The Role of Cargo Binding Strength in Polymer-Mediated Intracellular

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

The Role of Cargo Binding Strength in Polymer-Mediated Intracellular Protein Delivery Nicholas D. Posey,† Christopher R. Hango,† Lisa M. Minter,‡,§ and Gregory N. Tew*,†,‡,§ †

Department of Polymer Science and Engineering, ‡Department of Veterinary and Animal Sciences, and §Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States

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ABSTRACT: Delivering proteins into the intracellular environment is a critical step toward probing vital cellular processes for the purposes of ultimately developing new therapeutics. Polymeric carriers are widely used to facilitate protein delivery with guanidinium-rich macromolecules leading the way within this category. Although binding interactions between natural proteins and synthetic polymers have been studied extensively, the relationship between polymer−protein binding and intracellular delivery is seldom explored. Elucidating the role of cargo binding in delivery is a promising direction that is expected to provide new insights that further optimize intracellular protein delivery. Herein, model polymeric carriers called protein transduction domain mimics (PTDMs) were studied for their ability to bind to a variety of protein cargoes, including an antibody, where the proteins encompassed a range of sizes (∼16−151 kDa) and isoelectric points (4.7−11.4). The PTDM−protein complexes were also delivered into Jurkat T cells in an attempt to establish a general correlation between binding ability and delivery outcomes. Binding assays resulted in a vast range of dissociation constants (Kd), which spanned from 3.5 to 4820 nM and indicated a variety of binding strengths between PTDM and protein. More significantly, PTDMs preferentially bound certain types of proteins over others, such as the antibody fragment over the whole antibody. Furthermore, increased PTDM−protein binding affinity did not correlate with protein delivery, suggesting that the successful internalization of complexes is independent of binding equilibrium. Although binding did not correlate with internalization here, the potential for binding affinity to impact other aspects of delivery, like cargo functionality inside the cell, remains an open possibility.

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INTRODUCTION Delivering functional proteins, like antibodies, into the intracellular environment both in vitro and in vivo is a burgeoning approach for the study and treatment of numerous diseases.1,2 Though extremely promising, the intracellular delivery of antibodies is elusive as all current antibody-based therapies are confined to extracellular activity and cell surface targets.2 Proteins are attractive drug candidates because their specific function and precise target recognition diminish the occurrence of off-target effects and create the possibility for potent and promising therapeutics.1,3 Perhaps the greatest and most current example of protein-based therapeutics are antibodies, which have rapidly emerged as a class of drugs with ten new market approvals in the US and EU in 2017 alone, the most in any one year to date.4 In general, the potency of protein-based therapies in vivo is hindered by their lack of bioavailability, especially if administered orally or intravenously.1 The structural complexity of proteins further impedes their development and formulation into therapeutics.5 Even in the simplified case where proteins are delivered into cells in vitro, uptake is mitigated by the cellular membrane, which acts as a barrier against large, charged macromolecules.2,6,7 © XXXX American Chemical Society

For researchers intending to intracellularly deliver proteins, the cellular membrane is a serious imposition and consequently has been addressed by a multitude of membranepenetrating technologies including, but not limited to, microinjection, electroporation, microfluidics, virus-like capsules, macromolecular carriers, lipids, and other nanoparticles.8−10 One interesting membrane-penetrating strategy was inspired by a viral protein, called HIV-1 transactivator of transcription (tat), which exhibited self-promoted translocation across the cell membrane by virtue of a specific, short amino acid sequence. This sequence, which was found to be rich in basic residues like arginine, was termed a protein transduction domain (PTD) and was responsible for the protein’s membrane crossing activity.11,12 Though discovered first, HIV-1 tat now represents only one of many natural and synthetic cell-penetrating proteins and peptides (CPPs), which include molecules like transportan, Pep-1, penetratin, CADY, and MPG.7,13−18 Structure−activity relationships with CPPs have revealed the importance of the guanidinium functional Received: May 23, 2018 Revised: July 11, 2018

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DOI: 10.1021/acs.bioconjchem.8b00363 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

affinity binding was associated with delivery.10,15 That one report demonstrated binding and delivery of mostly small peptide cargoes and only two proteins, which limited the scope of the study, leaving unanswered questions about the relationship between binding and delivery for more complex cargoes. In a broader sense, studying protein binding with respect to delivery is important because it is not clear that binding to various types of protein cargo would be identical. Proteins are the most complex of all biomolecules, possessing many charged amino acid residues in various locations throughout their precisely folded structure, resulting in a unique conformation and 3D shape.54,55 Furthermore, proteins are complicated by their highly nonuniform surfaces brought about by a heterogeneous collection of neutral, charged, and hydrophobic surface patches.56 A comparison between proteins and nucleic acids further highlights the complexity of proteins as biopolymers and as bioactive cargoes. For example, nucleic acid-based cargoes are always highly negatively charged due to their regularly spaced backbone phosphate groups, unlike proteins, which have heterogeneous, patchy surfaces. Given the complexity of protein cargo and that the binding−delivery relationship is severely understudied, studying protein binding and clarifying its impact on protein delivery would greatly benefit the field. For the present study, a model, noncovalent polymer− protein delivery system with known protein binding and delivery capabilities was required to focus on isolating the relationship between binding and delivery. Previous work by our group has established such a system, using PTDMs based on a ring-opening metathesis polymerization (ROMP) scaffold, which can deliver functional protein cargo in difficult-to-transfect cell types, where the structures of the PTDMs used in this study are shown in Figure 2A.2,30,32,35,36 This delivery platform has demonstrated delivery with a variety of protein cargoes, though quantitative binding has only been determined with respect to EGFP, limiting our understanding of the protein-binding landscape.2,30,32,35,36,39 For example, the increase in binding strength between EGFP and one series of PTDMs, which varied the distribution of hydrophobic groups or PTDM architecture, appeared to correlate with an increase in EGFP internalization into T cells; however, it was not clear whether increased internalization was due to changes in binding strength, PTDM hydrophobic group distribution, or both.36 That initial report, in conjunction with the results of a separate protein-delivery experiment shown in Figure 2B using PTDM1 and EGFP, provided motivation to understand the importance of binding to delivery.35 In Figure 2B, “Complexed” refers to the fact that separate solutions of PTDM and EGFP were mixed together and allowed to complex (bind) prior to delivery, whereas “NonComplexed” denotes that separate PTDM and EGFP solutions were not mixed together and not allowed to complex prior to delivery. Instead, each solution was added separately to the cells causing PTDM and EGFP to encounter each other once in the presence of cells and media containing other serum proteins. Removing this initial complexation step eliminated the opportunity for PTDM−protein binding to occur prior to delivery, resulting in a marked decrease in protein internalization as compared to when PTDM−protein complexes were formed prior to delivery (Figure 2B).

group, contained in arginine residues, for the CPP’s ability to penetrate the membrane.19−21 PTDs and CPPs are used to deliver cargo by either covalent attachment to or noncovalent complexation with the cargo, though in the case of protein delivery, covalent attachment is most common.8,10,29,15,22−28 More recently, CPPs and PTDs have been mimicked using synthetic polymers, called CPPMs or PTDMs, that incorporate guanidinium side chains as the critical design element.6,13,35,36,19,20,22,30−34 For protein delivery applications, CPPMs, like CPPs, can either be covalently bound to or noncovalently complexed with their cargo to facilitate delivery.19,30,32 Although covalent polymer−protein conjugation has the advantage of stabilizing the protein cargo, it usually involves reactions with the protein’s functional groups, potentially impacting the protein’s function; furthermore, choosing the most appropriate covalent conjugation method from a variety of techniques and executing that additional synthetic step impedes their widespread implementation.37 Therefore, noncovalent protein delivery strategies, which do not require conjugation procedures, serve as promising alternatives to their covalent protein−polymer conjugate counterparts. Given the importance of the noncovalent interactions required for this alternative strategy, the cargo binding step becomes critical for complex formation and is of great interest to the field.10,24,36,38−41 Although many reports concerning polymer−protein binding interactions exist, including coacervation and polyelectrolyte complexation studies, they either do not study intracellular delivery or focus on nondeliveryrelated applications.42,43,52,44−51 Furthermore, despite many reports containing information on both protein binding and delivery, the direct relationship between these two aspects of delivery is rarely addressed or explicitly correlated.10,15,23,24,36,38,40 Most protein cargo alone cannot penetrate the cellular membrane, making internalization dependent on complex formation with a cell-penetrating carrier.10,49 Understanding binding as an equilibrium process, instead of a simple requirement that needs to be fulfilled, should create new opportunities for delivery optimization. A higher binding constant (Kb), or lower dissociation constant (Kd), indicative of stronger carrier−protein affinity, would ensure that more protein was incorporated in complexes,53 increasing the chance that it would be delivered (Figure 1). The impact of cargo binding on cellular uptake has been alluded to before in the CPP literature but was only explored in one report that demonstrated two main points: (1) a lack of binding between CPPs and their cargo resulted in no delivery and (2) high-

Figure 1. Generic binding scheme that emphasizes binding as an equilibrium process, showing the relationship among Keq, Kd, and Kb, and highlighting the role of binding in complex formation and its impact on delivery. B


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Figure 2. (A−D) Adapted from our previously published literature.30,35,36 (A) Structures of previously published PTDMs used in this study.30,35,39 (B) Uptake of EGFP into T cells demonstrating the necessity of the 30 min binding/complexation step prior to delivery, where “NonComplexed” means that EGFP was not complexed with its respective carrier prior to cellular uptake (Reprinted from Biochimica et Biophysica Acta (BBA) − Biomembranes, 1858, Coralie M. Backlund, Toshihide Takeuchi, Shiroh Futaki, Gregory N. Tew, Relating structure and internalization for ROMPbased protein mimics, 1443−1450, Copyright 2016, with permission from Elsevier).35 (C) Cellular uptake data demonstrating the ability of a block copolymer PTDM to deliver various protein cargoes into T cells (Reprinted from Journal of Controlled Release, 254, Federica Sgolastra, Coralie M. Backlund, E. Ilker Ozay, Brittany M. deRonde, Lisa M. Minter, Gregory N. Tew, Sequence segregation improves noncovalent protein delivery, 131−136, Copyright 2017, with permission from Elsevier).36 (D) Direct comparison of PTDM1 versus PTDM2 for EGFP cellular uptake into T cells (Adapted from ref 30 with permission of The Royal Society of Chemistry, http://dx.doi.org/10.1039/C6PY01615D).30

Additional studies have also detailed the ability of PTDMs to deliver other types of fluorescently labeled protein cargo, some of which are highlighted in Figure 2C, although binding was not quantified.2,32,36 The fact that PTDMs have delivered multiple types of protein cargo motivated the selection of this platform as a model delivery system to further examine the relationship between binding and delivery. Finally, it is important to note that both PTDM1 and PTDM2 (structures shown in Figure 2A) are capable of delivering protein, though not to the same level, as shown in Figure 2D, where the difference in performance was attributed to the discrepancy in hydrophobicity between the two PTDMs.30,39 However, their relative binding abilities were not measured, suggesting that differences in binding strength might have also affected the results, providing motivation for the studies reported here. Given this platform’s well-documented history of protein binding and delivery, it was chosen for use in this work’s deeper investigation of binding and delivery with respect to a wider range of protein cargoes. Specifically, this study utilized PTDM1 and PTDM2 to bind to a larger selection of protein cargoes to determine if differences in the attributes of the proteins would lead to variability in binding affinities. Only two PTDMs were used in this study, as opposed to our previous work, so that the emphasis of our experiments could be placed on the role that protein identity played in affecting binding as opposed to the role that polymer chemistry plays, which has already been thoroughly explored. Changing the focus here to attributes of the proteins instead of the PTDMs was critical because an in-depth analysis of our PTDMs binding to various proteins had not been carried out to date, and it was not clear that binding would be the same in all cases given the surface heterogeneities of each protein. Differences in binding affinity

for all PTDM−protein complexes were quantified by determining Kd using fluorescence-based, equilibrium binding experiments and then correlated to internalization outcomes. In this report, we first demonstrated that the PTDM1 exhibited preferential protein binding dependent on the relative attributes of the proteins and second, that differences in binding affinity over several orders of magnitude did not correlate with the internalization despite the preliminary evidence suggesting otherwise.35,36

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RESULTS AND DISCUSSION Experimental Design and Selection of Binding Pairs. To probe for differences in PTDM binding affinities, we selected a variety of proteins with a range of characteristics for use in fluorescence-based equilibrium binding experiments. The PTDM−protein pairs selected for binding experiments are depicted in Figure 3 along with the chemical structures of the PTDMs. PTDM1 was used to bind to all fluorescent proteins, whereas PTDM2, the less hydrophobic variant, was only used in binding experiments with the net negatively charged proteins. The names of the proteins are color coded in Figure 3 such that proteins with a net negative charge at pH 7.2 are orange, proteins with a net neutral charge are gray, and proteins with a net positive charge are purple. The charge assignments are based on the proteins’ isoelectric points (pIs), which were obtained from their respective manufacturers and reported in Table 1. The protein names colored in black in Figure 3 are for polyclonal antibodies with a broad range of pIs, which can be estimated using literature (Table 1).57 Additional characteristics of each protein, including the average number of fluorescent FITC labels per protein, their approximate molecular weights, and their pIs, along with their C


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occurred.36,39 EGFP undergoes quenching due to bindinginduced changes in its conformation, causing the perturbation of the specific residues in the local, electronic microenvironment, destabilizing the fluorophore.59 PTDM1 Binding to BSA-FITC, Streptavidin-FITC, and Avidin-FITC. The results of the binding experiments with PTDM1 and BSA-FITC, streptavidin-FITC, and avidin-FITC are shown in Figure 4, where the binding curves, as a function of increasing PTDM concentration, are shown in Figure 4A− C, and their corresponding fractional saturation plots are shown in Figure 4D−F. The curves were fit using eq 1 fractional saturation = =

SL − S0 Ssat − S0

([L T] + n[R T] + Kd) −

([L T] + n[R T] + Kd)2 − 4[L T]n[R T] 2n[R T]

(1) Figure 3. PTDM−protein binding pairs selected for the binding study, where arrows show which proteins were bound by which PTDM in separate, respective experiments. Protein names are color coded according to their net charge at pH 7.2, where net negative charge is orange, net neutral charge is gray, and net positive charge is purple.

where SL is the measured fluorescence signal from a solution containing a fixed concentration of PTDM, S0 is the fluorescence signal of the protein-only positive control (∼200 nM), Ssat is the fluorescence signal at saturation, n is the number of binding sites, LT is the total PTDM concentration, RT is the total protein concentration (∼200 nM), and Kd is the dissociation constant. Eq 1 represents a multiple, independent binding sites model and defines fractional saturation as the fraction of total binding sites occupied on the receptor molecule (protein) by the ligands (PTDMs). Accompanying n values from all fitting procedures are shown in Table S1.36,53,60 The results of the binding experiments using PTDM1 with BSA-FITC, streptavidin-FITC, and avidin-FITC are presented together because they possessed similar sizes (∼54−71 kDa) differing net charges under the experimental conditions (Table 1). PTDM1 was used here as a constant to isolate the effect of changing the protein cargo while maintaining polymer structure. Binding was tightest with the net negatively charged BSA-FITC yielding a calculated Kd in the single digit nM range (Kd = 3.5 nM) (Figure 4D). Binding to the net neutral protein, streptavidin-FITC, resulted in a calculated Kd that was significantly higher, in the triple digit nM range (Kd = 382 nM), indicative of weaker binding (Figure 4E). Finally, binding to the net positively charged avidin-FITC resulted in a calculated Kd in the thousands of nM, or μM, range (Kd = 2,700 nM), representing the weakest binding of the three proteins (Figure 4F).

full and abbreviated names, are found in Table 1. All proteins used in this study, except EGFP, had FITC dye labels unless otherwise noted in the text. Although this selection of proteins is not fully comprehensive, it represents a broad selection of commercially available, dye-labeled proteins that enabled this comparative binding study. Equilibrium Fluorescence-Based Binding Experiments. All binding experiments were carried out in PBS at pH ∼7.2 and 25 °C, where the quenching of either fluorescently dye-labeled proteins or EGFP was monitored as a function of increasing PTDM concentration in 96-well plates. The resultant decrease in fluorescence intensity from each well in the PTDM titration series was normalized and used to construct a binding curve that yielded Kd upon fitting. Fluorescence quenching was selected because it is highly sensitive at extremely low concentrations.10,24,53,58 For example, one report used peptides with FITC labels and monomeric red fluorescent protein (mRFP) to measure binding constants with CPPs.10 In other reports, where the binding to the inherently fluorescent EGFP was studied, increasing PTDM concentration led to fluorescence quenching, which suggested that a binding interaction had

Table 1. Summary of Protein Information and Dissociation Constants (Kd) protein name

no. of FITC molecules per proteine

∼MW (kDa)g

isoelectric points (pIs) or rangeh

net charge at pH 7.2

BSA-FITCa EGFPb streptavidin-FITC IgG-F(ab′)2-FITCc IgG-FITCd avidin-FITC lysozyme-FITC

11 − 3 2.7 3.8 3 3.5f

71 32.7 54 111 151 67 16

4.7 6.2 6.8−7.5 8.7−9.3i 5.9−9.0i 10−10.5 11.4

negative negative neutral − − positive positive

dissociation constant Kd (nM)j 3.5 ± 10.5 ± 382 ± 248 ± 4,820 ± 2,700 ± −

1.9 5.1 56 39 830 630

Bovine serum albumin. bEnhanced green fluorescent protein. cImmunoglobulin G fragment. dImmunoglobulin G. eReported by the manufacturer. Represents average of range of 2−5 FITC molecules per protein reported by manufacturer. gApproximate molecular weight taking into account the average number of FITC molecules per protein. hProvided by manufacturer unless otherwise noted. iIsoelectric point ranges for similar antibodies (polyclonal, anti-colchicine IgG and F(ab) (a fragment) produced in goat).57 jDissociation constants determined from nonlinear, least-squares regression fitting of the fractional saturation plots for each PTDM1−protein binding curve using eq 1. Numbers shown with ± the standard error associated with the fit. a f

D


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Figure 4. Comparison of (A) BSA-FITC, (B) streptavidin-FITC, and (C) avidin-FITC binding curves generated from binding experiments with PTDM1 (average of three independent trials) using equilibrium fluorescence quenching experiments. Respective fits of the fractional saturation plots using eq 1 for each protein (D) BSA-FITC, (E) streptavidin-FITC, and (F) avidin-FITC with the calculated Kd values shown with ± the standard error associated with the fitting. All graph error bars represent ± one standard deviation. NOTE: x-axis ranges are not the same for each protein because the saturation point for each protein varies.

For this set of proteins, PTDM1’s protein binding affinity substantially increased as the pI of the protein decreased, resulting in a clear PTDM binding preference for the net negatively charged BSA-FITC over the net neutral or net positive proteins. Interestingly, streptavidin-FITC and avidinFITC have similar 3D structures, yet PTDM1 demonstrated a clear binding preference for the neutral protein streptavidinFITC over the positive protein avidin-FITC. These results were consistent with other reports that have demonstrated selective peptide and protein binding on the basis of charge or electrostatic “complementarity” using polystyrene-based amphiphilic, random copolymers.61,62 In a separate example, a positively charged, arginine-based CPP exhibited similarly strong binding preferences for negatively charged cargo.10 However, in our case, PTDM1 was still able to bind to its positively charged protein cargo, whereas the CPP was unable to bind to a high pI, positively charged peptide cargo. PTDM1 was likely still able to bind a net positively charged protein due to negatively charged protein surface patches, which are known to facilitate binding with charged polymers.50 By studying binding to proteins with varying net charges in this particular size range, clear PTDM binding preferences were established. PTDM1−protein complexation was simultaneously assessed by measuring the turbidity, or % transmittance (%T) of 700 nm light, in the same solutions used in the fluorescence quenching experiments. A concurrent decrease in %T with increasing PTDM concentration, as compared to a PTDM1only solution (Figure S3), was observed for each binding pair as the fluorescence decreased except in one case (lysozymeFITC, Figures S4−S13 and Table S2). The decrease in %T is consistent with aggregation and the formation of a suspension providing additional evidence that complexes had formed.

To confirm that quenching occurred as a result of macromolecular binding and to evaluate whether small molecules with guanidinium ions could produce the same observed fluorescence quenching, BSA-FITC was titrated with increasing concentrations of a solution containing the guanidinium-based monomer used to synthesize these PTDMs (Figure S14). This resulted in no significant quenching over the same guanidinium ion concentration range for the corresponding PTDM titrations (Figure S15), suggesting that the observed FITC quenching in the preceding PTDM binding experiments was not due merely to the presence of guanidinium ions in solution. Finally, to determine if the tight binding to BSA-FITC or generalized fluorescence quenching was a result of using an FITC dye label, BSA labeled with Alexa Fluor 488 (AF488) was used in an analogous binding experiment with PTDM1. In this experiment, the binding curve and subsequent fitting also resulted in saturation at similarly low PTDM concentrations with a low Kd indistinguishable from that of BSA-FITC (Figures S16 and S17). PTDM1 Binding Experiments with EGFP and Lysozyme-FITC. To broaden the binding study, binding experiments with EGFP (32.7 kDa, pI = 6.2, net negative) and lysozyme-FITC (16 kDa, pI = 11.4, net positive) were also conducted with the results shown in Figures S18 and S19 and Table 1. The binding of PTDM1 to EGFP yielded a low dissociation constant (Kd = 10.5 nM) indicative of tight binding (Figure 4D). Although titrating lysozyme-FITC with PTDM1 resulted in FITC fluorescence quenching, no complexes were formed as no appreciable decrease in %T was observed, unlike all other proteins (Figures S4−S13). Furthermore, internalization studies discussed later demonstrated that lysozyme could not be delivered. Lysozyme is the E


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for these binding experiments. The binding of PTDM1 to the antibody fragment was much tighter than the binding to the whole antibody given that its curve was steeper in its initial decline and saturated at a lower PTDM concentration than the binding curve for the whole antibody (Figure 6A). Fitting the fractional saturation plots with eq 1 confirmed that the antibody fragment exhibited tighter binding to PTDM1 than the whole antibody with calculated Kd’s of 248 and 4,820 nM, respectively (Figure 6B). In this case, the estimated pI range for the antibody fragment is 8.7−9.3 based on its similarity to another polyclonal antibody fragment also produced in goat,57 which presumably would impart an overall net positive charge on the antibody fragment at pH ∼7.2. However, the binding of the antibody fragment by PTDM1 was similar in strength (Kd = 248 nM) to the binding of the net neutrally charged streptavidin-FITC (Kd = 382 nM). In contrast, the whole antibody, also polyclonal, had a much broader estimated pI range of 5.9−9.057 that presumably would cause a mixture of net negative, neutral, and positive charges, making it surprising that it exhibited such weak binding, similar to the purely net positively charged avidin-FITC (Table 1). If the pI of the protein was the sole factor in determining the binding strength of the PTDM, then the Kd from the whole antibody binding experiment would have been more similar to streptavidin-FITC, while the Kd from the antibody fragment binding experiment would have been larger given its higher pI range. Because this was not the case, it is clear that pI alone does not dictate binding strength or preferential binding. However, binding still occurred in the case of the antibodies because polyelectrolyte−protein binding is not solely dependent on net charge (pI) but rather is facilitated by charged surface patches on the protein, which can be opposite to the net charge of the protein, or by hydrophobic interactions.43,50 Preferential binding between the antibody and its fragment did not follow the previously observed pI trend, which may be due to one of several factors. For example, the size of the cargo could have affected the binding trend with respect to pI because both the antibody (∼150 kDa) and its fragment (∼110 kDa) are significantly larger than any of the previous proteins (Table 1). Furthermore, the shapes of the antibody and its fragment are markedly distinct from the other proteins. Finally, while it is

smallest and the most positively charged protein, based on its pI, studied here.63 The high concentration of surface charge coupled with a low abundance of negatively charged amino acids, 9 out of 129 total, likely prevented the binding of the cationic PTDM1. Though BSA-FITC and EGFP had different sizes, they still exhibited equally tight binding with PTDM1, emphasizing the importance of protein pI over protein size. To illustrate this observed correlation between pI and Kd, the Kd for each protein was plotted against its pI (Figure 5). In general, as the pI decreased, the binding strength of PTDM1 to those proteins increased as evidenced by the concurrent decrease in Kd over several orders of magnitude.

Figure 5. Plot of Kd values for each protein binding with PTDM1, labeled with the name of the protein, as a function of pI showing tighter binding for the proteins with lower pIs. Orange text denotes a net negative charge; gray text denotes a net neutral charge, and purple text denotes a net positive charge on the protein. Streptavidin-FITC had a manufacturer-reported pI range of 6.8−7.5, which was averaged for the purposes of plotting it here.

PTDM1 Binding to IgG-FITC and IgG-F(ab′)2-FITC. While the first set of proteins strongly suggests the importance of protein net charge for preferential binding, the second set of proteins illustrates a case of preferential binding that does not seem to depend on protein net charge. Specifically, a whole antibody (IgG-FITC) (∼151 kDa) and its corresponding fragment (IgG-F(ab′)2-FITC) (∼110 kDa) (Table 1), which did not have the whole antibody’s constant region, were used

Figure 6. (A) Comparison of IgG-FITC (closed circles) and IgG-F(ab′)2-FITC (open squares) binding curves using PTDM1 (average of three independent trials) using equilibrium fluorescence quenching experiments. (B) Respective fits of the fractional saturation plots for each protein with Kd determined from fitting with eq 1 and shown with ± the standard error associated with the fit. All error bars represent ± one standard deviation. F


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Figure 7. Comparison of respective fits of the fractional saturation curves (average of three independent trials) using (A) EGFP with PTDM1, (B) EGFP with PTDM2, and (C) BSA-FITC with PTDM2, where eq 1 was used to fit the curve and calculate Kd, which is shown in the graph with ± the standard error associated with the fit. All error bars represent ± one standard deviation.

FITC was also studied (Figure 7C and Figure S21). The calculated Kd for PTDM2 binding to BSA-FITC was 18.5 nM, again indicative of tight binding, and was similar to PTDM1’s Kd for binding to BSA-FITC (Figure 4D). Analogous to PTDM2 binding to EGFP, the fit of the fractional saturation plot for PTDM2 binding to BSA-FITC did not capture all of the data points as accurately as it did in the case of PTDM1 binding to BSA-FITC. In both cases where PTDM2 was used, the binding curves and the corresponding fractional saturation plots exhibited multiple slopes. Although the accurate determination of Kd was hindered for PTDM2, PTDM2 still exhibited tight binding to EGFP and BSA-FITC based on the features of its binding curves, like steep quenching, which were similar to PTDM1’s binding curves. BSA-FITC was chosen because it has a hydrophobic binding pocket containing two tryptophan residues.64 This hydropobic binding pocket was expected to make BSA-FITC more sensitive to a decrease in PTDM hydrophobicity. In the end, even the less hydrophobic PTDM2 exhibited equally strong binding to BSA-FITC when compared to its PTDM1 counterpart. Although PTDM2’s binding curves are more complex than any of PTDM1’s binding curves, demonstrated most clearly with EGFP, these studies with PTDM2 again point to the importance of electrostatics in PTDM binding given that high binding affinity was maintained despite the decrease in PTDM hydrophobicity. It is possible, however, that the architecture and backbone of the PTDMs used here still provided enough requisite hydrophobicity for binding such that changes to side chain hydrophobicity had little impact on binding, consistent with past studies.36,39 Within the polymer architecture studied here, decreasing or increasing the PTDM hydrophobicity through side chain modification does not significantly influence the protein binding, though such modifications can increase a PTDM’s ability to deliver protein intracellularly (Figure 2D).30,39 Intracellular Uptake of PTDM−Protein Complexes into Jurkat T Cells. Taking advantage of PTDM1’s binding preferences, cellular uptake experiments were carried out to understand how disparate binding strengths generally correlated with internalization outcomes. Because cellular uptake in this system can be influenced by a variety of factors, especially PTDM hydrophobicity,30,33,35,36,39 it was critical that the same PTDM was used in all cases here to elucidate a broader correlation between PTDM−protein Kd and internalization. Internalization outcomes in this model system were determined by flow cytometry. Internalization was deemed

possible to estimate the net charges of the antibody and its fragment, they do not have one set pI, making it difficult to analyze their binding with respect to pI like the other proteins. Although the protein binding survey above generally demonstrates the importance of electrostatics and net charge to preferential binding, the close structural comparison between the antibody and its fragment suggests that other factors also play a role given that preferential binding occurred in this pair despite their lack of defined pIs. Though the exact reasons for preferential binding are not clear, PTDM1 still exhibits clear and substantial binding preferences for some types of protein cargo over others with Kd’s ranging over 3 orders of magnitude for the six proteins studied here. Comparative Binding Studies with PTDM2. PTDM hydrophobicity is critical for promoting intracellular delivery, and Figure 2D shows an example of this where PTDM1 had increased EGFP delivery compared to PTDM2, its less hydrophobic counterpart. In this previous study, it was unclear whether the discrepancy in PTDM performance was also related to the relative cargo binding abilities of each PTDM. Therefore, to determine if decreased PTDM hydrophobicity results in diminished binding affinity, while holding PTDM block architecture constant, PTDM2 was selected for binding experiments with EGFP and BSA-FITC, which has a hydrophobic binding pocket.64 In a previous report, where PTDM1 was the least hydrophobic PTDM, increasing the overall PTDM hydrophobicity in a series of polymers did not lead to an increase in EGFP binding affinity, implying that polymer hydrophobicity, above a threshold, did not increase binding affinity.39 As a comparison, PTDM1 binding to EGFP is shown in Figure 7A and Figure S18, and the binding curves with PTDM2 can be found in Figures S20 and S21. The fitted fractional saturation plot for PTDM2 binding to EGFP, with the calculated Kd value, can be found in Figure 7B. Similar to PTDM1, PTDM2 appeared to bind EGFP tightly due to the steep quenching and onset of saturation at low PTDM concentrations, which resulted in a calculated Kd of 10.6 nM (Figure 7B and Figure S20). However, the fit of PTDM2’s fractional saturation with eq 1 did not capture all of the data points as accurately as it did with the fit of PTDM1’s data points, as evidenced by the larger error associated with the resultant Kd value. Close examination of the PTDM2-EGFP fractional saturation plot also revealed multiple slope changes in the data points of the curve, unlike the data for PTDM1. Though the decrease in PTDM hydrophobicity did not influence EGFP binding affinity, PTDM2’s affinity for BSAG


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Figure 8. Intracellular uptake of FITC-labeled cargo (IgG, IgG fragment, lysozyme, avidin, and streptavidin) into Jurkat T cells at 37 °C after 4 h of incubation using PTDM1. (A) Percent positive cells for IgG, IgG fragment, and lysozyme. (B) MFI for IgG, IgG fragment, and lysozyme. (C) Percent positive cells for avidin and streptavidin. (D) MFI for avidin and streptavidin. All data represent 10,000 recorded events in a population of Jurkat T cells treated with PTDM1−protein complexes (60 nM protein +1,200 nM PTDM1) in complete media and analyzed using flow cytometry. Error bars represent the mean ± SEM for three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant, as calculated by one-way ANOVA followed by a Bonferroni post-test.

(Figure S13 and Table S2). In the case of the internalization of avidin and streptavidin, as shown in Figure 8C, both attempts were successful as evidenced by high % positive values (∼70%). Furthermore, the MFIs of streptavidin and avidin also appeared higher than both their controls, which further confirmed their successful delivery (Figure 8D). Overall, it appeared that despite large discrepancies in binding strength PTDM−protein Kd did not correlate with internalization. The case of the two antibody-based complexes provides compelling evidence that binding is uncorrelated to delivery outcomes. The internalization of the whole antibody complex (IgG) was successful despite the antibody’s weak binding. From this, it is clear that weak polymer−protein binding does not necessarily inhibit protein internalization, which expands upon the findings of a previous study with the CPP Pep-1, which could still deliver peptide cargo despite similarly weak binding.10 That previous study mostly focused on peptide carriers binding and delivering other small peptides, whereas the study here focused exclusively on synthetic carrier binding and delivering larger, more complex protein cargoes. Additionally, the antibody fragment’s binding affinity (Kd = 248 nM) was significantly stronger than the whole antibody’s binding affinity (Kd = 4820 nM), yet stronger cargo binding, by an order of magnitude, did not enhance internalization. Similarly, as seen in Figure 8C and D, the delivery of the avidin and streptavidin complexes were also deemed successful despite the disparate binding strengths (Kd = 2,700 nM for avidin and Kd = 382 nM for streptavidin).

either successful or unsuccessful because some proteins had more FITC labels than others (Table 1), whereas EGFP had an entirely different fluorophore, making comparisons based on fluorescence brightness problematic, which could have biased more in-depth comparisons of the flow cytometry data beyond merely successful or unsuccessful. After quantifying the binding affinities for all PTDM− protein pairs, Jurkat T cells were treated with the same PTDM−protein complexes in an attempt to correlate the impact of disparate binding affinities on cellular uptake. PTDM1 was used to facilitate the uptake of the various proteins where the percentage of the cell population receiving FITC-labeled cargo (% positive cells) appears in Figure 8A (for IgG, IgG fragment, and lysozyme) and Figure 8C (for avidin and streptavidin), and the median fluorescence intensities (MFIs) of the entire live cell population for those proteins appears in Figure 8B (for IgG, IgG fragment, and lysozyme) and Figure 8D (for avidin and streptavidin). The internalization of EGFP using PTDM1 and PTDM2 has been previously reported and was used here as a positive control (Figure S22).30,35,39 All other data for cellular uptake experiments, including cell viability and histograms, can be found in Figures S22−S24. As seen in Figure 8A and B, the internalization of the antibody, IgG, and its corresponding fragment appeared equally successful, as evidenced by both having high % positive (>80%) and MFI values as compared to the controls. In contrast, the internalization of lysozyme, by MFI or % positive values, was unsuccessful due to the lack of complex formation H


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Bioconjugate Chemistry Both of these results expand upon the findings of a previous report that demonstrated that changing the peptide carrier resulted in a roughly 3-fold increase in the binding affinity for the same whole protein cargo, but it did not result in a difference in delivery outcomes.10 Unlike this previous report, the same carrier was used here in all experiments to avoid the impact of changing carriers on internalization outcomes. Moreover, the increases in binding affinities in our case were by an order of magnitude, much greater than the aforementioned report, yet did not significantly impact cargo internalization. The present study clearly demonstrates that enhancing binding strength, even by an order of magnitude, has no correlation to internalization. Overall, PTDM−protein binding was uncorrelated with internalization outcomes within the range of Kd’s studied here except in the case of lysozyme-FITC. These results expound upon the findings of Kurzawa et al., who found a correlation between a total lack of binding and failed delivery using peptide cargoes and two types of protein cargo.10 The two studies taken together support the concept of a minimum binding threshold below which complex formation is impeded and delivery suffers. The PTDM−protein Kd’s calculated here are likely above that threshold, which is why the binding was uncorrelated with internalization. Perhaps all binding, apart from lysozyme, was sufficient to facilitate complex formation for successful internalization, and binding affinities weaker than what were explored here would be required before a correlated decrease in internalization could be observed.

of the role of binding in the delivery of functional proteins, like enzymes and antibodies, as it pertains to cargo release and bioavailability. Therefore, fundamentally understanding biophysical interactions, including binding strength, between polymers and their cargo will continue to be important as the field of bioactive cargo delivery evolves.

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EXPERIMENTAL SECTION Materials. All proteins were purchased with dye labels and used directly without further purification or modification. Albumin, fluorescein isothiocyanate conjugate from bovine (BSA-FITC), anti-mouse IgG (whole molecule)-FITC produced in goat, affinity isolated antibody (IgG-FITC), and antimouse IgG (Fab specific) F(ab′)2 fragment-FITC antibody produced in goat, affinity isolated antibody, buffered aqueous solution (IgG F(ab′)2-FITC) were purchased from SigmaAldrich. Albumin from bovine serum (BSA), Alexa Fluor 488 conjugate (BSA-AF488) was purchased from Thermo Fisher Scientific/Invitrogen/Molecular Probes, Inc. Lyophilized, powdered enhanced green fluorescent protein (EGFP) was purchased from BioVision, Inc. Lysozyme FITC labeled from bovine (lysozyme-FITC) was purchased from Nanocs, Inc. Avidin, FITC conjugate (avidin-FITC) and streptavidin protein FITC (specifically Thermo Fisher’s recombinant streptavidin, which is ∼53 kDa and near neutral isoelectric point) (streptavidin-FITC) were purchased from Thermo Fisher Scientific, Inc. Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific, and DMSO-d6 was purchased from Cambridge Isotopes Laboratory, Inc. PBS buffer, 10×, pH 7.4, was purchased from Thermo Fisher Scientific, Inc. and was diluted to 1× with Milli-Q water and adjusted to pH 7.2 prior to use. Trifluoroacetic acid (TFA) was purchased from Alfa Aesar and used without further purification. Methanol and dichloromethane were purchased from Fisher Scientific and used without further purification. Though not synthesized again here, all materials required for the synthesis of PTDM1 and PTDM2 have been previously reported.30,35,39 Materials for intracellular delivery into Jurkat T cells included Gibco phosphate-buffered saline (PBS) pH 7.4 10×, which was purchased from Life Technologies and diluted to 1× before use along with Gibco trypsin-EDTA (0.25%) and Gibco RPMI 1640 GlutaMAX medium, which were purchased from Life Technologies. EquaFETAL bovine serum (a bioequivalent of FBS) was purchased from Atlas Biologicals. Sodium pyruvate solution (100 mM), penicillin/streptomycin (10K/10K), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 1×), and nonessential amino acids (NEAA, 10 mM, 100×) were purchased from Lonza or Fisher Scientific. 7AAD viability stain was purchased from BD Biosciences. Instrumentation. Fluorescence-based binding assays were conducted using a BioTek Instruments Synergy Mx plate reader with Gen5 1.10 software. Flow cytometry experiments were conducted with a BD LSRFortessa Dual with 18 color analysis capabilities and five lasers set at excitation wavelengths of 355, 405, 488, 561, and 640 nm. The 1H NMR spectrum was recorded at 500 mHz using a Bruker Ascend (500 mHz) Nuclear Magnetic Resonance Spectrometer retrofitted with a cryoprobe (500 mHz). PTDMs. PTDM1 and PTDM2 were selected for this study from a collection of previously synthesized PTDMs,30,35,39 and their structures are depicted in Figure 2A. The full synthesis and characterization of these polymers have been previously reported.30,35,39 The theoretical molecular weights of these

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CONCLUSIONS The role and importance of PTDM−protein complex Kd were explored with respect to intracellular protein delivery. The PTDMs used here exhibited substantial binding preferences for some proteins over others with calculated Kd’s spanning 3 orders of magnitude. In general, the tightest binding was observed for the proteins with low pIs and net negative charges, whereas the weakest binding was observed for higher pIs. Although protein pI appeared to play a significant role in establishing preferential protein binding, the case of the two antibody-based cargoes suggested that pI alone does not solely dictate binding and that other factors should be considered in the future, such as size or shape. Intracellular uptake studies clearly demonstrated that binding was not correlated to internalization outcomes. Overall, the readiness and ability of the PTDM to bind protein seems to be of little importance for promoting protein internalization despite the broad Kd range studied here. It is possible that, once a minimum binding affinity threshold is crossed, binding no longer influences internalization and that the range of Kd’s studied here is above that threshold. The case of lysozyme confirms that PTDMs must exhibit cargo binding to promote intracellular internalization. The comparison of PTDM1 and PTDM2 also emphasizes the lack of correlation between binding and delivery as both had similar Kd’s here but previously reported differences in EGFP internalization. Furthermore, the cellular uptake was quantified here by focusing on the overall internalization of the protein without regard to its function. Having shown here that internalization outcomes can be similar, despite varying cargo binding strengths, this study opens up the possibility for future studies in which cargo binding strength can be directly correlated with cargo activity levels in the intracellular environment postdelivery. This is anticipated to lead to a deeper understanding I


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Fitting Binding Curves. The normalized binding curves were generated, as described above, by plotting the relative fluorescence intensity of a given well on the y-axis versus PTDM concentration on the x-axis. Each data point represented the average of 3 normalized measurements where the error bars represented ± one standard deviation. The binding curves were converted to fractional saturation plots which were then fit with the following equation, eq 1, meant for fluorescence-based binding experiments in which only the total concentration of ligand ([LT] = [PTDMT]) is known and not the free ligand concentration, which some other models require.

polymers, based on their idealized stoichiometric structures (PTDM1 = 5,899.25 g/mol and PTDM2 = 5,138.25 g/mol) were used for calculations to create 1 mM stock solutions in DMSO in accordance with previously published procedures.35,39 PTDM stocks were stored at −20 °C in DMSO between uses and were allowed to thaw and reach room temperature prior to use. Fluorescence-Based Equilibrium Binding Assays. To determine the dissociation constants for PTDM−protein complexes, equilibrium fluorescence quenching assays were conducted, according to previously published procedures, at 25 °C in PBS at pH ∼7.2, where the fluorescence of each dyelabeled protein was monitored and recorded as a function of increasing PTDM concentration (from 0 up to 80 μM, depending on the protein) until saturation was achieved.36,39 In general, each well in the titration for a given dye-labeled protein was designed to contain a total volume of 200 μL of a PBS solution with a total protein concentration of ∼200 nM. These experiments were carried out in a 96-well polystyrene plate, where each well had black sides and flat clear bottoms. In general, PTDM stock solutions at 1 mM in pure DMSO were serially diluted in PBS to produce subsequent stock solutions of 100 μM (10% v/v DMSO) and 10 μM (1% v/v DMSO). Variable volumes from either of these less concentrated stocks were added to the wells such that each subsequent well in the series contained an increasing amount of PTDM. Because the use of PTDM stock solutions introduced residual DMSO in varying amounts, additional DMSO was added to each well in the titration series such that all wells, for a given protein, contained approximately the same concentration (% v/v) of DMSO set by the maximum volume of DMSO introduced by the most concentrated PTDM stock solution. Once all wells had received the calculated volume of the appropriate PTDM stock solution, which was added last, they were gently mixed by pipetting up and down and stirring with the tip of the pipet. The 96-well plate was allowed to incubate at room temperature for 30 min to achieve equilibrium prior to fluorescence measurements. The maximum absorption and emission wavelength of each FITC-labeled protein in PBS at pH ∼7.2 was determined using UV−vis and fluorescence spectroscopy, respectively, with the BioTek Mx plate reader. During the binding experiments, the fluorescence intensity of each well in the titration for a given protein was measured and normalized by the fluorescence of a positive control well that contained only ∼200 nM of the desired protein in PBS with a matching DMSO concentration. Each 96-well plate also used a negative control well consisting of PBS and DMSO only. The background noise of this negative control well was subtracted from all other wells before the fluorescence data were normalized to the positive control. In addition, the absorbance of 700 nm light, calculated using the path length correction feature of the plate reader, was measured for each well and converted to % transmittance (% T) to assess the solubility of the complexes after they formed. The absorbance of a blank negative control well (PBS and DMSO only) was first subtracted from the absorbance measurements of the other wells prior to the calculation of %T. The binding experiments were repeated in triplicate for each protein, and in the case of binding to EGFP, additional triplicate data points at new concentrations were added to existing curves to provide more information in the critical part of the binding curve and to improve the fitting.

fractional saturation = =

SL − S0 Ssat − S0

([L T] + n[R T] + Kd) −

([L T] + n[R T] + Kd)2 − 4[L T]n[R T] 2n[R T]

(1)

Eq 1 represents a multiple independent binding sites model where n is equal to the number of binding sites, LT is the total PTDM concentration in a well, RT is the total protein concentration in a well (∼200 nM), and Kd is the dissociation constant.36,53,60 Fractional saturation can be defined as the fraction of total sites occupied and can be determined indirectly through fluorescence measurements. The first part of eq 1 relates fluorescence measurements and fractional saturation, where SL is the measured fluorescence signal at a fixed concentration of ligand (PTDM), S0 is the fluorescence signal of the protein-only positive control, and Ssat is the fluorescence signal at saturation. For these binding experiments, the Ssat value was calculated by taking the average of the fluorescence intensity values of the last two data points in a given titration curve in the saturation region. Nonlinear leastsquares regression fitting with eq 1 was completed with OriginPro 2015 software using the nonlinear curve fitting module and the Levenberg−Marquardt iteration algorithm and yielded values of n and Kd along with the error associated with each of the parameters (n and Kd had initial guess values = 1 prior to fitting and were bound by the conditions of being greater than or equal to zero). All n values for each protein can be found in the Supporting Information along with further discussion about the use of eq 1 as compared to a previous report (Table S1 and Figure S1).39 Cellular Uptake in Jurkat T Cells. The following general procedure for fluorescent protein uptake in Jurkat T cells was based on previously published procedures.30,35,36,39 Polymers were dissolved in DMSO to make 1.0 mM stock solutions and were stored at −20 °C. On the day of each experiment, Jurkat T cells were harvested and resuspended in complete RPMI containing 10% (v/v) EquaFETAL bovine serum (a bioequivalent of FBS), 5 mM HEPES, 100 U/mL penicillin, 100 U/mL streptomycin, nonessential amino acids, and sodium pyruvate. Cells were seeded at a density of 4.0 × 105 cells/0.8 mL in a 24-well plate (800 μL per well). Proteins were diluted to 1 μM working concentrations, and polymers were diluted to 0.1 mM working concentrations with phosphate-buffered saline (PBS, pH 7.4). For making the polymer−protein complexes, appropriate volumes of protein (corresponding to 60 nM final delivery concentration) and polymer (corresponding to a 20:1 molar ratio of polymer:protein) were mixed in PBS to achieve a total volume of 200 μL. These complexes were incubated in the dark at rt for 30 min prior to adding them dropwise to each well, resulting in J


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Bioconjugate Chemistry final delivery volumes of 1 mL. The cells were incubated at 37 °C in 5% CO2 for 4 h. After the incubation period, the cells were harvested, transferred to microfuge tubes, centrifuged, resuspended in 200 μL of 0.25% trypsin-EDTA, and allowed to incubate for 3 min to remove any surface-bound fluorescent protein. Trypsin was neutralized by adding 1 mL of RPMI, and the cells were centrifuged followed by resuspension in 200 μL of FACS buffer (0.2% BSA in PBS) containing 5 μL of 7aminoactinomycin D (7-AAD) stain to assess viability. Then, 10,000 cells were collected by flow cytometry for analysis of fluorescent protein internalization and viability. Any additional materials and methods information pertaining to this manuscript can be found in the Supporting Information.

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(4) Kaplon, H., and Reichert, J. M. (2018) Antibodies to Watch in 2018. mAbs 10, 183−203. (5) Mitragotri, S., Burke, P. A., and Langer, R. (2014) Overcoming the Challenges in Administering Biopharmaceuticals: Formulation and Delivery Strategies. Nat. Rev. Drug Discovery 13, 655−672. (6) Sgolastra, F., deRonde, B. M., Sarapas, J. M., Som, A., and Tew, G. N. (2013) Designing Mimics of Membrane Active Proteins. Acc. Chem. Res. 46, 2977−2987. (7) Walrant, A., Cardon, S., Burlina, F., and Sagan, S. (2017) Membrane Crossing and Membranotropic Activity of Cell-Penetrating Peptides: Dangerous Liaisons? Acc. Chem. Res. 50, 2968−2975. (8) Yang, N. J., and Hinner, M. J. (2015) Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins. Site-Specific Protein Labeling. Methods in Molecular Biology (Gautier, A., and Hinner, M. J., Eds.) pp 29−53, Chapter 3, Humana Press, New York. (9) Fu, A., Tang, R., Hardie, J., Farkas, M. E., and Rotello, V. M. (2014) Promises and Pitfalls of Intracellular Delivery of Proteins. Bioconjugate Chem. 25, 1602−1608. (10) Kurzawa, L., Pellerano, M., and Morris, M. C. (2010) PEP and CADY-Mediated Delivery of Fluorescent Peptides and Proteins into Living Cells. Biochim. Biophys. Acta, Biomembr. 1798, 2274−2285. (11) Frankel, A. D., and Pabo, C. O. (1988) Cellular Uptake of the Tat Protein from Human Immunodeficiency Virus. Cell 55, 1189− 1193. (12) Vives, E., Brodin, P., and Lebleu, B. A. (1997) Truncated HIV1 Tat Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus. J. Biol. Chem. 272, 16010−16017. (13) deRonde, B. M., and Tew, G. N. (2015) Development of Protein Mimics for Intracellular Delivery. Biopolymers 104, 265−280. (14) Heitz, F., Morris, M. C., and Divita, G. (2009) Twenty Years of Cell-Penetrating Peptides: From Molecular Mechanisms to Therapeutics. Br. J. Pharmacol. 157, 195−206. (15) Deshayes, S., Morris, M., Heitz, F., and Divita, G. (2008) Delivery of Proteins and Nucleic Acids Using a Non-Covalent Peptide-Based Strategy. Adv. Drug Delivery Rev. 60, 537−547. (16) Pooga, M., Hallbrink, M., Zorko, M., and Langel, Ü . (1998) Cell Penetration by Transportan. FASEB J. 12, 67−77. (17) Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The Third Helix of the Antennapedia Homeodomain Translocates through Biological Membranes. J. Biol. Chem. 269, 10444−10450. (18) Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan Peptides: The Penetratin System for Intracellular Delivery. Trends Cell Biol. 8, 84−87. (19) Wender, P. A., Cooley, C. B., and Geihe, E. I. (2012) Beyond Cell Penetrating Peptides: Designed Molecular Transporters. Drug Discovery Today: Technol. 9, e49−e55. (20) Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L., and Rothbard, J. B. (2000) The Design, Synthesis, and Evaluation of Molecules That Enable or Enhance Cellular Uptake: Peptoid Molecular Transporters. Proc. Natl. Acad. Sci. U. S. A. 97, 13003−13008. (21) Mitchell, D. J., Kim, D. T., Steinman, L., Fathman, C. G., and Rothbard, J. B. (2000) Polyarginine Enters Cells More Efficiently than Other Polycationic Homopolymers. J. Pept. Res. 56, 318−325. (22) Futaki, S., and Nakase, I. (2017) Cell-Surface Interactions on Arginine-Rich Cell-Penetrating Peptides Allow for Multiplex Modes of Internalization. Acc. Chem. Res. 50, 2449−2456. (23) Keller, A. A., Mussbach, F., Breitling, R., Hemmerich, P., Schaefer, B., Lorkowski, S., and Reissmann, S. (2013) Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells. Pharmaceuticals 6, 184− 203. (24) Deshayes, S., Heitz, A., Morris, M. C., Charnet, P., Divita, G., and Heitz, F. (2004) Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 through Conformational Analysis. Biochemistry 43, 1449−1457.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00363. Binding curves, fractional saturation plots, fitting parameters n and Kd, NMR spectrum, UV−visible spectrum, % transmittance plots, % quenching calculations, cell viability, cellular uptake histograms, analyzed cellular uptake data, procedures, and notes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Gregory N. Tew: 0000-0003-3277-7925 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Amy Burnside, director of the Flow Cytometry Core Facility at the University of Massachusetts Amherst, where all flow cytometry data were collected. The authors also thank Dr. Coralie Backlund and Ms. Ronja Otter for providing PTDM2 for use in this study and Mr. Michael Kwasny for reviewing and providing feedback on earlier versions of this manuscript. The authors acknowledge grants NSF DMR-1308123, NSF NRT-1545399, and GAANN DoED P200A150276.

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ABBREVIATIONS PTD, protein transduction domain; PTDM, protein transduction domain mimic; CPP, cell-penetrating peptide; CPPM, cell-penetrating peptide mimic

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REFERENCES

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