features in the fluorescence spectra represent a subpopulation of dianion that is in an environment different than the main population, as fluorescein is known to exhibit spectral shifts in response to changes in polarity and hydrogenbonding interactions;63,64 however, the absorbance, excitation, and emission spectra usually shift in the same direction, which is not consistent with the hypsochromic shoulder in the excitation spectrum and bathochromic broadening of the emission spectrum. If not from the monoanion, these features would arise from multiple emitting species, multiple local environments in the SCPN, or some combination thereof. The other secondary dye species in the SCPN-P7 appeared to be a nonfluorescent or weakly fluorescent H-like dimer or aggregate, as indicated by the much larger hypsochromic shoulder (* in Figure 4Ai) in the absorption spectrum versus the excitation spectrum. Fluorescein dimers and trimers have been reported to have peaks in their absorption spectra at ca. 457, 470, and 505 nm,65 which is consistent with the hypsochromic shoulder (*). The hypsochromic shoulder was unaffected by dilution of the SCPN between 0.25 and 75 μM (data not shown), supporting an intraparticle interaction. The SCPN quantum yield value (0.12) was about one-fifth the measured value for FITC. The monoanion is reported to have a molar absorption coefficient and quantum yield that are ∼2.5-fold smaller than the corresponding values for the dianion.62 The presence of a minority subpopulation of monoanion may account for some of the apparent decrease in quantum yield, but not all of it, which further supports the formation of some intraparticle H-dimers or trimers. Lifetime. Figure 4B shows that SCPN-P7 had a biexponential fluorescence decay with a slow lifetime component (∼4.3 ns) that was similar to the lifetime for fluorescein and FITC alone. We attribute this component to the dianion, where the combination of low quantum yield and largely unchanged lifetime was consistent with nonfluorescent H-dimers within the SCPN. The fast lifetime component (∼1.8 ns) of the SCPN-P7 fluorescence decay may be due to the presence of fluorescein monoanion. A fluorescence lifetime of 3.0 ns is expected from the monoanion, but the apparent elevation in its pKa also suggests that a change in its lifetime from the local environment of the SCPN is possible. Alternatively, the short lifetime component could reflect that a fraction of dianions (and perhaps monoanions, if present) engage in some form of energy transfer to sinks within the SCPN, or that a subpopulation of dianion is in an environment that causes a faster nonradiative relaxation rate. Given that the SCPN and reference materials were measured under the same conditions, the possible presence of dye monoanion and probable presence of H-like aggregates is attributable to folding of the SCPN and its degree of labeling. Plane-to-plane stacking of the FITC molecules may be a passive side effect of the interactions between the BTA, and/or the FITC may actively engage in hydrogen bonding with the BTA. The latter is consistent with the possible presence of dye monoanion, as hydrogen-bonding interactions with the FITC phenol group may elevate the phenol pKa from its usual value of 6.0−6.4.

Following immersion of the avidin-functionalized and control filter papers in a solution of SCPN-P6 for 1 h, binding was monitored by the emission of the SCPN-bound FITC dye. Fluorescence emission was clearly indicative of binding of SCPNs to the avidin-functionalized paper, whereas the control paper showed only weak fluorescence from nonspecific adsorption (Figure 3). This data is evidence that functionalization of the α-terminus by CuAAC is a viable bioconjugation strategy for SCPNs in aqueous solution at micromolar concentrations. Following this successful trial, the conjugation of folic acidPEG100-alkyne onto the camptothecin-bearing SCPN P4 was performed to produce a model SCPN P7 bearing both targeting and therapeutic cargo (Scheme 1B). Folic acid (FA) targets cells bearing the folate receptor protein (FRP), which is known to be commonly overexpressed on human cancer cells.44−46 A long water-soluble polyether was used here to connect the FA to the SCPN, as studies by Wooley and coworkers have demonstrated the importance of keeping the targeting group available at the surface of polymer particles used in theranostic applications.59 Photophysical Properties. In order to develop fluorescent SCPNs as a viable platform for cellular labeling, an understanding of their photophysical properties, including the behavior of dye molecules in the SCPN microenvironment, is first required. Using the bioconjugated SCPN-P7, selected photophysical properties were compared to fluorescein, FITC, and FITC-labeled bovine serum albumin (BSA-FITC) as reference materials (Table 1). The BSA-FITC sample was selected as a model for FITC conjugated to amine groups on a macromolecule with defined structure and folding, albeit that the folding analogy is imperfect (the BSA is folded prior to labeling; the SCPN is folded after labeling). Nevertheless, the BSA-FITC is a useful reference because the properties of FITC (and other fluorescein derivatives) change between folded and unfolded BSA.60,61 Spectra and Quantum Yield. Figure 4A shows that fluorescein, FITC, and BSA-FITC had approximately the same absorption, fluorescence excitation, and fluorescence emission band shapes with small (≤10 nm) spectral shifts between them; however, SCPN-P7 exhibited several spectral features distinct from those of the reference materials. In particular, the absorption spectrum of SCPN-P7 had a very pronounced hypsochromic shoulder; a similar but much less pronounced feature was present in its excitation spectrum, and its emission spectrum was more spectrally broad, suggesting a weak bathochromic spectral feature. This data cumulatively suggested the presence of multiple dye species and/or environments in the SCPN. The main dye species was simply conjugated FITC with spectral properties similar to the reference materials. The reference material spectra were dominated by the properties of the dye dianion, which is the brightest form of fluorescein and generally dominant at pH ≥ 7. One possible secondary dye species within the SCPN-P7 appeared to be the dye monoanion, as suggested by the shoulder in the fluorescence excitation spectrum († in Figure 4Aii) and broadening of the emission spectrum (‡ in Figure 4Aiii). Although inner filter effects (e.g., in the form of spectral shifts) can occur with the SCPN samples, we confirmed that these do not account for the increased width of the emission spectrum nor the shoulder in the excitation spectrum. The monoanion is normally only observed at pH < 6, but it has an absorption peak and shoulder F


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ACS Applied Nano Materials Additional Fluorescence Measurements. Other evidence for inclusion of FITC within the folded interior of the SCPN came from fluorescence anisotropy, photobleaching, quenching, and unfolding measurements. The fluorescence anisotropy measured from SCPN-P7 was approximately an order of magnitude larger than the anisotropy of the fluorescein and FITC (see Table 1), indicating a significant loss of rotational freedom within the nanoparticle. Figure 4C shows that the majority of fluorescent dye associated with the SCPN-P7 bleached at least 4-fold more slowly than the fluorescein and FITC, suggesting protection within the interior of the SCPN. A minority of fluorescent dye in the SCPN-P7 bleached at close to the same rate as the fluorescein and FITC, presumably representing a subpopulation of dyes at or near the surface of the folded nanoparticle. These results are consistent with recent work by Zimmerman and co-workers, who have demonstrated that incorporation of fluorescent dyes into polymer particles can greatly improve their stability under continuous irradiation.66 Collisional quenching experiments with iodide ion were also consistent with protection of a majority of fluorescent dye molecules within the SCPN-P7 interior. The bimolecular quenching rate constants derived from the Stern−Volmer plots in Figure 4D were approximately 6-fold smaller with the SCPN-P7 than with fluorescein and FITC. Overall, the photophysical properties of FITC associated with SCPN-P7 were most analogous to those of BSA-FITC, which is also a folded structure. With respect to unfolding, Figure 5 and Figure S11 show normalized spectral characterization data for SCPN-P7 in aqueous solvent and 50% v/v DMF (aq), with fluorescein and FITC as reference materials. The fluorescein and FITC show spectral shifts with the added DMF but retain the same spectral shapes. In contrast, the absorbance and excitation

spectra for the SCPN show decreases in hypsochromic shoulder intensities with the addition of DMF, and the emission spectrum narrows slightly with less of a bathochromic shoulder (see Figure S11). All of these results indicate that the ability of DMF to disrupt hydrogen bonding and better solvate hydrophobic groups converts a larger fraction of the dye to dianion (i.e., less H-aggregates and monoanion), presumably by fully or partially unfolding the SCPN. Consistent with this hypothesis, the brightness of the SCPN sample increased with added DMF whereas the brightness measurements of fluorescein and FITC either decreased or were approximately unchanged (Figure 5B). Cellular Labeling and Imaging. SK-BR-3 cells were chosen for cell labeling studies because of their elevated levels of human epidermal growth factor receptor 2 (HER2) expression.67 The HER2 receptor is located on the extracellular membrane of SK-BR-3 cells and can be used as a tag for fluorescent cell labeling. The biotinylated SCPN-P6 was used as a fluorescent probe for labeling SK-BR-3 cells in a sandwich binding format with NeutrAvidin and biotinylated anti-HER2 antibody. The samples with all three components of the sandwich displayed significantly more fluorescence intensity versus the negative controls (Figure 6), demonstrating that

Figure 6. Fluorescent labeling of SK-BR-3 breast cancer cells with SCPN-P6. Top row: bright-field images. Middle row: fluorescence images (the pixel intensity calibration scale is indicated on the right). Bottom row: merged bright-field and fluorescence images. The sample is in the leftmost column. The “+” indicates that the cells were incubated with the respective material. Scale bars are 100 μm.

bioconjugated, fluorescent SCPNs are a viable tool for selective cell labeling. We note, however, that the magnitude of the contrast versus the negative controls was lower when older stock solutions of SCPN were used (see Figure S12 for an example using the same stock solution of SCPN-P6, obtained several weeks after that shown in Figure 6). Further studies will be needed to elucidate if this variation was inherent to the position of the biotin label on the polymer or inherent to the cultured cells, or whether the SCPNs experienced some degradation in aqueous solution over time.

Figure 5. SCPN unfolding measurements in DMF (50% v/v with PBS) and PBS (0.5×) at 24−26 °C. (A) Fluorescence excitation spectra for SCPN-P7, FITC, and fluorescein. See Figure S11 for absorbance and emission spectra. (B) Integrated fluorescence emission intensities for the same materials. G


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ACS Applied Nano Materials Cellular Viability Assay. CPT-SCPN-N3 (P4) or CPTSCPN-FA (P7) was dosed to SK-BR-3 cells, which are known to overexpress folate receptors and were thus chosen as a model cell type for viability assays. For both materials, the cell viability was approximately constant across SCPN concentrations ranging from picomolar to micromolar (Figure 7).

such that its release by esterase activity was inefficient. If that was the case, then the next-generation design of the SCPN should also include a mechanism for the SCPNs to unfold in the endolysosomal system. When we accept that the camptothecin was unable to exert a biological effect, our results are consistent with other studies that have little or no acute cytotoxicity for SCPNs,74 including other SCPNs with BTA and PEG side chains,26 and PEGylated SCPNs based on other folding motifs75,76 or cross-linking chemistries.77 Microinjection Results. Given the tolerance to the presence of significant concentrations of SCPNs in the extracellular environment, we next examined the viability of cells after direct injection of SCPNs into the cytosol. A549 cells were chosen for the microinjection because of their good adherence to the growth substrate and tolerance of the microinjection procedure. As shown by the bright-field, fluorescence, and merged images in Figure 8 and Figure S13, the morphology of the cells injected with SCPN-P7 was unchanged postinjection and the SCPNs were evenly distributed throughout the cytosol but excluded from the

Figure 7. SK-BR-3 cell viability assay with CPT-SCPN-N3 (P4) and CPT-SCPN-FA (P7). The nanoparticle concentrations were in the range 10 pM−16 μM (8 × 10−7−1.3 mg/mL) in PBS buffer (see Supporting Information for recipe) and incubated with cells at 37 °C. Cell viability is expressed as a percentage of the negative control (cells that were not incubated with the SCPN). Data points and error bars are the average and standard deviation of three replicates. The dashed lines are to guide the eye. The solid trendlines show that viability does not decrease as the concentration of SCPN increases.

This trend, which reflected a lack of acute cytotoxicity, suggested (i) that the SCPN was quite benign to the cells, and (ii) that the SCPN-conjugated camptothecin was unavailable to exert its biological effects, or otherwise had greatly reduced potency. Typical IC50 values for free camptothecin are in the range 679 ± 92 nM.68 Release of the camptothecin from the SCPN was hypothetically possible through acidic or enzymatic hydrolysis of the ester linkage after uptake via the endolysosomal system, as implemented with other nanoparticle materials.69−71 There are several possible reasons that we saw no acute cytotoxic effect. The first possibility is that the folateconjugated SCPNs were not efficiently taken up by the cells, which precluded the drug from exerting an effect. Inefficient uptake may have resulted if the folate remained tightly associated with or sterically occluded by the folded SCPN, or if the display of a single folate did not meet the threshold for ligand display to induce uptake at the SCPN concentrations tested. An example of the latter is the observation that a minimum number of cell-penetrating peptides (∼25) need to be conjugated to a semiconductor quantum dot to induce uptake.72,73 Quantum dots are similar in size to our SCPN, and so the display of multiple folate molecules may have been necessary. Another possibility is that there was indeed uptake of the SCPN, but that it was not observed by fluorescence microscopy because of rapid loss of the fluorescence signal in the endolysosomal system, similar to what was observed in microinjection experiments (vide infra). Full degradation of the SCPNs should have resulted in release of the CPT drug and a cytotoxic effect, and thus was unlikely. However, if only the fluorescent dye component of the SCPN was degraded (e.g., by rapid photobleaching), then another possibility for the absence of toxicity is that the hydrophobic CPT was tightly associated with or sterically occluded by the folded SCPN,

Figure 8. Bright-field, fluorescence, and merged images of SCPN-P7 injected into A549 cells. The microinjected cells are highlighted with a dashed outline. Images were taken 1 and 30 min after the injection. Scale bars are 50 μm. The images shown for each time point were acquired with the same microscope settings but processed with different color scales. The fluorescence in the 1 min image is from the SCPN-P7; the fluorescence in the 30 min image is cellular autofluorescence. (More images taken at different time points are shown in Figure S13 in the Supporting Information.) H


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ACS Applied Nano Materials nucleus. The cells were then imaged at different intervals for up to 1 h following the injection (images up to 30 min are shown in Figure S13). Curiously, the fluorescence from the SCPN-P7 faded after injection and became indistinguishable from the background autofluorescence of adjacent noninjected cells within ∼10 min. This process is tentatively attributed to exocytic trafficking of the SCPNs, degradation of the dye within the cytosol, and/or greatly accelerated photobleaching of the dye within the cytosol. Regarding the latter, the illumination in these experiments was not continuous (samples were illuminated for image acquisition, but not between time points), and Figure 4C suggests a photobleaching half-life on the order of 30−40 min in buffer, such that a photobleaching process, if it is the cause of the signal loss, must be accelerated by an order of magnitude or more within the cytosol. More importantly, no significant changes in cell morphology were observed postinjection. Blebbing, fragmentation, and nuclear condensation are characteristic of cell death (apoptosis or necrosis), and these features did not develop within the time frame of the experiment. Another important observation was that, prior to fading, the fluorescence signal was approximately uniform across the cytosol with no signs of aggregation of the SCPNs. In our experience with the cytosolic microinjection of other materials, poor intracellular colloidal stability will quickly yield visible (i.e., microscopic) aggregates and the material will only be partially distributed in the cytosol. The results in Figure 8 (and Figure S13) thus indicated that the SCPNs had good colloidal stability within the cytosol.

mode. DLS analysis was performed on a Wyatt Technology DynaPro Titan. SEC experiments were performed on a Malvern OMNISEC GPC instrument equipped with refractive index, viscometry, light scattering, and photodiode array detectors. Atomic force microscopy (AFM) images were obtained using an Asylum Instruments Cypher S AFM system in tapping mode at scan rates of 3.0 Hz. Absorbance and fluorescence excitation, emission, and anisotropy measurements were made with an Infinite M1000 Pro multifunction plate reader. Fluorescence lifetime measurements were made using a FluoroCube time correlated single photon counting instrument. SK-BR-3 and A549 cells were cultured in supplemented media plus antibiotic and antimycotic at 37 °C under an atmosphere of 95% air/ 5% CO2 and subcultured weekly. For experiments, cells were seeded in the wells of a tissue-cultured-treated microtiter plate or, for microinjection, a fibronectin-coated glass bottom culture dish. Microinjection was done with an Eppendorf Injectman 4 system. Cell imaging was done with an Olympus IX83 inverted epifluorescence microscope (see Supporting Information for technical details). Cell viability was measured using a commercial MTS cell proliferation assay kit.

CONCLUSION In summary, a strategy has been developed for the α and ω chain-end functionalization of SCPNs to give heterotelechelic polymer nanoparticles suitable for bioconjugation. Fluorescein dyes in the SCPN microenvironment were found to be more resistant to photobleaching than free dye, experienced excitation and emission broadening, and were less bright because of effects associated with folding of the polymer chain. Bioconjugation of these SCPNs was performed with either biotin or folate, and biotin-functionalized SCPN-P6 was used successfully in the selective labeling of cellulose substrates as well as SK-BR-3 cells. Heterotelechelic SCPN-P7 had essentially no impact on cell viability when present in the extracellular environment at concentrations up to 16 μM, and minimal uptake of the SCPNs by endocytosis was observed. Direct microinjection of SCPN-P7 into A549 cells revealed a loss of fluorescence with time, again with little impact on cell viability. This work demonstrates that SCPNs can be used as a viable platform for bioconjugation and cell labeling, opening the door to the development of SCPNs for a broader scope of targets. Future work will look to improve the ease and robustness of the end-group functionalization reactions, rationally design enhancements to the SCPN structure to benefit bioimaging (e.g., uptake by receptor-mediated endocytosis), and optimize loading and release for SCPNs with therapeutic and contrast agent cargoes for applications in theranostics.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b02149. Detailed experimental methods and additional results, including a full list of materials, experiment conditions, synthetic procedures, characterization methods, cell experiment protocols, 1H and 19F NMR spectra, gel permeation chromatography data, additional photophysical data, and additional cell labeling data (PDF)

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ASSOCIATED CONTENT

S Supporting Information *

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

W. Russ Algar: 0000-0003-3442-7072 Zachary M. Hudson: 0000-0002-8033-4136 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), the British Columbia Knowledge Development Fund (BCKDF), and the University of British Columbia (UBC) for support of their research programs. The authors also gratefully acknowledge the following sources of support: an NSERC Undergraduate Student Research Award (D.N.F.B.), NSERC CREATE NanoMat studentships (M.V.T and H.-Y.T.), a UBC FourYear Fellowship (H.K. and H.-Y.T.), NSERC postgraduate scholarships (H.K. and N.R.P), a Michael Smith Foundation for Health Research Scholar Award and an Alfred P. Sloan Fellowship (W.R.A), and support from the Canada Research Chairs program (Tier 2, W.R.A and Z.M.H.). Fluorescence lifetime measurements were made at the UBC Laboratory for Advanced Spectroscopy and Imaging Research (LASIR).

EXPERIMENTAL SECTION

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Detailed synthetic procedures and experimental details may be found in the Supporting Information. 1 H, 19F, and 13C NMR spectra were acquired on a Bruker AVIII HD 400 MHz spectrometer. Mass spectra were measured on a Bruker HCT Ultra ion-trap mass spectrometer in electrospray ionization

REFERENCES

(1) Elsabahy, M.; Heo, G. S.; Lim, S.-M.; Sun, G.; Wooley, K. L. Polymeric Nanostructures for Imaging and Therapy. Chem. Rev. 2015, 115, 10967−11011.

I


Article

ACS Applied Nano Materials (2) Dahlman, J. E.; Barnes, C.; Khan, O. F.; Thiriot, A.; Jhunjunwala, S.; Shaw, T. E.; Xing, Y.; Sager, H. B.; Sahay, G.; Speciner, L.; Bader, A.; Bogorad, R. L.; Yin, H.; Racie, T.; Dong, Y.; Jiang, S.; Seedorf, D.; Dave, A.; Singh Sandhu, K.; Webber, M. J.; Novobrantseva, T.; Ruda, V.; Lytton-Jean, A. K. R.; Levins, C. G.; Kalish, B.; Mudge, D. K.; Perez, M.; Abezgauz, L.; Dutta, P.; Smith, L.; Charisse, K.; Kieran, M. W.; Fitzgerald, K.; Nahrendorf, M.; Danino, D.; Tuder, R. M.; von Andrian, U. H.; Akinc, A.; Panigrahy, D.; Schroeder, A.; Koteliansky, V.; Langer, R.; Anderson, D. G. In Vivo Endothelial SiRNA Delivery Using Polymeric Nanoparticles with Low Molecular Weight. Nat. Nanotechnol. 2014, 9, 648−655. (3) Xiao, Q.; Zheng, X.; Bu, W.; Ge, W.; Zhang, S.; Chen, F.; Xing, H.; Ren, Q.; Fan, W.; Zhao, K.; Hua, Y.; Shi, J. A Core/Satellite Multifunctional Nanotheranostic for in Vivo Imaging and Tumor Eradication by Radiation/Photothermal Synergistic Therapy. J. Am. Chem. Soc. 2013, 135, 13041−13048. (4) Sowers, M. A.; McCombs, J. R.; Wang, Y.; Paletta, J. T.; Morton, S. W.; Dreaden, E. C.; Boska, M. D.; Ottaviani, M. F.; Hammond, P. T.; Rajca, A.; Johnson, J. A. Redox-Responsive Branched-Bottlebrush Polymers for in Vivo MRI and Fluorescence Imaging. Nat. Commun. 2014, 5, 5460. (5) Lyu, Y.; Fang, Y.; Miao, Q.; Zhen, X.; Ding, D.; Pu, K. Intraparticle Molecular Orbital Engineering of Semiconducting Polymer Nanoparticles as Amplified Theranostics for in Vivo Photoacoustic Imaging and Photothermal Therapy. ACS Nano 2016, 10, 4472−4481. (6) Kim, H.-C.; Park, S.-M.; Hinsberg, W. D. Block Copolymer Based Nanostructures: Materials, Processes, and Applications to Electronics. Chem. Rev. 2010, 110, 146−177. (7) Bucella, S. G.; Luzio, A.; Gann, E.; Thomsen, L.; McNeill, C. R.; Pace, G.; Perinot, A.; Chen, Z.; Facchetti, A.; Caironi, M. Macroscopic and High-Throughput Printing of Aligned Nanostructured Polymer Semiconductors for MHz Large-Area Electronics. Nat. Commun. 2015, 6, 8394. (8) Xie, G.; Krys, P.; Tilton, R. D.; Matyjaszewski, K. Heterografted Molecular Brushes as Stabilizers for Water-in-Oil Emulsions. Macromolecules 2017, 50, 2942−2950. (9) Barnes, J. C.; Bruno, P. M.; Nguyen, H. V.-T.; Liao, L.; Liu, J.; Hemann, M. T.; Johnson, J. A. Using an RNAi Signature Assay To Guide the Design of Three-Drug-Conjugated Nanoparticles with Validated Mechanisms, In Vivo Efficacy, and Low Toxicity. J. Am. Chem. Soc. 2016, 138, 12494−12501. (10) Hanlon, A. M.; Lyon, C. K.; Berda, E. B. What Is Next in Single-Chain Nanoparticles? Macromolecules 2016, 49, 2−14. (11) De-La-Cuesta, J.; González, E.; Pomposo, A. J. Advances in Fluorescent Single-Chain Nanoparticles. Molecules 2017, 22, 1819. (12) Single-Chain Polymer Nanoparticles: Synthesis, Characterization, Simulations, and Applications; Pomposo, J. A., Ed.; Wiley-VCH: Weinheim, 2017. (13) Mavila, S.; Eivgi, O.; Berkovich, I.; Lemcoff, N. G. Intramolecular Cross-Linking Methodologies for the Synthesis of Polymer Nanoparticles. Chem. Rev. 2016, 116, 878−961. (14) Foster, E. J.; Berda, E. B.; Meijer, E. W. Metastable Supramolecular Polymer Nanoparticles via Intramolecular Collapse of Single Polymer Chains. J. Am. Chem. Soc. 2009, 131, 6964−6966. (15) Hosono, N.; Gillissen, M. A. J.; Li, Y.; Sheiko, S. S.; Palmans, A. R. A.; Meijer, E. W. Orthogonal Self-Assembly in Folding Block Copolymers. J. Am. Chem. Soc. 2013, 135, 501−510. (16) Tuten, B. T.; Chao, D.; Lyon, C. K.; Berda, E. B. Single-Chain Polymer Nanoparticles via Reversible Disulfide Bridges. Polym. Chem. 2012, 3, 3068−3071. (17) Beck, J. B.; Killops, K. L.; Kang, T.; Sivanandan, K.; Bayles, A.; Mackay, M. E.; Wooley, K. L.; Hawker, C. J. Facile Preparation of Nanoparticles by Intramolecular Cross-Linking of Isocyanate Functionalized Copolymers. Macromolecules 2009, 42, 5629−5635. (18) Sanchez-Sanchez, A.; Arbe, A.; Colmenero, J.; Pomposo, J. A. Metallo-Folded Single-Chain Nanoparticles with Catalytic Selectivity. ACS Macro Lett. 2014, 3, 439−443.

(19) Heiler, C.; Offenloch, J. T.; Blasco, E.; Barner-Kowollik, C. Photochemically Induced Folding of Single Chain Polymer Nanoparticles in Water. ACS Macro Lett. 2017, 6, 56−61. (20) Heiler, C.; Bastian, S.; Lederhose, P.; Blinco, J. P.; Blasco, E.; Barner-Kowollik, C. Folding Polymer Chains with Visible Light. Chem. Commun. 2018, 54, 3476−3479. (21) Liu, Y.; Pauloehrl, T.; Presolski, S. I.; Albertazzi, L.; Palmans, A. R. A.; Meijer, E. W. Modular Synthetic Platform for the Construction of Functional Single-Chain Polymeric Nanoparticles: From Aqueous Catalysis to Photosensitization. J. Am. Chem. Soc. 2015, 137, 13096− 13105. (22) Sanchez-Sanchez, A.; Fulton, D. A.; Pomposo, J. A. PHResponsive Single-Chain Polymer Nanoparticles Utilising Dynamic Covalent Enamine Bonds. Chem. Commun. 2014, 50, 1871−1874. (23) Huerta, E.; Stals, P. J. M.; Meijer, E. W.; Palmans, A. R. A. Consequences of Folding a Water-Soluble Polymer Around an Organocatalyst. Angew. Chem., Int. Ed. 2013, 52, 2906−2910. (24) Gillissen, M. A. J.; Voets, I. K.; Meijer, E. W.; Palmans, A. R. A. Single Chain Polymeric Nanoparticles as Compartmentalised Sensors for Metal Ions. Polym. Chem. 2012, 3, 3166−3174. (25) Rothfuss, H.; Knöfel, N. D.; Roesky, P. W.; Barner-Kowollik, C. Single-Chain Nanoparticles as Catalytic Nanoreactors. J. Am. Chem. Soc. 2018, 140, 5875−5881. (26) Liu, Y.; Pujals, S.; Stals, P. J. M.; Paulöhrl, T.; Presolski, S. I.; Meijer, E. W.; Albertazzi, L.; Palmans, A. R. A. Catalytically Active Single-Chain Polymeric Nanoparticles: Exploring Their Functions in Complex Biological Media. J. Am. Chem. Soc. 2018, 140, 3423−3433. (27) Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Formulation of Functionalized PLGA-PEG Nanoparticles for in Vivo Targeted Drug Delivery. Biomaterials 2007, 28, 869−876. (28) Cheng, Y.; Samia, A. C.; Meyers, J. D.; Panagopoulos, I.; Fei, B.; Burda, C. Highly Efficient Drug Delivery with Gold Nanoparticle Vectors for in Vivo Photodynamic Therapy of Cancer. J. Am. Chem. Soc. 2008, 130, 10643−10647. (29) Zhao, Y.; Trewyn, B. G.; Slowing, I. I.; Lin, V. S.-Y. Mesoporous Silica Nanoparticle-Based Double Drug Delivery System for Glucose-Responsive Controlled Release of Insulin and Cyclic AMP. J. Am. Chem. Soc. 2009, 131, 8398−8400. (30) Kim, J.; Kim, H. S.; Lee, N.; Kim, T.; Kim, H.; Yu, T.; Song, I. C.; Moon, W. K.; Hyeon, T. Multifunctional Uniform Nanoparticles Composed of a Magnetite Nanocrystal Core and a Mesoporous Silica Shell for Magnetic Resonance and Fluorescence Imaging and for Drug Delivery. Angew. Chem., Int. Ed. 2008, 47, 8438−8441. (31) Bagalkot, V.; Zhang, L.; Levy-Nissenbaum, E.; Jon, S.; Kantoff, P. W.; Langer, R.; Farokhzad, O. C. Quantum Dot−Aptamer Conjugates for Synchronous Cancer Imaging, Therapy, and Sensing of Drug Delivery Based on Bi-Fluorescence Resonance Energy Transfer. Nano Lett. 2007, 7, 3065−3070. (32) Heredia, K. L.; Grover, G. N.; Tao, L.; Maynard, H. D. Synthesis of Heterotelechelic Polymers for Conjugation of Two Different Proteins. Macromolecules 2009, 42, 2360−2367. (33) Boyer, C.; Liu, J.; Bulmus, V.; Davis, T. P.; Barner-Kowollik, C.; Stenzel, M. H. Direct Synthesis of Well-Defined Heterotelechelic Polymers for Bioconjugations. Macromolecules 2008, 41, 5641−5650. (34) Lorenzo, M. M.; Decker, C. G.; Kahveci, M. U.; Paluck, S. J.; Maynard, H. D. Homodimeric Protein−Polymer Conjugates via the Tetrazine−trans-Cyclooctene Ligation. Macromolecules 2016, 49, 30− 37. (35) Reader, P. W.; Pfukwa, R.; Jokonya, S.; Arnott, G. E.; Klumperman, B. Synthesis of α,ω-Heterotelechelic PVP for Bioconjugation, via a One-Pot Orthogonal End-Group Modification Procedure. Polym. Chem. 2016, 7, 6450−6456. (36) Wiss, K. T.; Krishna, O. D.; Roth, P. J.; Kiick, K. L.; Theato, P. A Versatile Grafting-to Approach for the Bioconjugation of Polymers to Collagen-like Peptides Using an Activated Ester Chain Transfer Agent. Macromolecules 2009, 42, 3860−3863. J


Article

ACS Applied Nano Materials (37) Stella, B.; Arpicco, S.; Peracchia, M. T.; Desmaële, D.; Hoebeke, J.; Renoir, M.; D’Angelo, J.; Cattel, L.; Couvreur, P. Design of Folic Acid-Conjugated Nanoparticles for Drug Targeting. J. Pharm. Sci. 2000, 89, 1452−1464. (38) Horcajada, P.; Chalati, T.; Serre, C.; Gillet, B.; Sebrie, C.; Baati, T.; Eubank, J. F.; Heurtaux, D.; Clayette, P.; Kreuz, C.; Chang, J.-S.; Hwang, Y. K.; Marsaud, V.; Bories, P.-N.; Cynober, L.; Gil, S.; Férey, G.; Couvreur, P.; Gref, R. Porous Metal-Organic-Framework Nanoscale Carriers as a Potential Platform for Drug Delivery and Imaging. Nat. Mater. 2010, 9, 172−178. (39) Zhang, Z.; Wang, L.; Wang, J.; Jiang, X.; Li, X.; Hu, Z.; Ji, Y.; Wu, X.; Chen, C. Mesoporous Silica-Coated Gold Nanorods as a Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418−1423. (40) Sanson, C.; Diou, O.; Thévenot, J.; Ibarboure, E.; Soum, A.; Brûlet, A.; Miraux, S.; Thiaudière, E.; Tan, S.; Brisson, A.; Dupuis, V.; Sandre, O.; Lecommandoux, S. Doxorubicin Loaded Magnetic Polymersomes: Theranostic Nanocarriers for MR Imaging and Magneto-Chemotherapy. ACS Nano 2011, 5, 1122−1140. (41) Wang, Y.; Black, K. C. L.; Luehmann, H.; Li, W.; Zhang, Y.; Cai, X.; Wan, D.; Liu, S.-Y.; Li, M.; Kim, P.; Li, Z.-Y.; Wang, L. V.; Liu, Y.; Xia, Y. Comparison Study of Gold Nanohexapods, Nanorods, and Nanocages for Photothermal Cancer Treatment. ACS Nano 2013, 7, 2068−2077. (42) Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Drug/DyeLoaded, Multifunctional Iron Oxide Nanoparticles for Combined Targeted Cancer Therapy and Dual Optical/Magnetic Resonance Imaging. Small 2009, 5, 1862−1868. (43) Lee, N. S.; Sun, G.; Neumann, W. L.; Freskos, J. N.; Shieh, J. J.; Dorshow, R. B.; Wooley, K. L. Photonic Shell-Crosslinked Nanoparticle Probes for Optical Imaging and Monitoring. Adv. Mater. 2009, 21, 1344−1348. (44) Shrestha, R.; Elsabahy, M.; Luehmann, H.; Samarajeewa, S.; Florez-Malaver, S.; Lee, N. S.; Welch, M. J.; Liu, Y.; Wooley, K. L. Hierarchically Assembled Theranostic Nanostructures for SiRNA Delivery and Imaging Applications. J. Am. Chem. Soc. 2012, 134, 17362−17365. (45) Sun, G.; Cui, H.; Lin, L. Y.; Lee, N. S.; Yang, C.; Neumann, W. L.; Freskos, J. N.; Shieh, J. J.; Dorshow, R. B.; Wooley, K. L. Multicompartment Polymer Nanostructures with Ratiometric DualEmission pH-Sensitivity. J. Am. Chem. Soc. 2011, 133, 8534−8543. (46) Eberhardt, M.; Mruk, R.; Zentel, R.; Théato, P. Synthesis of Pentafluorophenyl(meth)acrylate Polymers: New Precursor Polymers for the Synthesis of Multifunctional Materials. Eur. Polym. J. 2005, 41, 1569−1575. (47) Moad, G.; Rizzardo, E.; Thang, S. H. Living Radical Polymerization by the RAFT Process. Aust. J. Chem. 2005, 58, 379−410. (48) Thomas, D. B.; Convertine, A. J.; Myrick, L. J.; Scales, C. W.; Smith, A. E.; Lowe, A. B.; Vasilieva, Y. A.; Ayres, N.; McCormick, C. L. Kinetics and Molecular Weight Control of the Polymerization of Acrylamide via RAFT. Macromolecules 2004, 37, 8941−8950. (49) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Camptothecin: Current Perspectives. Bioorg. Med. Chem. 2004, 12, 1585−1604. (50) Garcia-Carbonero, R.; Supko, J. G. Current Perspectives on the Clinical Experience, Pharmacology, and Continued Development of the Camptothecins. Clin. Cancer Res. 2002, 8, 641−661. (51) Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. Camptothecin Induces Protein-Linked DNA Breaks via Mammalian DNA Topoisomerase I. J. Biol. Chem. 1985, 260, 14873−14878. (52) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.; Pommier, Y. Structure-Activity Study of the Actions of Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for a Specific Receptor Site and a Relation to Antitumor Activity. Cancer Res. 1989, 49, 1465−1469. (53) Pommier, Y.; Kohlhagen, G.; Kohn, K. W.; Leteurtre, F.; Wani, M. C.; Wall, M. E. Interaction of an Alkylating Camptothecin Derivative with a DNA Base at Topoisomerase I-DNA Cleavage Sites. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 8861−8865.

(54) Beaumont, K.; Webster, R.; Gardner, I.; Dack, K. Design of Ester Prodrugs to Enhance Oral Absorption of Poorly Permeable Compounds: Challenges to the Discovery Scientist. Curr. Drug Metab. 2003, 4, 461−485. (55) Deshmukh, M.; Chao, P.; Kutscher, H. L.; Gao, D.; Sinko, P. J. A Series of α-Amino Acid Ester Prodrugs of Camptothecin: In Vitro Hydrolysis and A549 Human Lung Carcinoma Cell Cytotoxicity. J. Med. Chem. 2010, 53, 1038−1047. (56) Chen, M.; Moad, G.; Rizzardo, E. Thiocarbonylthio End Group Removal from RAFT-Synthesized Polymers by a Radical-Induced Process. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 6704−6714. (57) Liu, Y.; Turunen, P.; de Waal, B. F. M.; Blank, K. G.; Rowan, A. E.; Palmans, A. R. A.; Meijer, E. W. Catalytic Single-Chain Polymeric Nanoparticles at Work: From Ensemble towards Single-Particle Kinetics. Mol. Syst. Des. Eng. 2018, 3, 609−618. (58) Petryayeva, E.; Algar, W. R. Proteolytic Assays on QuantumDot-Modified Paper Substrates Using Simple Optical Readout Platforms. Anal. Chem. 2013, 85, 8817−8825. (59) Pan, D.; Turner, J. L.; Wooley, K. L. Folic Acid-Conjugated Nanostructured Materials Designed for Cancer Cell Targeting. Chem. Commun. 2003, 2400−2401. (60) Lindhoud, S.; Westphal, A. H.; Visser, A. J. W. G.; Borst, J. W.; van Mierlo, C. P. M. Fluorescence of Alexa Fluor Dye Tracks Protein Folding. PLoS One 2012, 7, e46838. (61) Hungerford, G.; Benesch, J.; Mano, J. F.; Reis, R. L. Effect of the Labelling Ratio on the Photophysics of Fluorescein Isothiocyanate (FITC) Conjugated to Bovine Serum Albumin. Photochem. Photobiol. Sci. 2007, 6, 152−158. (62) Sjö back, R.; Nygren, J.; Kubista, M. Absorption and Fluorescence Properties of Fluorescein. Spectrochim. Acta, Part A 1995, 51, L7−L21. (63) Klonis, N.; Clayton, A. H. A.; Voss, E. W., Jr.; Sawyer, W. H. Spectral Properties of Fluorescein in Solvent-Water Mixtures: Applications as a Probe of Hydrogen Bonding Environments in Biological Systems. Photochem. Photobiol. 1998, 67, 500−510. (64) Martin, M. M. Hydrogen Bond Effects on Radiationless Electronic Transitions in Xanthene Dyes. Chem. Phys. Lett. 1975, 35, 105−111. (65) Arbeloa, I. L. Dimeric and Trimeric States of the Fluorescein Dianion. Part 1.Molecular Structures. J. Chem. Soc., Faraday Trans. 2 1981, 77, 1725−1733. (66) Li, Y.; Bai, Y.; Zheng, N.; Liu, Y.; Vincil, G. A.; Pedretti, B. J.; Cheng, J.; Zimmerman, S. C. Crosslinked Dendronized Polyols as a General Approach to Brighter and More Stable Fluorophores. Chem. Commun. 2016, 52, 3781−3784. (67) Menendez, J. A.; Vellon, L.; Mehmi, I.; Oza, B. P.; Ropero, S.; Colomer, R.; Lupu, R. Inhibition of Fatty Acid Synthase (FAS) Suppresses HER2/Neu (ErbB-2) Oncogene Overexpression in Cancer Cells. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 10715−10720. (68) Luzzio, M. J.; Besterman, J. M.; Emerson, D. L.; Evans, M. G.; Lackey, K.; Leitner, P. L.; McIntyre, G.; Morton, B.; Myers, P. L.; Peel, M.; Sisco, J. M.; Sternbach, D. D.; Tong, W.-Q.; Truesdale, A.; Uehling, D. E.; Vuong, A.; Yates, J. Synthesis and Antitumor Activity of Novel Water Soluble Derivatives of Camptothecin as Specific Inhibitors of Topoisomerase I. J. Med. Chem. 1995, 38, 395−401. (69) Cengelli, F.; Grzyb, J. A.; Montoro, A.; Hofmann, H.; Hanessian, S.; Juillerat-Jeanneret, L. Surface-Functionalized Ultrasmall Superparamagnetic Nanoparticles as Magnetic Delivery Vectors for Camptothecin. ChemMedChem 2009, 4, 988−997. (70) Ding, Y.; Zhou, Y.-Y.; Chen, H.; Geng, D.-D.; Wu, D.-Y.; Hong, J.; Shen, W.-B.; Hang, T.-J.; Zhang, C. The Performance of ThiolTerminated PEG-Paclitaxel-Conjugated Gold Nanoparticles. Biomaterials 2013, 34, 10217−10227. (71) Sangtani, A.; Petryayeva, E.; Wu, M.; Susumu, K.; Oh, E.; Huston, A. L.; Lasarte-Aragones, G.; Medintz, I. L.; Algar, W. R.; Delehanty, J. B. Intracellularly Actuated Quantum Dot−Peptide− Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway. Bioconjugate Chem. 2018, 29, 136−148. K


Article

ACS Applied Nano Materials (72) Delehanty, J. B.; Medintz, I. L.; Pons, T.; Brunel, F. M.; Dawson, P. E.; Mattoussi, H. Self-Assembled Quantum Dot−Peptide Bioconjugates for Selective Intracellular Delivery. Bioconjugate Chem. 2006, 17, 920−927. (73) Delehanty, J. B.; Bradburne, C. E.; Boeneman, K.; Susumu, K.; Farrell, D.; Mei, B. C.; Blanco-Canosa, J. B.; Dawson, G.; Dawson, P. E.; Mattoussi, H.; Medintz, I. L. Delivering Quantum Dot-Peptide Bioconjugates to the Cellular Cytosol: Escaping from the Endolysosomal System. Integr. Biol. 2010, 2, 265−277. (74) Kröger, A. P. P.; Paulusse, J. M. J. Single-Chain Polymer Nanoparticles in Controlled Drug Delivery and Targeted Imaging. J. Controlled Release 2018, 286, 326−347. (75) Cheng, C.-C.; Lee, D.-J.; Liao, Z.-S.; Huang, J.-J. StimuliResponsive Single-Chain Polymeric Nanoparticles towards the Development of Efficient Drug Delivery Systems. Polym. Chem. 2016, 7, 6164−6169. (76) Koda, Y.; Terashima, T.; Sawamoto, M.; Maynard, H. D. Amphiphilic/Fluorous Random Copolymers as a New Class of NonCytotoxic Polymeric Materials for Protein Conjugation. Polym. Chem. 2015, 6, 240−247. (77) Wong, E. H. H.; Lam, S. J.; Nam, E.; Qiao, G. G. Biocompatible Single-Chain Polymeric Nanoparticles via Organo-Catalyzed RingOpening Polymerization. ACS Macro Lett. 2014, 3, 524−528.

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