Comparing Zwitterionic and PEG Exteriors of Polyelectrolyte Complex Micelles

A series of model polyelectrolyte complex micelles (PCMs) was prepared to investigate the consequences of neutral and zwitterionic chemistries and distinct charged cores on the size and stability of nanocarriers. Using aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization, we synthesized a well-defined diblock polyelectrolyte system, poly(2-methacryloyloxyethyl phosphorylcholine methacrylate)-block-poly((vinylbenzyl) trimethylammonium) (PMPC-PVBTMA), at various neutral and charged block lengths to compare directly against PCM structure–property relationships centered on poly(ethylene glycol)-block-poly((vinylbenzyl) trimethylammonium) (PEG-PVBTMA) and poly(ethylene glycol)-block-poly(l-lysine) (PEG-PLK). After complexation with a common polyanion, poly(sodium acrylate), the resulting PCMs were characterized by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). We observed uniform assemblies of spherical micelles with a diameter ~1.5–2× larger when PMPC-PVBTMA was used compared to PEG-PLK and PEG-PVBTMA via SAXS and DLS. In addition, PEG-PLK PCMs proved most resistant to dissolution by both monovalent and divalent salt, followed by PEG-PVBTMA then PMPC-PVBTMA. All micelle systems were serum stable in 100% fetal bovine serum over the course of 8 h by time-resolved DLS, demonstrating minimal interactions with serum proteins and potential as in vivo drug delivery vehicles. This thorough study of the synthesis, assembly, and characterization of zwitterionic polymers in PCMs advances the design space for charge-driven micelle assemblies.


Introduction
Polyelectrolyte complex assemblies have the unique ability to interface with an array of biologics [1][2][3][4]. This liquid-liquid phase separation process upon mixing oppositely-charged polyelectrolyte solutions can be advantageous for partitioning biomolecules into compartmentalized domains [5][6][7][8]. When the complex domain is confined to the core of polyelectrolyte complex micelles (PCMs) with neutral-charged block polycation and/or polyanion architectures, therapeutically relevant cargo can be captured, protected by the outer hydrophilic corona, and released in response to changes in pH or ionic strength. In terms of polymer selection for the outer stealth layer, poly(ethylene glycol) (PEG; synonymously polyethylene oxide or PEO) is one of the most common ways to facilitate steric stabilization from aggregation and facilitate biocompatibility. PEG-containing PCMs can self-assemble into discrete nanoparticles with controlled size and morphologies at dilute polymer conditions [9,10] and resemble ordered gels at higher polymer concentrations [11][12][13][14]. However, certain limitations of PEG have become identified in the past decade. For biological applications, this includes hypersensitivity after drug administration, deteriorating oxidative stability over time, and accelerated systemic clearance upon repeated dosage [15][16][17]. Thus, there has been substantial effort to explore PEG alternatives that can instill colloidal stability in PCM carriers in aqueous environments while mitigating some of these drawbacks [18][19][20][21][22][23].
One promising alternative includes polyampholytes, or polyelectrolytes with both cations and anions along the chain. Polyzwitterions are polyampholytes with paired cation and anion moieties per repeat unit along the electrostatically neutral chain [24,25]. The 2methacryloyloxyethyl phosphorylcholine (MPC) methacrylate monomer, in particular, has become transformative as a building block for polyzwitterions in emerging biomaterials and polyelectrolytes [49]. These results have advanced our overall understanding of how to tailor the static and dynamic properties of polyelectrolyte complex assemblies.
The objective of this work is to evaluate and compare the formulation and stability of polyelectrolyte-based nanocarriers, comprising PMPC and PEG as hydrophilic coronas and chemically distinct charged cores. Using aqueous RAFT polymerization, we have previously established a synthetic framework to prepare designer block polyelectrolytes from PEG macromolecular RAFT chain transfer agents [50]. Specifically, the diblock polycation PEG-blockpoly((vinylbenzyl)trimethylammonium chloride) (PEG-PVBTMA) has been paired with various synthetic polyanion architectures, including poly(sodium acrylate) (PAA) [50][51][52][53], PEG-blockpoly(styrene sulfonate) [50,54], and DNA oligonucleotides [55,56], to form assemblies of varying size, shape, and responsivity to salt. Because of the ease and versatility of RAFT chemistry for the production of stimuli-responsive materials [57], these endeavors have enabled us to establish structure-property relationships with special attention to effects of modulating charged block length, chemical identity, and polyelectrolyte pairing.  poly(2-methacryloyloxyethyl phosphorylcholine methacrylate)-blockpoly(vinylbenzyl)trimethylammonium chloride) (PMPC-PVBTMA). Poly(sodium acrylate) (PAA) was used as the polyanion for all cases. See Table 1 for polymer length and characterization.

Materials
Certain commercial equipment and materials are identified in this paper in order to specify adequately the experimental procedure. In no case does such identification imply recommendations by the National Institute of Standards and Technology (NIST) nor does it imply that the material or equipment identified is necessarily the best available for this purpose.
The following chemicals were reagent grade and used as received unless otherwise For RAFT polymerizations, monomers VBTMA and NaA were used as received. MPC monomer was filtered with diethyl ether to remove trace inhibitor, vacuum dried, and stored under dried nitrogen until use. Acetate buffer solution (pH 5.2) was prepared from 0.1 M acetic acid and 0.1 M sodium acetate trihydrate (42/158, v/v). Water was filtered from a Milli-Q water purification system at a resistivity of 18.2 MΩ-cm at 25 °C.

RAFT Synthesis of PEG-PVBTMA, PAA, and PMPC-PVBTMA
We synthesized PEG-PVBTMA with aqueous RAFT polymerization from a PEG macromolecular chain transfer agent (macro CTA), according to parallel synthesis and characterization procedures described by Ting et al. [30] previously. PAA was synthesized using BuPA as a RAFT chain transfer agent (CTA) in aqueous settings and characterized. Detailed molecular characterization is provided in the Supplementary Materials Section S1.

H NMR measurements were collected using a Bruker AVANCE III HD 400 Mhz
NanoBay spectrometer with 16 transients to minimize signal-to-noise. 1 H NMR spectra were processed and analyzed using iNMR (Version 5.5.7).
A representative 1 H NMR spectrum of the PMPC homopolymer is shown in Figure 2

Thermogravimetric Analysis (TGA)
TGA was conducted using a TA Instruments Discovery TGA equipped with an infrared furnace, auto-sampler, and a gas delivery module. Nitrogen was used as the purge gas set at a flow rate of 10 mL/min; a heating rate of 10 °C/min was set for all samples. TA TRIOS software (Version 2.2) was used to analyze the thermal transitions.

Micelle Preparation
PCMs were prepared via rapid mixing of water, NaCl or MgCl2, and polyelectrolyte solutions. Water and salt were first mixed with the neutral-cationic diblock polymer, mixed thoroughly with a vortexer, and followed by the polyanion and additional rapid mixing. The polyelectrolytes were added from a 5 mg/mL stock for a final concentration of 3 mM charge concentration for both the cation and anion. For PEG-PLK and PEG-PVBTMA systems, reversing the order had no effect on the final assembly structure. For PMPC-PVBTMA, we unexpectedly

DLS measurements were made using a Brookhaven Instruments BI-200SM Research
Goniometer System with an incident laser (λ = 637 nm) at room temperature. A dust-free decalin bath was used to match the refractive index of glass. The angular dependence of D was acquired by plotting q 2 versus decay rate Γ. A linearity over a range of scattering angles is a good indication of isotropic scatters. The average hydrodynamic radius of scatterers under Brownian diffusion was calculated by the Stokes-Einstein relationship. The correlation function at each angle was fitted to a first-order single exponential using a MATLAB code. The size distribution was obtained using the REPES algorithm. Kinetics experiments were done on the same instrument by recording data every 5 min for up to 16 h in a sealed sample container to minimize evaporation. For APS experiments, micelle samples were irradiated in a thin-wall glass capillary flow cell with a photon energy of 14 keV. Data were reduced in MATLAB at the beamline. Background subtraction and fitting were performed using the multi-level modeling macros distributed with the Irena software package [58] for Igor Pro as described in Ref [56].
For SSRL experiments, the sample-to-detector distance was set to 3.5 m with measurements collected at a photon energy of 9 keV. Aliquots of 30 µL of the polyelectrolyte micellar suspension were loaded onto the automated fluid sample loader at the beamline. At least 20 consecutive 1 s exposures were collected first from the buffer background (water, or water/salt mixtures), followed by samples. During data collection, solutions were oscillated in a stationary quartz capillary cell to maximize the exposed volume and reduce the radiation dose per unit volume. The SAXS data were radially integrated, analyzed for radiation damage, and buffer subtracted using the automated data reduction pipeline available at the beam line. Only data that did not show any evidence of radiation damage were included in the final average for each sample.

Micelle Salt Dependence
salt concentrations disrupt complex formation as large amounts of counterions compete with ion pairing. This is noticeable as SAXS and DLS intensity decrease and nanoparticles lose shape and dissolve into solution. The salt resistance of a polyelectrolyte complex can be used as a measure of ion pair stability in the assembly material.

Micelle Stability Tests
Micelle solutions were prepared in water with no salt at 4.5 mg/mL of total polymer. These solutions were then diluted 5x in 100% Fetal Bovine Serum (FBS), and DLS measurements were taken at θ = 90° scattering angle at 25 °C for 2 min collection time every 5 min. The autocorrelation function and light scattering intensity were recorded for an 8 hr period to observe changes in the size of detectable particles in the presence of FBS media.

PMPC-PVBTMA Synthesis and Characterization
We have previously demonstrated the consequences of modifying the chemical nature of the core in PCMs for the complexation of oligonucleotides with PLK or PVBTMA units [55].
Other studies that employ block polyelectrolytes with PEG as the neutral block have also highlighted analogous design considerations [13,59]. However, far fewer PCM studies have focused on the fundamental PCM properties of zwitterionic block polyelectrolytes that form coreshell structured micelles. To this end, we sought to prepare direct polycation analogs to PEG-PLK and PEG-PVBTMA and evaluate aspects of PCM formulation with PAA as a model polyanion.
For nomenclature throughout this work, subscripts next to the neutral and charged polymer block represent the molar mass and number-average degree of polymerization, respectively.

Resistance, and Stability
In order to elucidate the effect these physical and chemical differences have on micelle structure, we used the six polymers from Table 1 to separately complex with PAA(50) as a common polyanion, to form micelles in water. PCMs and zPCMs were assembled via direct dissolution and rapid mixing into the desired salt solution. Three polymer chemistries are being compared (PEG-PLK, PEG-PVBTMA, and PMPC-PVBTMA) at two lengths each (i.e., a 10 kg/mol neutral block with ~100 unit cation versus a 5 kg/mol neutral block and ~50 unit cation).
For nomenclature, these systems will be referred to as neutral(10k)-charged(100) and neutral(5k)charged (50). First, we screened the assembly size and size distribution of PEG-PVBTMA and PMPC-PVBTMA systems as a function of salt with a single-angle dynamic light scattering (DLS) instrument. In a low salt regime from 0-75 mM NaCl, no difference in salt resistance was detected (Supplementary Figure S-9). At 100 mM NaCl, we observed an increase in the polydispersity of the zPCMs for both neutral(10k)-charged(100) and neutral(5k)-charged(50) lengths. In comparison, the PCMs for both systems with an outer PEG corona exhibited no change in aggregation through 200 mM NaCl.
To better understand this salt-driven transition in PCM and zPCM assembly, small angle X-ray scattering (SAXS) was used to more precisely quantify the micelle shape and the size of the core and corona, providing complete structural morphology for each system. Figure 5 shows SAXS results for each neutral(10k)-charged(100) system in 100 mM NaCl. For all six systems, we observe micelle formation at 100 mM NaCl. PEG-PVBTMA and PMPC-PVBTMA systems form spherical micelles, apparent from the flat slope at low q, while PEG-PLK forms worm-like micelles (q -2 dependence) when complexed with PAA. We also notice a consistent core size difference where PMPC-PVBTMA is the largest and PEG-PLK is the smallest, determined by SAXS fitting results displayed in Table 2 in the next section below. This was consistent with the DLS screening results. Furthermore, longer cationic blocks form larger micelle cores throughout this study, consistent with previous reports [52,56] of micelle size being solely dependent on the charged block length of the block copolymer and independent of homopolymer length.   Table 2. The data for neutral(5k)-charged(50) systems show similar trends and can be found in Supplementary Figures S40-S45. For PEG-PLK PCMs, minimal structural differences or changes in scattering intensity were seen across all conditions, signifying strong complexation. PEG-PVBTMA formed PCMs at 100 and 250 mM NaCl and 50 mM MgCl2 but not at higher salts. PMPC-PVBTMA exhibited the weakest complexation, with well-formed zPCMs only at 100 mM NaCl. Additionally, both PVBTMA systems revealed a loss in intensity and increase in size dispersity with increased salt, which was not present in the PEG-PLK system. PLK has been shown to form stronger ion pairs with DNA in previous literature [55], and here we observe a consistent trend with PAA. We find a drastic discrepancy in salt stability due to the neutral versus zwitterionic block. PMPC-PVBTMA is unable to form micelles at 50 mM MgCl2 and forms extremely polydisperse poorly formed micelles at 250 mM NaCl, conditions that are favorable for micelle formation when using the PEG variant. Additional SAXS data and models are available in Supplementary Figures S40-S45.

PCMs and zPCMs in Biologically Relevant Media
Since micelle stability is crucial for systemic circulation and controlled release, we investigated the structural integrity of prepared PCMs and zPCMs in biologically relevant media, FBS. The temporal evolution of the micelles in full serum was monitored with static scattering intensity (I). As previously shown by Lin and coworkers [62], this technique provides a straightforward way to detect micelle-protein interactions shown in Equation 1: K is the prefactor constant from the optics setup, c is the sample mass concentration, and been previously shown to affect nanoparticle stabilization and delivery efficacy [33]. Nevertheless, because proteins adsorption onto nanoparticle carriers in biological settings is a known design consideration for micelle assemblies [63,64], the prepared PCMs and zPCM show good overall resistance to destabilization by serum.

Discussion
In alignment with greater efforts reported recently on elucidating the interplay between electrostatics and other non-covalent associations on polyelectrolyte complexation [65,66], this work provides a central platform for designing charged micelle-based nanocarriers with fundamental structure-property relationships. Neutral blocks in PCMs and zPCMs force nanophase separation and provide a protective corona around a charged polymer core, which often contains a sequestered cargo. Understanding the physical effects attributed to zwitterionic and neutral polymer coronas, and chemically distinct charged polymer cores, expands the design space and versatility of PCMs and zPCMs. Altogether, we have prepared a series of polyelectrolytebased nanocarriers from PEG-PLK, PEG-PVBTMA, and PMPC-PVBTMA with PAA, characterized the self-assembled micelle structure in water as a function of salt, and evaluated long-term stability in biologically relevant media. Aqueous RAFT polymerization was used to synthesize a well-defined zwitterionic diblock polyelectrolyte system PMPC-PVBTMA at various block lengths. Upon complexation with PAA, the zPCMs exhibited ~40 nm spherical micelles.
These uniform assemblies were slightly larger than the PCMs formed from PEG-PLK and PEG-PVBTMA (~20-30 nm).
Characterizing micelle structure and disassembly in the presence of monovalent and divalent salts provide a measure of complex stability. Our results show that complexes formed by PEG-PLK are much more stable against salt, followed by PEG-PVBTMA which is slightly more resistant to salt than PMPC-PVBTMA, the least stable system. This result is consistent for both neutral(5k)-charged (50) and neutral(10k)-charged(100) block lengths. Overall, this study examines neutral and zwitterionic chemistries used as polymer coronas in PCMs to protect sequestered cargo and to control particle size, stability, and environmental responsiveness.