Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(Amino Acids) and Their Potential Applications in Ph-Dependent Drug Delivery

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Article Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(amino acids) and Their Potential Applications in pH-Dependent Drug Delivery

Zoë Folchman-Wagner 1 , Jennica Zaro 2 and Wei-Chiang Shen 1,* 1 Department of Pharmaceutical Sciences, University of Southern California School of Pharmacy, 1985 Zonal Avenue, Los Angeles, CA 90089, USA; [email protected] 2 Department of Pharmaceutical Sciences, West Coast University School of Pharmacy, 590 Vermont Ave, Los Angeles, CA 90004, USA; [email protected] * Correspondence: [email protected]

Received: 28 April 2017; Accepted: 27 June 2017; Published: 30 June 2017

Abstract: Polyelectrolyte complexes (PECs) are self-assembling nano-sized constructs that offer several advantages over traditional nanoparticle carriers including controllable size, biodegradability, biocompatibility, and lack of toxicity, making them particularly appealing as tools for drug delivery. Here, we discuss potential application of PECs for drug delivery to the slightly acidic tumor microenvironment, a pH in the range of 6.5–7.0. Poly(L-glutamic acid) (En), poly(L-lysine) (Kn), and a copolymer composed of histidine-glutamic acid repeats ((HE)n) were studied for their ability to form PECs, which were analyzed for size, polydispersity, and pH sensitivity. PECs showed concentration dependent size variation at residue lengths of E51/K55 and E135/K127, however, no complexes were observed when E22 or K21 were used, even in combination with the longer chains. (HE)20/K55 PECs could encapsulate daunomycin, were stable from pH 7.4–6.5, and dissociated completely between pH 6.5–6.0. Conversely, the E51-dauno/K55 PEC dissociated between pH 4.0 and 3.0. These values for pH-dependent particle dissociation are consistent with the pKa’s of the ionizable groups in each formulation and indicate that the speciﬁc pH-sensitivity of (HE)20-dauno/K55 PECs is mediated by incorporation of histidine. This response within a pH range that is physiologically relevant to the acidic tumors suggests a potential application of these PECs in pH-dependent drug delivery.

Keywords: polyelectrolyte complexes; drug delivery; pH-sensitive; histidine

1. Introduction Polyelectrolyte complexes (PECs) are structures that form spontaneously upon combination of oppositely charged macromolecules in solution. The formation and physical characteristics of these complexes is driven by multiple factors including, but not limited to, molecular weight and ionic strength of each component, charge density, concentration, polymer chain rigidity, pH, and mixing intensity [1,2]. The type and concentration of salt in solution is also important as this can result in a neutralization of polymer charge [2,3]. The PECs are formed via an entropic process that has been previously described [4]. Brieﬂy, the complex formation, while largely driven by electrostatics, also involves hydrogen bonding and Van der Waals interactions [1]. The mechanism can be broken into roughly three steps, the ﬁrst of which is initial complex formation driven by electrostatic interactions, followed by formation of new bonds within this complex, and, lastly, the aggregation of multiple system complexes. The size of PECs is highly controllable, and can be affected by multiple factors including polymer molecular weight and concentration [4]. This property allows for uncomplicated production of PECs within a size range appropriate for drug delivery applications [5].

Molecules 2017, 22, 1089; doi:10.3390/molecules22071089 www.mdpi.com/journal/molecules Molecules 2017, 22, 1089 2 of 14

PECs have several advantages over alternative complexes or nanoparticles in that they have a highly controllable size, avoid toxic cross-linkers, and are biodegradable, biocompatible, and non-toxic [4,6]. Many iterations of PEC work exists in the literature [7–13], including extensive efforts using chitosan [6,14–19], and the complexes have been applied to ﬁelds that include drug delivery, gene delivery, and microencapsulation of both cells and tissues [1]. Due to their optimal physicochemical and biological properties, PECs are an ideal area of exploration for development and analysis of potential drug carriers. Owing to the highly controllable formation of PECs, it is of further interest to perform a closer examination of the self-assembly process and properties of PECs formed in a poly(amino-acid) solution. In addition to these advantages, PECs also present a unique opportunity in the potential for modular design enabled by the array of properties inherent to amino acids. One possibility is to take advantage of the pH-dependent ionization in various amino acid side groups for the design of PECs whose assembly/disassembly properties are pH-sensitive. Previous studies in our lab have demonstrated that complexes between imidazole-modiﬁed poly-glutamic acid and polylysine, Kn, exhibited a response to environmental pH from 5 to 7 on complex formation [20]. Additionally, our lab has previously developed a pH-responsive peptide composed of repeats of glutamic acid and histidine, (HE)n [21]. The pH-responsiveness of this copolymer has been demonstrated at several repeat lengths including (HE)15 [22], (HE)10 [23], and (HE)8–12 [24]. At physiologic pH of ~7.4, glutamic acid is negatively charged, while histidine remains unprotonated, giving (HE)n a net negative charge. As the pH is lowered, the imidazole group in histidine (pKa = 6.5) becomes protonated, resulting in neutralization of the (HE)n copolymer. Thus, (HE)n may be used as a highly sensitive pH responsive switch, rapidly converting from charged to uncharged within the small pH window of 6.5–7. Here, we report the use of a longer version of the (HE)n copolymer than previously reported, with 20 repeats it is designated as (HE)20. In this study, we evaluated the combination of pH responsive (HE)n with Kn to create a pH-sensitive PEC, capable of forming at neutral pH due to electrostatic interaction of the negative glutamic acid and the positive lysine. At slightly acidic pH, both the histidine (cationic) and glutamic acid (anionic) residues in (HE)n are ionized, resulting in a net-neutral charge of the (HE)n copolymer and loss of electrostatic interaction with lysine. The physicochemical factors necessary for formation of PEC from Kn and En as well as Kn and the (HE)n copolymer, including the ultimate pH sensitivity of these PECs, were investigated. The chemotherapeutic drug daunomycin [25] was added to the PEC formed with Kn and (HE)n as proof of concept for utility as a drug delivery tool.

2. Materials and Methods

2.1. Poly(L-glutamic acid)/Poly(L-lysine) PECs

En and Kn were purchased from Alamanda Polymers (Huntsville, AL, USA). The degree of polymerization (DPn), as determined by nuclear magnetic resonance spectroscopy, was for En, n = 22 (molecular weight (MW) = 3300) and n = 51 (MW = 7700) and for Kn, n = 21 (MW = 4400) and n = 55 (MW = 11,500). Aqueous solutions were prepared and filtered through 0.22 µm filters (Argos Technologies, Elgin, IL, USA). Larger molecular weight En (MW 15,000–50,000) and Kn (MW 15,000–30,000) with average DPn of 135 and 127, respectively, were purchased from Sigma Aldrich (St. Louis, MO, USA). En and Kn were mixed starting at En concentrations of 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, or 0.125 mg/mL with Kn at 1:1, or 1:2 molar charge in ddH2O. PECs were formed by drop-wise addition of En into a stirred solution of Kn.En solution was added in 50 or 100 µL aliquots to 500 µL or 1 mL of Kn at 30 s intervals until the full volume of 500 µL or 1 mL was added. The resulting dispersion was stirred for an additional 10 min and then analyzed using dynamic light scattering (DLS) (Dynapro Plate Reader, Wyatt, Santa Barbara, CA, USA) to determine particle size. Size readings were performed in quadruplicate, where data was shown as average size diameter reading over the four wells ± standard deviation (nm), and outliers were identiﬁed using Grubb’s test with α = 0.05. Studies on size and polydispersity of PECs were performed by incubating PEC Molecules 2017, 22, 1089 3 of 14 dispersions at either 25 ◦C or 4 ◦C for various periods of time followed by analysis of size variance and polydispersity index (PDI) using DLS.

2.2. Recombinant Expression of (HE)20 Peptide

The pH-responsive (HE)20 peptide was produced recombinantly as a glutathione S-transferase (GST)-(HE)n fusion protein containing a thrombin cutting site between the GST and (HE)n domains as previously described [23]. Briefly, plasmids containing the GST-(HE)20 gene were transformed into Escherichia coli expression strain BL21 and the recombinant protein was expressed as previously described [21,23,24]. The resultant bacterial pellets were resuspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.25 mg/mL lysozyme. After incubation for 30 min on ice, phenylmethylsulfonyl fluoride (1 mM) and Triton X-100 (1%, v/v) were added to lyse the bacteria. The lysate was centrifuged (15,000× g for 30 min at 4 ◦C) and the supernatant was loaded onto a glutathione (GSH) agarose column pre-balanced with PBS. The GSH column was washed with 1% Triton X-100 in PBS followed by PBS, and then thrombin (Sigma) was loaded onto the agarose followed by incubation for 16 h at room-temperature. After thrombin cleavage, the (HE)20 peptide was eluted with PBS and purified by nickel-nitriloacetic acid (Ni-NTA) agarose chromatography using 250 mM imidazole, pH 8.0 as the elution buffer [21]. (HE)20 peptide was then dialyzed (molecular weight cut-off (MWCO) 3500 Da) against PBS to remove imidazole, and the purified (HE)20 peptide was analyzed by SDS-PAGE with Coomassie blue staining to determine purity and confirm size. The final sequence of the peptide was (HE)10-F-(HE)10, abbreviated as “(HE)20”.

2.3. Formulation of PEC with Daunomycin, (HE)20, and PLys55 Daunomycin HCl was purchased from Cayman Chemical (Ann Arbor, MI, USA). PECs with daunomycin, (HE)20, and K55 were made by combining 1–0.125 mg/mL daunomycin with 1–0.125 mg/mL (HE)20 at equal concentrations ((HE)20-dauno). The (HE)20-dauno was then added dropwise to a stirred solution of K55 at 1:1 (HE)20-dauno:K55 molar charge ratio. Resulting PECs were analyzed using DLS to determine particle size as well as fluorescence (excitation/emission of 480/590 nm) to determine daunomycin concentration and incorporation efficiency [26]. The encapsulation efficiency was determined by comparing the ratio of fluorescence intensity between free daunomycin to that of the (HE)20-dauno/K55 PEC.

2.4. Formulation of PEC with Daunomycin, (HE)20-dauno, and K55 in ZnCl2·2NH4Cl solution

ZnCl2, NH4Cl, and tricine were purchased from Sigma-Aldrich (St. Louis, MO, USA). ZnCl2·2NH4Cl was formed by mixing ZnCl2 and NH4Cl in a 1:2 molar ratio in ddH2O, followed by dilution into a solution of 10 mg/mL tricine as chelator. The ZnCl2·2NH4Cl/tricine solution, at a 1:2 molar ratio to histidine in (HE)20-dauno, was then used to dissolve K55 (K55-Zn). Then, 1 mg/mL daunomycin was added to an equivalent concentration of (HE)20-dauno dissolved in 10 mg/mL tricine and the pH increased to 8.0. The (HE)20-dauno solution was then added dropwise to a stirred solution of K55-Zn in tricine at a 1:1 (HE)20-dauno:K55 molar charge ratio. The resulting PECs were stirred for an additional 10 min and then analyzed by DLS.

2.5. Formulation of PEC with Daunomycin, E51, and K55 in ZnCl2·2NH4Cl solution

One milligram per milliliter daunomycin was added to 1 mg/mL E51 solution (E51-dauno) in ddH2O prior to PEC formation. ZnCl2 was prepared as described above and used to dissolve K55 (K55-Zn). The E51-dauno solution was then added dropwise to a stirred solution of K55-Zn in tricine at a 1:1 molar charge ratio. The resulting PECs were stirred for an additional 10 min, diluted into 10 mg/mL Tricine, and then analyzed by DLS. Molecules 2017, 22, 1089 4 of 14 Molecules 2017, 22, 1089 4 of 14

2.6. pH Sensitivity of E 51-dauno/K/K55-Zn55-Zn PECPEC and and (HE) (HE)20-dauno20-dauno/K55-Zn/K 55-Zn

To determinedetermine thethe pHpH sensitivity sensitivity of of the the PEC PEC formed formed from from E51-dauno E51-dauno/K/K55-Zn andand (HE) (HE)20-dauno20-dauno/K55-Zn/K55-Zn, the, PECthe PEC were were formed formed in ddH in ddH2O or2 O10 ormg/mL 10 mg/mL tricine tricine solutions. solutions. The (HE) The20-dauno (HE)/K20-dauno55-Zn PEC/K were55-Zn formedPEC were as describedformed as above described in ddH above2O and in ddH then2O diluted and then into diluted 10 mg/mL into 10Tricine. mg/mL The Tricine. pH was The adjusted pH was using adjusted 1 N HCl,using and 1 N HCl,monitored and monitored with an with Orion an OrionPerpHecT PerpHecT ROSS ROSS combination combination pH pH Micro Micro Electrode Electrode from ThermoFischer ScientiﬁcScientific (Waltham,(Waltham, MA, MA, USA). USA). PEC PEC were were analyzed analyzed at at intervals intervals of of 1 or1 or 0.5 0.5 pH pH units units by byDLS; DLS; PEC PEC degradation degradation was was determined determined by aby DLS a DLS reading reading of 0.00of 0.00 nm nm or anor ‘incomplete’an ‘incomplete’ reading. reading.

3. Results and Discussion

3.1. PEC Formed from K n andand E Enn PEC showed a high degree of concentrationconcentration and molecular weight dependence in ultimate hydrodynamic diameter.diameter. TheThe PECPEC formed formed from from E 135E135/K/K127127 were signiﬁcantlysignificantly largerlarger thanthan the the E 51E51/K/K5555 and 2X-E51/K/K5555 atat all all concentrations concentrations tested tested (Figure (Figure 11).). Further, as thethe concentrationconcentration ofof KKn and EEnn decreased, there was a corresponding decrease inin thethe sizesize ofof thethe PECPEC (Figure(Figure1 1).).

PEC Size 1 mg/mL

300 0.5 mg/mL 0.25 mg/mL 0.125 mg/mL 200

100 NP Diameter (nm) Diameter NP

0 E /K E /K 2X-E /K 135 127 51 55 51 55

Figure 1. Size summary of poly(L-Lysine) (Kn) and poly(L-Glutamic Acid) (En) polyelectrolyte Figure 1. Size summary of poly(L-Lysine) (Kn) and poly(L-Glutamic Acid) (En) polyelectrolyte complexes (PECs). PECs were formulated at 1:1 or 1:2 molar charge ratios of En:Kn with the indicated complexes (PECs). PECs were formulated at 1:1 or 1:2 molar charge ratios of En:Kn with the degree of polymerization (DPn) and beginning from an En concentration of 1 mg/mL. Decreasing the indicated degree of polymerization (DPn) and beginning from an En concentration of 1 mg/mL. DPn of poly(L-Lysine) and poly(L-Glutamic Acid) from 127/135 to 55/51 resulted in a statistically Decreasing the DPn of poly(L-Lysine) and poly(L-Glutamic Acid) from 127/135 to 55/51 resulted in a significantstatistically signiﬁcantdecrease in decrease diameter in diameter at all atconcen all concentrationstrations tested. tested. All All formulations formulations demonstrated demonstrated concentration dependence in in diameter. Data Data show shownn are hydrodynamic diamet diameterer as determined by dynamic lightlight scatteringscattering (DLS) (DLS) and and are are the the averages averages over over 3–4 3–4 wells wells± standard ± standard deviation. deviation. Results Results were wereanalyzed analyzed by two-way by two-way ANOVA. ANOVA.

This trend was observed for the 1:1 charge ratio of E135/K127, the 1:1 charge ratio of E51/K55, and This trend was observed for the 1:1 charge ratio of E135/K127, the 1:1 charge ratio of E51/K55, the 1:2 charge ratio of E51/K55. The concentration dependence of PECs was expected based on and the 1:2 charge ratio of E /K . The concentration dependence of PECs was expected based well-established models of aggregation51 55 by Debye models and Derjaguin, Landau, Verwey, and on well-established models of aggregation by Debye models and Derjaguin, Landau, Verwey, Overbeek Theory, as well as previous studies [27,28]. PEC formulated with a two-fold excess of and Overbeek Theory, as well as previous studies [27,28]. PEC formulated with a two-fold excess of positive charge (2X-E51/K55) were slightly smaller than those formed with an equal charge ratio. This positive charge (2X-E /K ) were slightly smaller than those formed with an equal charge ratio. This size difference was significant51 55 at all concentrations except 0.5 mg/mL. When shorter poly amino-acid size difference was signiﬁcant at all concentrations except 0.5 mg/mL. When shorter poly amino-acid chains were used, K21 and E22, no PEC formation could be seen at a 1:1 charge ratio for En chains were used, K and E , no PEC formation could be seen at a 1:1 charge ratio for E concentration concentration of 1 21mg/mL 22or 2 mg/mL. When these shorter chains were combinedn with longer of 1 mg/mL or 2 mg/mL. When these shorter chains were combined with longer repeats, E51/K21 repeats, E51/K21 and E22/K55, there remained no complex formation at a 1:1 charge ratio or 1:1 molar and E /K , there remained no complex formation at a 1:1 charge ratio or 1:1 molar ratio. This result ratio. This22 55result suggests that there is a critical limitation for PEC size using these poly amino-acids. suggests that there is a critical limitation for PEC size using these poly amino-acids. As some of As some of the driving factors for PEC formation include molecular weight, charge density, and the driving factors for PEC formation include molecular weight, charge density, and hydrophobicity, hydrophobicity, it is reasonable to suggest that the DPn of 21 and 22 did not have sufficient charge density and/or hydrophobicity to nucleate the formation of PEC. The lack of PEC formation even

Molecules 2017, 22, 1089 5 of 14

Molecules 2017, 22, 1089 5 of 14 it is reasonable to suggest that the DPn of 21 and 22 did not have sufﬁcient charge density and/or hydrophobicitywhen paired with to nucleate a long poly the formationamino-acid of further PEC. Thesupports lack ofthis PEC claim formation that there even is a whenbottom paired limit withto a longPEC polyformation amino-acid when working further with supports these short this claim chain thatamino-acids. there is aThe bottom details limit of this to process PEC formation are as whenyet workingunclear, as with the theseprocess short of chainPEC formation amino-acids. is mu Thelti-step. details It is of well this processestablished are asthat yet increasing unclear, as theconcentration process of PEC of poly-electrolytes formation is multi-step. in solution decreases It is well the established Debye length that of increasing the system, concentration resulting in a of poly-electrolytesreduction in electrostatic in solution repulsion decreases [29,30]. the DebyeIt is, therefore, length ofpossible the system, that the resulting short chain in length a reduction of K21 in electrostaticand E22 did repulsion not have [29 enough,30]. It is,charge therefore, density possible to decrease that the the short Debye chain length, length resulting of K21 and in Ehigh22 did notelectrostatic have enough repulsion. charge density to decrease the Debye length, resulting in high electrostatic repulsion. ◦ ◦ TheThe E 51E51/K/K5555 andand 2X-E 2X-E51/K5551/K55 were were analyzed analyzed for size for and size polydispersity and polydispersity at 25 °C and at 25 4 °CC (Figures and 4 C (Figures2 and 23) and at various3) at various concentrations concentrations from 1 fromto 0.125 1 to mg/mL 0.125 mg/mLE51. E 51.

E /K PEC ABE /K PEC 51 55 51 55 25˚C Polydispersity 600 25˚C Size 1.0 1 mg/mL 1 mg/mL 0.5 mg/mL 0.8 0.5 mg/mL 400 0.25 mg/mL 0.25 mg/mL 0.6 0.125 mg/mL 0.125 mg/mL 0.4 200 Polydispersity

NP Diameter (nm) Diameter NP 0.2

0 0.0 02468 02468 Days of Incubation Days of Incubation

CD E51/K55 PEC E51/K55 PEC 600 4C Size 1.0 4C Polydispersity 1 mg/mL 1 mg/mL 0.5 mg/mL 0.8 0.5 mg/mL 400 0.25 mg/mL 0.25 mg/mL 0.6 0.125 mg/mL 0.125 mg/mL 0.4 200 Polydispersity

NP Diameter (nm) Diameter NP 0.2

0 0.0 02468 02468 Days of Incubation Days of Incubation ◦ ◦ FigureFigure 2. Stability2. Stability of Eof51 E/K51/K55 55PEC PEC formed formed from from 1:1 1:1 charge charge ratio ratio at 25 at C25and °C and 4 C. 4Panels °C. Panels (A,B )(A show,B) show the size ◦ andthe polydispersity, size and polydispersity, respectively, resp ofectively, PEC formed of PEC from formed E51 and from K55 Eat51 and a 1:1 K charge55 at a 1:1 ratio charge at 25 ratioC over at 25 7 °C days. Atover 1 mg/mL 7 days. and At 0.51 mg/mL mg/mL and there 0.5 wasmg/mL an increasethere was in an size increase and polydispersity in size and polydispersity after 24–72 h ofafter incubation, 24–72 followedh of incubation, by plateau. followed The 0.25 by and plateau. 0.125 mg/mLThe 0.25 formulationsand 0.125 mg/mL showed formulations little change showed in diameter little change over 7 daysin anddiameter a slight increaseover 7 days in polydispersity; and a slight Panels increase (C, Din) showpolydispersity; the size and Panels polydispersity, (C,D) show respectively, the size ofand PEC polydispersity, respectively, of PEC formed from◦ E51 and K55 at a 1:1 charge ratio at 4 °C over 7 days. formed from E51 and K55 at a 1:1 charge ratio at 4 C over 7 days. No plateau was reached in diameter for theNo 1 mg/mLplateau was or 0.5 reached mg/mL in formulations,diameter for the while 1 mg/ themL 0.25 or and0.5 mg/mL 0.125 mg/mL formulations, formulations while the showed 0.25 and little 0.125 mg/mL formulations showed little change. The polydispersity showed an increase after 24 h in change. The polydispersity showed an increase after 24 h in the 1 mg/mL formulation, followed by plateau, the 1 mg/mL formulation, followed by plateau, while the lower concentrations remained nearly while the lower concentrations remained nearly constant with minor fluctuations. Data were obtained by constant with minor fluctuations. Data were obtained by DLS, and are displayed as averages over 3–4 DLS, and are displayed as averages over 3–4 wells ± standard deviation. wells ± standard deviation.

AtAt the the high high end end of of the the concentration concentration range, range, with with E Ennconcentration concentration ofof 11 mg/mL,mg/mL, the the E E5151/K/K55 55PECPEC showedshowed a dramatica dramatic increase increase in in diameter diameter afterafter 24 h of 25 °C◦C incubation, incubation, nearly nearly doubling doubling in insize, size, and and werewere then then constant constant for for 7 7 days days (Figure (Figure2 A).2A). The The polydispersity polydispersity of these PEC PEC showed showed the the same same initial initial increase,increase, then then proceeded proceeded with with a a downward downward trend trend up up to to the the last last time-point time-point (Figure(Figure2 2B).B).When When storedstored at ◦ 4 atC, 4 the °C, E the51/K E5551/KPEC55 PEC at1 at mg/mL 1 mg/mL E51 Ehad51 had a graduala gradual and and sustained sustained increase increase in in size size up up to to the the last last time point,time with point, no with clear no plateauclear plateau reached reached (Figure (Figure2C). 2C The). The polydispersity polydispersity of of the the 1 1 mg/mL mg/mL formulation formulation at at 4 ◦4C °C increased increased after after 24 24 h h and and was was mostmost stablestable for the following 6 6 days days (Figure (Figure 2D).2D). A A similar similar trend trend was seen in the PEC formed at an E51 concentration of 0.5 mg/mL, however, the size increase in both was seen in the PEC formed at an E51 concentration of 0.5 mg/mL, however, the size increase in bothstorage storage temperatures temperatures was was more more gradual, gradual, and and no noclear clear size size plateau plateau was was reached reached at 4 at °C. 4 ◦ C.The The polydispersitypolydispersity at at 0.5 0.5 mg/mL, mg/mL, when when stored stored at at 25 25◦ C,°C, showed showed no no change change untiluntil dayday 3,3, wherewhere therethere waswas an an increase from ~0.2 to ~0.35, followed by stabilization of the reading for the remaining 4 days. PEC

Molecules 2017, 22, 1089 6 of 14 increaseMolecules from 2017, 22 ~0.2, 1089 to ~0.35, followed by stabilization of the reading for the remaining 4 days.6 of PEC14 formed from 0.25 and 0.125 mg/mL were mostly constant in size at both storage conditions. Under formed from 0.25 and 0.125 mg/mL were mostly constant in size at both storage conditions. Under a a 25◦C incubation, the PEC were stable in size for approximately 5 days before increasing slightly. 25°C incubation, the PEC were stable in size for approximately 5 days before increasing slightly. At 4 At 4 ◦C, the PEC remained at a stable size for the full 7 days. The polydispersity was more varied °C, the PEC remained at a stable size for the full 7 days. The polydispersity was more varied for for these lower concentration PECs. The 0.5 mg/mL E displayed a trend similar to 1 mg/mL of these lower concentration PECs. The 0.5 mg/mL E51 displayed51 a trend similar to 1 mg/ml of increase increase followed by plateau, however, the 0.25 and 0.125 mg/mL E PEC maintained a fairly constant followed by plateau, however, the 0.25 and 0.125 mg/mL E51 PEC51 maintained a fairly constant ◦ ◦ polydispersitypolydispersity but but began began to to show show aa slightslight increaseincrease after 4 4 days days of of incubation incubation at at 25 25 °C.C. At At 4 °C 4 Cthe the polydispersitypolydispersity remained remained constant, constant, however,however, therethere was noticeably higher, higher, although although not not statistically statistically signiﬁcant,significant, deviation deviation between between days. days. SimilarSimilar trends trends in stabilityin stability were were observed observed for for the the PEC PEC formulated formulated with with a 2-fold a 2-fold molar molar charge charge excess of Kexcess55 over of K E5155 over(Figure E51 3(Figure). 3).

E /K PEC ABE /K PEC 51 55 51 55 25˚C Polydispersity 600 25˚C Size 1.0 1 mg/mL 1 mg/mL 0.5 mg/mL 0.8 0.5 mg/mL 400 0.25 mg/mL 0.25 mg/mL 0.6 0.125 mg/mL 0.125 mg/mL 0.4 200 Polydispersity

NP Diameter Diameter (nm)NP 0.2

0 0.0 02468 02468 Days of Incubation Days of Incubation

CD E51/K55 PEC E51/K55 PEC 600 4C Size 1.0 4C Polydispersity 1 mg/mL 1 mg/mL 0.5 mg/mL 0.8 0.5 mg/mL 400 0.25 mg/mL 0.25 mg/mL 0.6 0.125 mg/mL 0.125 mg/mL 0.4 200 Polydispersity

NP Diameter (nm)Diameter NP 0.2

0 0.0 02468 02468 Days of Incubation Days of Incubation ◦ ◦ FigureFigure 3. 3.Stability Stability of of E 51E51/K/K5555 PEC formed from from 1:2 1:2 charge charge ratio ratio at at 25 25 °CC and and 4 4°C.C. Panels Panels (A (,AB),B show) show ◦ thethe size size and and polydispersity, polydispersity, respectively, respectively, of of PEC PEC formed formed from from EE5151 and K 5555 atat a a2:1 2:1 charge charge ratio ratio at at25 25 °C C overover 7 days. 7 days. At 1At mg/mL 1 mg/mL and and 0.5 mg/mL0.5 mg/mL there there was was an increase an increase in size in aftersize 24after h of 24 incubation, h of incubation, followed byfollowed plateau. Thereby plateau. was little There change was in little polydispersity. change in The polydispersity. 0.25 and 0.125 The mg/mL 0.25 formulationsand 0.125 mg/mL showed littleformulations change in diametershowed little or polydispersity change in diameter over 7or days; polydispersity Panels (C ,overD) show 7 days; the Panels size and (C, polydispersity,D) show the size and polydispersity, respectively, of PEC formed from E51 and K55 at◦ a 2:1 charge ratio at 4 °C over respectively, of PEC formed from E51 and K55 at a 2:1 charge ratio at 4 C over 7 days. All formulations except7 days. 0.125 All mg/mLformulations showed except a gradual0.125 mg/mL increase showed in size a gradual over 7 increase days. The in size 0.125 over mg/mL 7 days. formulationThe 0.125 showedmg/mL no formulation significant change showed in no diameter significant during change the 7 dayin diameter incubation. during The 1the and 7 0.5day mg/mL incubation. formulations The 1 increasedand 0.5 inmg/mL polydispersity formulations for threeincrease daysd in before polydispersity reaching afor plateau, three da whileys before the 0.25 reaching and 0.125 a plateau, mg/mL formulationswhile the 0.25 remained and 0.125 stable mg/mL until day formulations 7, where there rema wasined an stable increase until in day the polydispersity.7, where there Datawas werean increase in the polydispersity. Data were obtained by DLS, and are displayed as averages over 3–4 obtained by DLS, and are displayed as averages over 3–4 wells ± standard deviation. wells ± standard deviation.

WithWith the the higher higher concentrationconcentration of poly poly amino-acid amino-acid solutions solutions (1 mg/mL (1 mg/mL and and0.5 mg/mL), 0.5 mg/mL), the ◦ thecomplexes complexes stored stored at at 25 25 °CC increased increased sharply sharply in insize size after after 24 24 h, h,and and then then remained remained constant constant for for the the remainingremaining 7 days 7 days (Figure (Figure3A). 3A). The The polydispersity polydispersity of of these these complexes complexes remained remained consistent consistent for for 7 7 days days of 25 of◦C 25 incubation °C incubation (Figure (Figure3B). Conversely, 3B). Conversely, for the for PEC the storedPEC stored at 4 ◦ atC, 4 the °C, complex the complex size increasedsize increased slowly upslowly to day up 3, andto day remained 3, and remained relatively relatively constant constant after that after (Figure that3 (FigureC). The 3C). polydispersity The polydispersity of the 4 of◦C the PEC was4 °C inconsistent PEC was inconsistent with that at with 25 ◦C, that showing at 25 °C, an showing increase an for increase 3 days, for followed 3 days, by followed plateau by (Figure plateau3D). ◦ The(Figure 0.5 mg/mL 3D). The E51 0.5concentration mg/mL E51 concentration PEC demonstrated PEC demonstrated a similar 24 a hsimilar increase 24 ath increase 25 C followed at 25 °C by plateau,followed while by theplateau, 4 ◦C while PEC increasedthe 4 °C PEC in size increased steadily infor size the steadily 7 days for of the 7 study. days Theof the polydispersity study. The of thepolydispersity 25 ◦C formulation of the 25 remained °C formulation fairly constantremained throughout fairly constant the 7throughout days, while the the 7 days, polydispersity while the of polydispersity of the 4 °C formulation increased slowly for 3 days before leveling off. The 0.25

Molecules 2017, 22, 1089 7 of 14

◦ the 4MoleculesC formulation 2017, 22, 1089 increased slowly for 3 days before leveling off. The 0.25 mg/mL concentration7 of 14 formulation showed a very gradual increase in diameter and reached a plateau after approximately 3 days,mg/mL while concentration the 4 ◦C formulation formulation continued showed toa showvery gradual small daily increase size in increases diameter with and noreached plateau. a The polydispersityplateau after for approximately both formulations 3 days, was while relatively the 4 °C constant,formulation with continued a sharp to increase show small after daily 7 days size at 4 ◦C. Lastly,increases the most with dilute no plateau. of the formulations,The polydispersity 0.125 for mg/mL, both formulations displayed was a fairly relatively constant constant, size for with all a 7 days sharp increase after 7 days at 4 °C. Lastly, the most dilute of the formulations, 0.125 mg/mL, of the experiment. Similarly, the 4 ◦C formulation did not change in size in the dispersion at ~80 nm displayed a fairly constant size for all 7 days of the experiment. Similarly, the 4 °C formulation did until day 7. There was no signiﬁcant change in polydispersity during the 25 ◦C incubation, while the not change in size in the dispersion at ~80 nm until day 7. There was no significant change in ◦ 4 Cpolydispersity incubation remained during the constant 25 °C incubation, until day while 7, where the 4 it°C increased incubation from remained ~0.3 toconstant ~0.5. until day 7, whereThe plateau it increased reached from in ~0.3 almost to ~0.5. all cases except the most dilute formulation suggests that there is a most stableThe form plateau of thereached En/K inn almostPEC. Theall cases last except step in th PECe most formation, dilute formulation as outlined suggests in the that introduction, there is is aggregationa most stable of multiple form of complexesthe En/Kn PEC. formed The last by electrostaticstep in PEC formation, interactions as outlined [1,4,30]. in This the is introduction, likely a dynamic processis aggregation that continues of multiple until ancomplexes equilibrium formed is reachedby electrostatic [30]. Theinteractions lack of plateau[1,4,30]. This in the is 4likely◦C storagea is likelydynamic due process to a decrease that continues in the thermodynamicuntil an equilibrium potential is reached of [30]. the system,The lack thus,of plateau increasing in the 4 the°C time storage is likely due to a decrease in the thermodynamic potential of the system, thus, increasing the to equilibrium. The PEC formulated with a 2-fold molar excess of Kn should have a large positive time to equilibrium. The PEC formulated with a 2-fold molar excess of Kn should have a large surface charge, thus, providing electrostatic stability to the system by repulsion between neighboring positive surface charge, thus, providing electrostatic stability to the system by repulsion between complexes.neighboring In most complexes. cases, thereIn most is cases, a decrease there inis a time decrease for equilibrium in time for equilibrium plateau to beplateau reached, to be which tendedreached, to occur which after tended 24 h to of occu incubation.r after 24 h of incubation.

3.2. Formulation3.2. Formulation of PECof PEC with with Daunomycin, Daunomycin, (HE)(HE)20, ,and and K K55 55

InitialInitial formulation formulation of of PEC PEC with with E Enn/K/Knn waswas crucial crucial in inestablishing establishing both both the technique the technique for and for and limitationslimitations of theof the complex complex formation. formation. PreliminaryPreliminary experiments experiments utilizing utilizing the the(HE) (HE)10 peptide10 peptide mixed mixed withwith K21 Kand21 and E22 Edid22 did not not form form complexes, complexes, as expected based based on on results results with with E22 Eand22 andK21. Therefore, K21. Therefore, the number of HE-repeats was increased to n = 20 ((HE)20). In these complexes, no PEC formation the number of HE-repeats was increased to n = 20 ((HE)20). In these complexes, no PEC formation was possiblewas possible without without the additionthe addition of daunomycin. of daunomycin. Once daunomycinOnce daunomycin was addedwas added into the into formulation, the formulation, the homogeneity of the resulting PEC was dependent on whether daunomycin was the homogeneity of the resulting PEC was dependent on whether daunomycin was added to the added to the polyanion ((HE)20) or polycation (K55) phase (Figure 4). polyanion ((HE)20) or polycation (K55) phase (Figure4).

A Comparison of Daunomycin Phase Addition B 250 2.5

200 2.0 Polydispersity

150 1.5

100 1.0

NP DiameterNP (nm) 50 0.5

0 0.0 (HE)20-dauno/K55 (HE)20/K55-dauno NP Diameter (nm) Polydispersity Figure 4. Comparison of daunomycin phase addition. (A) In two separate experiments daunomycin Figure 4. Comparison of daunomycin phase addition. (A) In two separate experiments daunomycin was dissolved in the histidine and glutamic acid peptide ((HE)20) solution ((HE)20-dauno) prior to PEC was dissolved in the histidine and glutamic acid peptide ((HE)20) solution ((HE)20-dauno) prior to formation or in the K55 solution (K55-dauno). The PEC formed from (HE)20-dauno and K55 were ~180 ± 10 nm PEC formation or in the K55 solution (K55-dauno). The PEC formed from (HE)20-dauno and K55 were in diameter with a polydispersity index (PDI) of 0.6 ± 0.04. Conversely the PEC formed from (HE)20 and ~180 ± 10 nm in diameter with a polydispersity index (PDI) of 0.6 ± 0.04. Conversely the PEC formed K55-dauno were ~130 ± 96 nm in diameter with a PDI of 0.96 ± 1.0. Data shown are averages of 4 wells ± fromstandard (HE)20 deviation;and K55-dauno (B) Structurewere ~130of encapsulated± 96 nm daunomycin. in diameter with a PDI of 0.96 ± 1.0. Data shown are averages of 4 wells ± standard deviation; (B) Structure of encapsulated daunomycin. When daunomycin was combined with (HE)20 prior to PEC formation ((HE)20-dauno/K55), the resulting complexes were approximately 180 nm with a PDI of 0.60. There was, additionally, low deviation When daunomycin was combined with (HE)20 prior to PEC formation ((HE)20-dauno/K55), between the samples tested. Conversely, when daunomycin was added to the polycation prior to the resulting complexes were approximately 180 nm with a PDI of 0.60. There was, additionally, PEC formation ((HE)20/K55-dauno), the resulting complexes were ~133 nm in diameter with a PDI of low deviation between the samples tested. Conversely, when daunomycin was added to the polycation nearly 1. Additionally, there was large variation between samples for the PEC formed from prior to PEC formation ((HE) /K ), the resulting complexes were ~133 nm in diameter with a (HE)20/K55-dauno, with standard20 deviation55-dauno of ~100 nm for PEC diameter and ~1.0 for PDI (Figure 4). PDI of nearlyThese 1.results Additionally, suggest that there the wasincorporation large variation of daunomycin between into samples the PEC for is the driven PEC formedby both from (HE)electrostatic20/K55-dauno and, with hydrophobic standard intera deviationctions. of At ~100 pH values nm for above PEC the diameter pKa of histidine and ~1.0 (6.5), for PDI the (HE) (Figure20 4). These results suggest that the incorporation of daunomycin into the PEC is driven by both electrostatic and hydrophobic interactions. At pH values above the pKa of histidine (6.5), the (HE)20 copolymer will Molecules 2017, 22, 1089 8 of 14 copolymer will have a net negative charge and is, therefore, capable of electrostatic interaction betweenhave a net the negative positively charge charged and is, therefore,daunomycin capable (pKa of 8.4 electrostatic [31]) and interaction the gamma-carboxyl between the positivelygroup of glutamiccharged daunomycin acid in (HE) (p20K.a Further8.4 [31]) andinteraction the gamma-carboxyl may be possible group due of glutamic to hydrophobic acid in (HE) interactions20. Further betweeninteraction the may hydrophobic be possible duedaunomycin to hydrophobic (logP interactions 1.8 [32]) betweenand the thehistidine hydrophobic residues daunomycin in the (HE) (logP20 copolymer.1.8 [32]) and The the polycationic histidine residues K55 lacks in the the (HE) potential20 copolymer. for electrostatic The polycationic interaction K55 withlacks daunomycin, the potential andfor electrostatic the higher charge interaction density with of daunomycin, the lysines would and the likely higher repel charge interactions density of with the lysines the drug. would The likely lack ofrepel discrete interactions PEC formation with the drug. upon The addition lack of discreteof daunomycin PEC formation to the uponpolycation addition K55 of phase daunomycin supports to the the probabilitypolycation K that55 phase the driving supports force the probabilityfor encapsulation that the of driving daunomycin force for in encapsulation the PECs is ofelectrostatic daunomycin and in hydrophobicthe PECs is electrostatic interactions and between hydrophobic daunomycin interactions and between (HE)20, daunomycinwhich form andprior (HE) to 20the, which addition form of prior the polyanionto the addition phase of and, the polyanion thus, result phase in drug and, thus,incorporation result in drugonce incorporationthe (HE)20/K55 once PECs the are (HE) formed.20/K55 BasedPECs onare the formed. heterogeneity Based on theof heterogeneitythe (HE)20/K55-dauno of the PEC, (HE) 20the/K 55-daunodaunomycinPEC, thewas daunomycin exclusively was added exclusively to the polyanionadded to the phase, polyanion (HE)20 phase,, in all (HE)further20, informulations. all further formulations. PEC formed fromfrom (HE)(HE)20-dauno20-dauno/K/K55 55showedshowed a adecrease decrease in in the the fluorescent fluorescent signal signal of of daunomycin (Figure 55)) at at all all concentrations concentrations test tested.ed. The fluorescence The fluorescence quenching quenching observed observed in Figure 5 in was Figure attributed5 was to encapsulationattributed to encapsulation of daunomycin of daunomycin within the withinPEC, thethus PEC, dampening thus dampening the fluorescent the fluorescent signal signal[33]. [The33]. fluorescenceThe fluorescence intensity intensity of free of freedaunomycin daunomycin was was compared compared to tothe the fluorescent fluorescent signal signal of of the (HE)20-dauno/K/K55 PEC,55 PEC, and and the the decrease decrease in insignal signal was was taken taken as as proportional proportional to to encapsulation encapsulation efficiency. efficiency. The loading amount (wt %) of daunomycin inin PECPEC solutionssolutions withwith concentrationsconcentrations ofof 1–0.1251–0.125 mg/mLmg/mL polyanion and an equal molar charge of polycation,polycation, was 36% at all formulations. The encapsulation efficiencyefficiency ranged from 58% to 85% (Table1 1).).

Figure 5. Fluorescence quenching of daunomycin in (HE)20-dauno/K55 PEC. Fluorescence measurements Figure 5. Fluorescence quenching of daunomycin in (HE)20-dauno/K55 PEC. Fluorescence were taken of the (HE)20-dauno solution alone and the (HE)20-dauno/K55 PEC. There is a significant measurements were taken of the (HE)20-dauno solution alone and the (HE)20-dauno/K55 PEC. There is decreasea signiﬁcant in fluorescence decrease in ﬂuorescencesignal from daunomycin signal from daunomycin at all concentrations at all concentrations of PEC. The of data PEC. shown The data are averagesshown are over averages 4 wells over ± standard 4 wells ± deviation.standard deviation.Results were Results analyzed were by analyzed the Student’s by the Student’st-test, witht-test, p < 0.001with pindicated< 0.001 indicated (*). (*).

Table 1. Encapsulation efficiency and loading content of daunomycin in (HE)20-dauno/K55 PEC. Table 1. Encapsulation efficiency and loading content of daunomycin in (HE)20-dauno/K55 PEC. Fluorescence measurements were taken of the daunomycin solution alone and the (HE)20-dauno/K55 PEC. Fluorescence measurements were taken of the daunomycin solution alone and the (HE)20-dauno/K55 ThePEC. ratio The ratiobetween between free freedrug drug and anddrug drug encapsulat encapsulateded within within the the PEC PEC was was used used to to determine determine the encapsulation efficiency efficiency and loading content. Daunomycin Encapsulation Loading Content, DaunomycinConcentration Concentration (mg/mL) (mg/mL) EncapsulationEfficiency, % Efficiency, %wt Loading % Content, wt % 11 68% 68%25% 25% 0.5 58% 21% 0.5 58% 21% 0.25 74% 27% 0.1250.25 74% 85%27% 31% 0.125 85% 31%

The (HE)20-dauno/K55 complexes also demonstrated a concentration dependent size decrease, with The (HE)20-dauno/K55 complexes also demonstrated a concentration dependent size decrease, with average diameter and polydispersity decreasing as the concentration prior to formulation decreased average diameter and polydispersity decreasing as the concentration prior to formulation decreased (Figure6). These PEC further demonstrate the degree of control available for the ultimate size of the (Figure 6). These PEC further demonstrate the degree of control available for the ultimate size of the complexes formed. complexes formed.

Molecules 2017, 22, 1089 9 of 14 Molecules 2017, 22, 1089 9 of 14

(HE)20-dauno/K55 PEC 400 1.0 *

* 0.8 Polydispersity 300 0.6 200 0.4 100

NP DiameterNP (nm) 0.2

0 0.0 1 mg/mL 0.5 mg/mL 0.25 mg/mL 0.125 mg/mL NP Diameter (nm) Polydispersity

Figure 6. 6. PECPEC formulated formulated from from (HE) (HE)20-dauno20-dauno and K55and PEC K formulated55 PEC formulated from (HE)20-dauno from/K (HE)55 demonstrated20-dauno/K55 significantdemonstrated concentration signiﬁcant dependence concentration in size dependence at concentrations in size below at concentrations 1 mg/mL. Polydispersity below 1 mg/mL. of the PECPolydispersity decreased along of the with PEC the decreased size, and was along statisti withcally the significant size, and at wasconcentrations statistically below signiﬁcant 1 mg/mL. at Dataconcentrations shown are below averages 1 mg/mL. of four Data wells shown ± standard are averages deviation. of four Results wells ± werestandard analyzed deviation. by two-way Results wereANOVA analyzed with p by < 0.05 two-way indicated ANOVA (*). with p < 0.05 indicated (*).

3.3. Formulation of PEC with Daunomycin, (HE) 20,, and and K K5555 inin ZnCl ZnCl2·2NH2·2NH4Cl4Cl Solution Solution

While the PEC diddid formform betweenbetween (HE)(HE)20-dauno20-dauno andand K K5555, the, the resulting resulting formulations formulations were were of of varying varying size with high polydispersity (Figure 66).). ToTo mitigatemitigate thisthis problem,problem, studiesstudies werewere performedperformed withwith thethe addition ofof zinczinc to to stabilize stabilize the the PEC PEC through through metal me iontal coordination ion coordination [34–36 ],[34–36], thus serving thus asserving a stabilizer as a stabilizerfor the PEC. for the Histidine PEC. Histidine is able to is coordinate able to coordinate divalent divalent metal ions, metal including ions, including zinc, with zinc, high with affinity high affinitythrough through two to six two histidine to six histidine residues [residues37]. Additionally, [37]. Additionally, there is evidence there is for evidence doxorubicin for doxorubicin interaction withinteraction zinc [38 with], specifically zinc [38], throughspecifically the twothrough keto-phenolate the two keto-phenolate substituents [39substituents], which are [39], maintained which are in maintaineddaunomycin. in The daunomycin. ability of zinc The to ability interact of strongly zinc to withinteract not onlystrongly histidine with butnotcarbonyl only histidine residues but is wellcarbonyl documented residues [is39 well–41] and,documented thus, provides [39–41] the and, chemical thus, provides basis for the interactions chemical betweenbasis for the interactions zinc ions, betweenthe (HE) 20thecopolymer, zinc ions, andthe encapsulated(HE)20 copolymer, daunomycin. and encapsulated Therefore, daunomycin. zinc was added Therefore, to the polycation zinc was addedphase toto increasethe polycation the number phase of to interactions increase the between number the of interactions (HE)20-dauno betweenand K55 tothe include (HE)20-dauno electrostatic, and K55 tohydrophobic, include electrostatic, and metal hydrophobic, ion coordination. and Zincmetal was ion addedcoordination. to solution Zinc in was the formadded of to zinc solution ammonium in the formchloride of zinc and dissolvedammonium in 10chloride mg/mL and tricine, dissolved which in was 10 usedmg/mL as a tricine, chelator. which The zinc was ammonium used as a chelator. chloride Theand tricinezinc ammonium solution was chloride used to and dissolve tricine K55 solution, followed was by PECused formation to dissolve as describedK55, followed above. by These PEC formationcomplexes as were described significantly above. smaller These incomplexes size and had were far significantly lower polydispersity smaller in (Figure size and7) illustrating had far lower the polydispersityeffect of the zinc (Figure ions in 7) providing illustrating additional the effect interaction of the zinc potential ions in betweenproviding the additional (HE)20-dauno interactionand K55. potentialThe (HE) 20-daunobetween/K 55-Znthe (HE)complexes20-dauno and continued K55. The to exhibit(HE)20-dauno a concentration/K55-Zn complexes dependent continued size (Figure to exhibit7). a concentration dependent size (Figure 7). 3.4. pH Sensitivity of E51-dauno/K55-Zn PEC and (HE)20-dauno/K55-Zn

3.4. pH Sensitivity of E51-dauno/K55-Zn PEC and (HE)20-dauno/K55-Zn The (HE)20-dauno/K55-Zn PEC exhibited a high degree of pH sensitivity, dissociating completely at a pHThe between (HE)20-dauno 6.0 and/K55-Zn 6.5 PEC (Figure exhibited8). The a complexeshigh degree were of pH stable sens initivity, size atdissociating pH values completely of 7.4, 7.0 andat a pH6.5, between with little 6.0 variation and 6.5 in (Figure diameter 8). The or increase complexes in deviation. were stable The in PEC size formulated at pH values with of E7.4,51-dauno 7.0 and/K55-Zn 6.5, withshowed little a highvariation degree in ofdiameter variation or in increase size from in pHdeviation. 7.4–4.0, The and dissociatedPEC formulated completely with E between51-dauno/K55-Zn pH showed3.0 and 4.0a high (Figure degree8). These of variation results in indicate size from that pH the 7.4–4.0, pH dependence and dissociated of the completely (HE) 20-dauno between/K55-Zn pHPEC 3.0 is andspecifically 4.0 (Figure mediated 8). These by the results histidine indicate present that in (HE)the 20pH. (HE) dependence20, as described of the above, (HE)20-dauno has a/K pK55-Zna of PEC 6.5 foris specificallythe imidazole mediated group inby histidine. the histidine At pHpresent 6.5, therein (HE) will20. be(HE) a 1:120, as ratio described of ionized above, to unionized has a pKa of histidine 6.5 for theresidues. imidazole The PECgroup formation in histidine. is dependent At pH 6.5, on there charge will interaction be a 1:1 rati betweeno of ionized the negative to unionized glutamic histidine acid of (HE)residues.20 and The the PEC positive formation lysine is of depe K55ndent. When on histidine charge interaction in (HE)20 begins between to the protonate, negative the glutamic overall acid charge of (HE)of the20 (HE)and the20 copolymer positive lysine is lost, of K resulting55. When in histidine loss ofthe in (HE) electrostatic20 begins interactions to protonate, that the held overall together charge the of thePEC. (HE) This20 resultcopolymer wasalso is lost, seen resulting in the E 51-daunoin loss /Kof the55-Zn electrostatic, which dissociates interactio atns a pHthat of held 3.0, together a value below the PEC. the ThispKa ofresult the gamma-carboxylwas also seen in groupthe E51-dauno of glutamic/K55-Zn, which acid in dissociates E51. The loss at a of pH charge of 3.0, on a the value (HE) below20 copolymer the pKa of at thepH gamma-carboxyl 6.0–6.5 will additionally group resultof glutamic in loss acid of the in electrostaticE51. The loss interactions of charge on present the (HE) in the20 copolymer (HE)20-daunomycin at pH 6.0– 6.5 will additionally result in loss of the electrostatic interactions present in the (HE)20-daunomycin

Molecules 2017, 22, 1089 10 of 14 Molecules 2017, 22, 1089 10 of 14 interaction discussed previously. previously. Theref Therefore,ore, it is likely that loss of the PEC will also result in a release of Molecules 2017, 22, 1089 10 of 14 daunomycin from interaction with with (HE) (HE)2020 andand the the accumulation accumulation of of free free drug drug in in solution. solution. interaction discussed previously. Therefore, it is likely that loss of the PEC will also result in a release of Comparison of (HE) /K PEC, daunomycin from interaction with (HE)20 and the accumulation20-dauno of 55free drug in solution. Formulated with and without zinc 400 1.0 * Comparison* of (HE)20-dauno/K55 PEC,

Formulated with and without zinc 0.8 Polydispersity 300400 1.0 * *

* 0.8 0.6Polydispersity 200300 * 0.4 * 0.6 200 100 * 0.4 NP Diameter (nm) Diameter NP 0.2 100 NP Diameter (nm) Diameter NP 0 0.2 0.0 1 mg/mL 0.5 mg/mL 0.25 mg/mL 0.125 mg/mL 0 0.0 1 mg/mLw/o Zinc, 0.5Diameter mg/mL (nm) 0.25 mg/mL 0.125w/o Zinc, mg/mL PDI w/w/o Zinc, Zinc, Diameter Diameter (nm) (nm) w/ow/ Zinc,Zinc, PDI PDI

w/ Zinc, Diameter (nm) w/ Zinc, PDI

Figure 7. Comparison of PEC formulated from (HE)20-dauno and K55 with and without zinc. PEC Figure 7. Comparison of PEC formulated from (HE)20-dauno and K55 with and without zinc. PEC Figure 7. Comparison of PEC formulated from (HE)20-dauno and K55 formulated from (HE)20-dauno/K55 with zinc were significantly smaller atwith all andconcentrations without zinc. tested PEC than formulated from (HE)20-dauno/K55 with zinc were signiﬁcantly smaller at all concentrations tested than formulated from (HE)20-dauno/K55 with zinc were significantly smaller at all concentrations tested than those without zinc. There There was a corresponding decrease in polydispersity that was most apparent at those without zinc. There was a corresponding decrease in polydispersity that was most apparent at the 1 mg/mLmg/mL and and 0.5 0.5 mg/mL mg/mL formulations, formulations, however, however, it it was was not not statistically significant. signiﬁcant. Data shown the 1 mg/mL and 0.5 mg/mL formulations, however, it was not statistically significant. Data shown are averages of four wells ± standard deviation. Results were analyzed by two-way ANOVA with are averagesare averages of four of four wells wells± standard± standard deviation. deviation. Results Results were were analyzed analyzed by bytwo-way two-way ANOVA ANOVA with with p < 0.0001 p < 0.0001 indicated indicated (*). (*).

(HE)(HE)20-dauno20-dauno/K/K5555 and and EE51-dauno/K/K5555 PEC, PEC, pH pH Response Response 500500 (HE) /K PEC (HE)20-dauno20-dauno55-Zn/K55-Zn PEC

400 E51-dauno/K55-Zn PEC 400 E51-dauno/K55-Zn PEC 300 300 200 200

NP Diameter (nm) Diameter NP 100

NP Diameter (nm) Diameter NP 100 0 8 7 6 5 4 3 2 0 pH 8 7 6 5 4 3 2 pH Figure 8. pH dependence of (HE)20-dauno/K55-Zn PEC and E51-dauno/K55-Zn PEC formulated with (HE) 20 Figure 8. pH dependence of (HE)20-dauno/K55-Zn PEC and E51-dauno/K55-Zn PEC formulated with demonstrate a high degree of pH sensitivity, with complete complex degradation between pH 6.5–6.0. (HE)Figure20 demonstrate8. pH dependence a high of degree (HE)20-dauno of pH/K sensitivity,55-Zn PEC and with E51-dauno complete/K55-Zn complex PEC formulated degradation with between (HE)20 Complex size stayed constant until pH 6.0. PEC formulated from E51 and K55 showed high variability pH 6.5–6.0. Complex size stayed constant until pH 6.0. PEC formulated from E and K showed demonstratein complex a high size overdegree the of pH pH range sensitivity, tested and with did complete not degrade complex until between degradation pH 4.0–3.0. between51 These pH55 data 6.5–6.0. high variability in complex size over the pH range tested and did not degrade until between pH Complexshow size that stayed the pH constant sensitivity unt exhibitedil pH 6.0. by PEC the (HE) formulated20-dauno/K55-Zn from PEC E51 is and specifically K55 showed mediated high byvariability the

in4.0–3.0. complexhistidine These size present data over show in the (HE) thatpH20 . therangeData pH shown tested sensitivity are and averages di exhibitedd not of degrade3–4 by wells the (HE) until± standard20-dauno between deviation./K pH55-Zn 4.0–3.0. PEC is These speciﬁcally data showmediated that by the the pH histidine sensitivity present exhibited in (HE) 20by. Datathe (HE) shown20-dauno are/K averages55-Zn PEC of is 3–4 specifically wells ± standard mediated deviation. by the

histidine4. Summary present in (HE)20. Data shown are averages of 3–4 wells ± standard deviation. 4. SummaryThis paper outlines the formulation of polyelectrolyte complexes composed of cationic and 4. SummaryThisanionic paper poly(amino outlines acids). the Formulations formulation of of PEC polyelectrolyte with poly(glutamic-acid) complexes and composed poly(lysine) of cationicshowed and concentration dependent size variation at lengths of E51/K55 and E135/K137, however, no PECs were anionicThis poly(amino paper outlines acids). the Formulations formulation of of PEC polyelectrolyte with poly(glutamic-acid) complexes composed and poly(lysine) of cationic showed and observed when E22 or K21 were used, even in combination with longer chain-length E51 or K55 at anionicconcentration poly(amino dependent acids). size Formulations variation at of lengths PEC wi ofth E51 poly(glutamic-acid)/K55 and E135/K137 and, however, poly(lysine) no PECs showed were charge ratios of 1:1, or 1:2. This result suggests a size limitation in PEC formation with these concentrationobserved when dependent E22 or K21 weresize variation used, even at in lengths combination of E51 with/K55 and longer E135 chain-length/K137, however, E51 orno K PECs55 at charge were observedratios of 1:1,when or 1:2.E22 or This K21 result were suggestsused, even a size in limitationcombination in PECwith formationlonger chain-length with these E poly(amino51 or K55 at charge ratios of 1:1, or 1:2. This result suggests a size limitation in PEC formation with these

Molecules 2017, 22, 1089 11 of 14 acids). While the specific formation process with these poly(amino acids) has yet to be elucidated, it is possible that the short chain length of K21 and E22 did not have enough charge density to decrease the Debye length, resulting in high electrostatic repulsion and a lack of successful PEC. Complexes formed with E51/K55 and a two-fold positive charge excess, 2X-E51/K55, were shown to exhibit a fairly rapid increase in size after 24 h followed by plateau. The change in size was most dramatic at the highest concentrations studied (1 mg/mL and 0.5 mg/mL), with a much smaller size increase at lower concentrations. To investigate the potential pH sensitivity of these PECs, a (HE)20 copolymer composed of repeats of histidine and poly(glutamic acid) was used in place of En for complex formation. Histidine has previously shown potential as a tool for pH-sensitive drug delivery [42–47] and the (HE)20 copolymer specifically has been shown to enable pH-dependent internalization of cell penetrating peptides [21,23], thus making it a promising candidate for formation of pH-sensitive PECs. The (HE)20 copolymer was mixed with K55 at 1:1 molar charge ratios and, in the presence of zinc, was able to encapsulate the anticancer drug daunomycin. These (HE)20-dauno/K55-Zn PEC showed a high degree of pH responsiveness, maintaining a ~400 nm diameter at pH values of 7.4–6.5, and dissociating completely between pH 6.5 and 6.0. In contrast, PEC formed with E51-dauno/K55-Zn did not dissociate until between pH 4.0 and 3.0. This large difference in pH response is likely mediated specifically by the incorporation of histidine in the (HE)20 copolymer. (HE)20 will have a net negative charge at pH values above ~6.5, however, histidine has a pKa of 6.5 and so at a pH of 6.5 will be 50% ionized. This ionization partially neutralizes the negative charge of (HE)20, and as the copolymer loses its net negative charge, the electrostatic interactions with Kn driving the PEC formation are lost, and the particle dissociates. The E51-dauno/K55-Zn particles, lacking histidine, do not dissociate until a pH between pH 4.0 and 3.0, which is close to the pKa of poly(glutamic acid). At pH 3.0 the poly(glutamic acid) residues start to become protonated, thus losing the charge-based interaction with Kn and falling apart. The use of pH-responsive platforms for drug delivery has garnered increased attention due to the fact that solid tumors possess a slightly acidic environment (pH 6.5–7) compared to that of healthy physiologic fluid (pH 7.4) [48–53]. This enables the use of stimulus-sensitive carriers, such as the (HE)20-dauno/K55-Zn PEC, that respond to the change in pH found in the tumor microenvironment by dissociation of the carrier complex. Further studies in the release profiles of these pH-responsive PECs may illustrate the use of these complexes in enabling site-specific drug release directly to the tumor microenvironment, ultimately resulting in higher specificity of drug action, lower side-effects, and reduced dosage. The (HE)20-dauno/K55-Zn PEC demonstrate a controllable size and pH response and offer a highly modular platform from which to continue further studies into the use of this technology for pH-dependent drug delivery to the tumor microenvironment.

5. Conclusions This paper outlines the production of polyelectrolyte complex nanoparticles formulated from poly(L-glutamic acid) and poly(L-lysine) at low concentrations from 1 to 0.125 mg/mL. The complexes formed are highly controllable in size, and a critical length limitation of >~50 repeats was noted for successful complex formation. The addition of histidine to these complexes, in the form of the copolymer (HE)20, resulted in polyelectrolyte nanoparticles that retained a high dependence on concentration for ultimate size. Additionally, the (HE)20/K55 nanoparticles, with encapsulated daunomycin and zinc, were highly pH-responsive, degrading completely between pH 6.5–6.0, demonstrating the effectiveness of histidine in conferring pH-sensitivity.

Acknowledgments: The authors would like to thank Likun Fei for his help with the expression of the (HE)20 peptide, and Hsin-Fang Lee for her assistance in the lab. This work was supported in part by grants (to J.Z.) from the NIH National Cancer Institute (R21 CA169841) and the University of Southern California (USC) Ming Hsieh Institute for Research of Engineering-Medicine for Cancer. Molecules 2017, 22, 1089 12 of 14

Author Contributions: Z.F.-W., J.Z. and W.-C.S. conceived and designed the experiments; Z.F.-W. performed the experiments; Z.F.-W., J.Z. and W.-C.S. analyzed the data; Z.F.-W. wrote the paper; Z.F.-W., J.Z. and W.-C.S. edited the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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