CONTENTS

1. INTRODUCTION

1.1. Historic Polyelectrolyte Complexes

1.2. Polyion Complexes: Polypeptides

1.3. Physical Characteristics of Polyion Complexes

1.4. Intrinsically Disordered Proteins, Polyampholytes, and Zwitterions

1.5. Rational Design of BIC Nanoparticles

1.6. Bio-inspired Cationic Polymers

1.7 Conclusions

2. PROTEIN THERAPEUTIC DELIVERY IN CONTEXT OF BIC

2.1. Protein Drugs

2.2. Polyelectrolyte Complexes and PIC Micelles Allowing Small Molecules Access to Enzymes or Targeting Release to Specific Environments

2.3. Charge Conversion

2.4. Enzyme Encapsulation

2.5. Signaling Proteins

2.6. A Special Focus on the Nanoformulation of Brain Derived Neurotrophic Factor (BDNF)

2.7. Conclusion

3. PEG APPLICATIONS, DRAWBACKS, AND ALTERNATIVE TECHNOLOGIES

3.1. PEG Function and Application

3.2. PEG Drawbacks

3.3. Poly(sarcosine) (PSR) Polymers

3.4. Poly(oxazoline) (POx) Polymers

3.5. Zwitterionic Polymers

3.6. Reformulation of Nano-BDNF

3.7. Conclusions

4. SUMMARY

1 INTRODUCTION

Polyelectrolyte complexes (PECs) might be simply considered as the products of electrostatic coupling of two oppositely charged polymers. These complexes can be dispersed particles, gels, or insoluble resins. This field has a rich history stretching back to the early 1900’s when coacervation of charged, colloidal systems was first observed. Later, PECs comprised of dense, uniform polymers were described by several independent groups which set the stage for thorough investigation of PECs. Primarily, the early part this review will focus on the scientific legacy established by Dr. Viktor Kabanov, a leader in science from the Soviet Union and, later, the Russian Federation followed by an extensive review of modern PECs. Then, we will present the challenges facing effective delivery of protein therapeutics and the application of PEC technology to address these challenges. Finally, we will consider one of the pillars of modern nanoparticle technology, poly(ethylene glycol) (PEG); both its utility and its deficiencies in the context of pharmaceutical formulations of nanosized PECs.

This review intends to connect the historical context of natural coacervates and PECs to recent efforts in nanoformulation. We will compare the physical forces which enable complexation and consider the similarities and differences of various PECs as well as the possible applications.

1.1 Historic Polyelectrolyte Complexes

PECs roughly defined might be considered the complexes such as particles, gels, or structures formed when two oppositely charged polymers are mixed. Early observations by Friedrich Meischner identified nitrogenous bases which associated with DNA in the heads of salmon sperm [1]. Albrecht Kossel then clarified that proteic protamines can condense DNA and investigated the electrostatic complexation of protamines with egg albumin [2, 3]. More systematic observations were then made in the 1920’s and 1930’s by scientists studying colloid systems. In particular, the work of H.G. Bungenberg de Jong and colleagues in describing the coacervation of gum arabic and gelatins [4], ribonucleic salts [5], and pectins [6] are landmark examples of the phenomena. These and further works are described in great depth in Colloid Sciences [7]. It is worth noting that these were some of the earliest observations of spontaneous organization of biological macromolecules which influenced early speculation on precellular organization of life [8] and now informs the study of non-organelle structures in cells [9].

The early examples of complex coacervation were accomplished with relatively low charge density, and irregular polyelectrolytes. With improved ability to synthesize high charge density and consistent polyelectrolytes came a widespread investigation into the physical properties of these new substances. During the 1950’s, active catalase was precipitated using poly(methacrylic acid) and bovine serum albumin was also isolated in its native form using several polyelectrolytes [10, 11]. Later, in 1956, Peterson and Sober developed cellulose ion exchange resins which laid the foundation for modern ion exchange chromatography [12]. Then in the 1960’s, a study of how poly(4-vinylpyridine) with different degrees of quaternization with ethyl bromide affected the binding between metal ions and DNA definitively showed that polybases could bind DNA and could even precipitate as a unified complex (Fig. 1a) [13]. In the words of the paper’s authors, a “spectacular” result. While the realization of bioactive polyelectrolyte complexes was still many years away, the promise of these new materials was immediately clear.

Fig. 1.
figure 1

Schematic representation of various PEC. Notably, we have illustrated one component as spherical though this component can be any number of morphologies including a second polymer. Also, the simple ladder model depicted in the figure is a vast oversimplification of real polyelectrolytes, their charge distributions, and their interactions. (a) The stoichiometric PEC has polyion chains mutually neutralized which leads to the complex precipitation out of solution. The complex is formed by oppositely charged polyelectrolytes of the same or different length. The definition of stoichiometry is the equality of the positive and negative charges in the PEC. (b) NSPEC has hydrophobic regions in the sites of polyion coupling of GPE (blue) to HPE (red). The complex remains in a dispersed state due to the solubilizing effect of the HPE chains that are non-neutralized. The hydrophobic regions of the neutralized GPE and HPE can aggregate resulting in core-shell-micelle-like structures. (c) The water soluble BIC is formed as a result of coupling of “doubly hydrophilic” block polyelectrolyte (containing water soluble non-nonionic and polyelectrolyte blocks) with an oppositely charged polyelectrolyte, which can be also a block polyelectrolyte. Such BICs can be either non-stoichiometric or stoichiometric. Commonly such BIC aggregate in a core-shell structures and remain stable in aqueous dispersion even when the complex is stoichiometric due to the solubilizing effects of the water soluble non-nonionic block.

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In the ensuing discussions of PECs, especially in the Russian context, we would be remiss if we did not direct the reader to Viktor Kabanov’s review article which discusses the nearly forty years he was an active researcher in the field [14]. Several of these topics are discussed in great depth and the review greatly informed the work here. The reader is again directed to V. Kabanov [14] for extensive descriptions of relevant phenomenon including how to determine the degree of conversion of a PEC and a discussion of free energy in relation to pH and Gibbs free energy.

During the 1970’s, the principles by which polymeric bases and acids interact were explored and formalized. Zezin et al. described an expansive study considering the permutations of both weak and strong polyacids and polybases [15]. Primarily, polyacids and polybases were mixed together then potentiometric titrations were performed. While strong polyelectrolytes bound and precipitated, weak polyelectrolytes exhibited more complicated behavior. Notably, investigators observed that the complexes exhibited cooperative binding between the partner polyelectrolytes. Furthermore, while weak pairs can be dissociated in high or low pH, if one partner is a strong polyelectrolyte the weak polyelectrolyte partner determines whether the complex breaks down in high or low pH. Lastly, this study also demonstrated that the secondary structure of poly(L-lysine) (PLK) or poly(L-glutamic acid) (PLE), i.e. the coil/helix transition, can be controlled via polyelectrolyte complexation [15].

This work [15] also observed precipitation of higher molecular weight structures which were later explained through the formation of hydrophobic regions where oppositely charged polyelectrolytes bound electrostatically and were stabilized by secondary bonding, postulated as hydrogen bonding stabilization [16]. This study also visualized the resultant fibrils, via electron micrograph, and noted the possible similarities to the coil/helix transitions of polypeptides which impart structure to biological systems. The cooperativity of the polyelectrolyte complexation was later confirmed using polyelectrolyte oligomers of varying lengths and measuring the propensity of association [17].

The early works discussed above laid out some of the critical properties of PECs though their application outside of specific precipitation [18] remained limited. In 1972, Tsuchida et al. published on the existence of interpolymer complexes comprised of poly(styrene sulphonate) and a polycation which solubilized the stoichiometric precipitate at a ratio of polyanion/polycation of 3 [19]. Tsuchida and colleagues contributed to a number of works exploring these interpolymer complexes concurrently with Russian groups [16, 19–23] Notably, in 1979, Kharenko et al. published a pair of papers describing nonstoichiometric, water soluble PECs (NSPECs) [24, 25] where an equilibrium exists, dependent on conditions, such that a longer polyelectrolyte binds to a shorter one, creating regions of hydrophobicity. These hydrophobic regions associate and form a core/shell structure where the shell is comprised of the free stretches of the longer polyelectrolyte (Fig. 1b). Later formalization described these reagents as the host polyelectrolyte (HPE) and the guest polyelectrolyte (GPE) [14]. It is worth noting that a number of groups were working on similar technologies at the same time. However, political, language, and social barriers limited collaborative efforts, especially in comparison to current times. This section of the review primarily focuses on the Russian tradition though there were significant contributions from groups in Japan and the United States.

Early studies identified the susceptibility of PECs to increasing ionic strength [26]. NSPECs demonstrated similar behavior in three regimes such that at low ionic strength I < I2 the NSPEC is unaltered and the solution is homogenous, in the second regime, I2 < I < I3, the system is heterogeneous and there is phase separation of the solution and in the third regime of high ionic strength, the polyelectrolytes have separated and are in solution. Notably, in regime one, the rise in ionic strength is accompanied by a decrease in radius of gyration and the second virial coefficient as the quality of solvent deteriorates [27]. These effects are heavily influenced by the identity of the polyelectrolytes, the low molecular weight electrolytes used, the ratios of components, and the length of the polymers involved [27–29]. Concurrent with these studies, polyelectrolyte complexation with model proteins were also being investigated which will be discussed further in part 2 of this review [30].

With the basic principles of PECs established, deeper investigations into the mechanisms of association, polyelectrolyte interchange, and the importance of non-electrostatic interactions were performed. While cooperativity had been established as an important aspect of how PECs associated, the minimum length was not determined. As it turns out, polymers as short as 6 units could instigate similar reactions as their longer chain counterparts. This was demonstrated when polybases of various molecular weight were complexed with heparin or poly(acrylic acid) [31]. Furthermore, it was confirmed that GPEs could switch over from one HPE to another HPE though the mechanism was unclear. The processes involving transfer of GPE between chemically identical HPEs were termed polyion exchange reactions. The processes of transfer of GPE between chemically dissimilar HPEs were termed polyion substitution reactions. With the advent of fluorescence quenching experiments, the formation of PECs could be confirmed as fluorescent units of a labelled HPE were quenched via interaction with a GPE. The importance of simple salt was reconfirmed and it was shown that the primary mechanism by which a GPE interchange proceeded was via contact and intermediate formation of a tripartite complex (Fig. 2) [32]. Further investigation confirmed the above and observed disproportionation whereby the GPE distributed unequally [33]. Also the kinetics of the PEC complexation or interchange is drastically affected by chain length, polymer identity, and ionic strength of solution [33]. Additionally, addition of a “stronger” HPE to an already formed NSPEC will completely convert to the complex with the new HPE [34]. These results were again confirmed and a later study showed that the rate limiting step of the polyion substitution was the transfer of the GPE to the second HPE when all three polymers combine [35].

Fig. 2.
figure 2

Reactions of polyelectrolyte interchange (presented here by the substitution) proceed through formation of three-component transition complex state in which the polyelectrolyte chain that is being interchanged transitions from one oppositely charged polyelectrolyte to another. Similar mechanisms are realized in NSPEC and BIC which are illustrated here.

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The discovery of the above phenomena brought about the question of what, energetically, drives the observed substitution reactions. The total Gibbs free energy of the system (ΔGT) can be represented as: \(\Delta {{G}_{{\text{T}}}} = \Delta {{G}_{{\text{P}}}} + \Delta {{G}_{{\text{C}}}}.~\) Where ΔGP represents the change in energy arising from the interaction of GPE-HPE1 changing to GPE-HPE2, ΔGC describes the difference in energy between the counterions in solution interacting with the exposed or occluded regions of HPE1 as opposed to HPE2. While ΔGC is entirely dictated by the identity of the HPEs, ΔGP is influenced heavily by length of the polymer. Izumrudov, et al. [36] described how chain length affects the propensity for the substitution reaction based on the entropy of the system.

With clearly established principles understood, other types of systems were explored as well. Polyampholytic polymers (polymers with both positive and negative sections) have also been used to generate PECs and NSPECs which could be used in similar applications to other PECs. Notably, many proteins and natural polymers are polyampholytes and the behavior of synthetic polyampholytic polymers informed their later application. Bekturov et al. reviewed the subject in depth [91] and we would direct the reader to their review for an in depth description of how polyampholytic polymers could be used in interpolyelectrolyte complexes.

After the thorough exploration of PEC complexation, the next generation of polymers, comprised of two or more distinct blocks, were developed. Block copolymers in the context of PECs generally have one polymer block which is an ionizable polyelectrolyte and a second which can be a non-ionizable, water soluble polymer. When complexed with an oppositely charged polyelectrolyte, even in stoichiometric amounts, the resulting complex is soluble due to the formation of a macromolecular superstructure where a hydrophobic core forms but is solubilized by the exterior water soluble block (Fig. 1c). These complexes have been termed a number of names in the literature; water soluble block ionomer complexes (BIC) [37, 38], polyion complex (PIC) micelles [39], and complex core coacervate micelles (C3M) [40, 41]. Several groups discovered these complexes near the same time and contributed much to the early work. Here we will use the term water soluble BIC (or BIC) advanced by A. Kabanov’s group, and PIC micelle (or PIC) advanced by Kataoka’s group interchangeably.

When considering these systems comprised of block copolymers, we can separate the discussion into distinct parts before reconvening to consider the polymers as a whole. This review will first explore what might be considered the associative blocks, those which drive formation of the complex whether complexation with a second reagent or via self-assembly. Generally, in the context of nanoparticle formulation for drug delivery, these form the core where a drug cargo may be located. Then, later in this review, we will consider the exterior block which, in the context of drug delivery, may comprise the corona of a nanoparticle and protect or promote clearance.

Early instances of complexation of block copolymers for pharmaceutical applications were demonstrated using well defined systems. Early on, A. Kabanov et al. demonstrated the utility of triblock copolymers PEG-poly(oxypropylene)-PEG whereby a micelle with a hydrophobic core was used to load and deliver low molecular weight drugs [42, 43]. This work demonstrated an interesting principle whereby hydrophobic cores could segregate hydrophobic drugs for delivery or slow release [44]. Generally, the delivery of low molecular weight drugs, especially using hydrophobic blocks, will be considered outside the scope of review though there may be occasional reference.

Later though, the same group described the complexation of PEG-b-spermine (a cationic polymer) with oligonucleotides [37]. Concurrently, Kataoka and colleagues described similar nanoparticles comprised of PEG-PLK and PEG-b-poly(α/β aspartic acid) which formed spherical particles with narrow distributions [39]. A nanoparticle comprised of PEG-PLK and short oligonucleotides soon followed with similar morphology, size, and distribution [45]. Subsequently, a nanoparticle comprised of poly(N-ethyl-4-vinylpyridinium) and PEG-b-poly(methacrylate) was described similarly though it was noted that the stability maintained across a wider range of pH and temperature than the correlating PECs [38]. Later, PEG-b-poly(ethyleneimine) (PEG-PEI) and PEG-b-poly(spermine) were complexed with DNA to form narrowly disperse, spherical, and stable nanoparticles [46]. Finally, Stuart et al. described a system of poly(dimethylamino)ethyl methacrylate)-b-poly-(glyceryl methacrylate) and poly(acrylic acid) which exhibited similar properties as the BIC micelles described above [40]. While we will not explore every polyion complex described in the literature, these selections were chosen to identity the seminal papers which inaugurated the field and realize, from the earliest examples, the therapeutic potential and opportunities were clear [47].

1.2 Polyion Complexes: Polypeptides

The utility of uniform polypeptide polyelectrolytes, which were easily manufactured to exacting specifications, was realized with the advent of water soluble BICs (PIC micelles) [39]. However, it was quickly realized that there were certain limitations which must be taken into account when designing a novel PIC. Harada et. al. demonstrated that complementary block copolymers of PEG-PLK and PEG-b-poly(α/β aspartic acid) needed the associative blocks to be of similar length [48]. Furthermore, if one complementary block copolymer was added to a milieu of both high molecular weight (MW) and low MW binding partners, it would selectively associate with the block copolymer of similar length. While this system required similarly sized binding partners, association with a homopolymer generally did not, though mixing ratio and method clearly mattered [49]. With these realizations, much effort was put into improving and diversifying the polypeptide polyelectrolyte toolbox.

Despite early success, stability issues remained as sensitivity to salt and pH was still present with polypeptide PIC micelles. One solution was to chemically crosslink the core of the micelle once associated. The PEG-PLK and PEG-b-poly(α/β aspartic acid) system was modified to include thiol groups on PEG-PLK which would crosslink and stabilize the core. The resulting particles were resistant to dissolution with sodium chloride but dissociated in reducing conditions [50]. This strategy would be used to great effect in the context of protein-block copolymer formulation [51] and we will revisit applications later in this review.

Polypeptide PICs were of immediate interest in gene transfer though association with DNA remained somewhat capricious [37, 45, 47]. Realizing the diversity of amino acids available to use for association, poly(L-histidine) was used to formulate a particle with DNA. Association was easy, DNA was condensed, and the resultant PIC was comparable to PEG-PLK/DNA PICs. Notably, because histidine is pH sensitive, the particle integrity was also susceptible to changes in pH [52]. Other groups decided to modify already proven biocompatible poly(amino acids). In particular PEG-b-poly(β-benzyl l-aspartate) was modified with diethylamine to yield PEG-b-poly(N-(N-(2-aminoethyl)-2-aminoethyl)aspartamide) (PEG-DET) which was less toxic and effected gene transfer [53]. Critically, PEG-DET minimally interacted with cell or organelle membranes at pH 7.4 but was disruptive at pH 5.0, the pH present in endosomal compartments [54]. PEG-DET has been used to encapsulate a variety of macromolecules including DNA [53, 54], RNA [55, 56], antibodies [57, 58], and enzymes [59].

The above examples were all PIC micelles, where a coacervate core forms a phase separated region. However, through careful selection of PEG inclusive block copolymers, it is possible to create a polymer vesicle. Kataoka and colleagues pioneered the technology of semipermeable polyion complex membrane vesicles (PICsomes) (Fig. 3) [60]. The first PICsomes were large though alteration of its component polymers with alkyl spacers lowered the size to more useful dimensions. Quickly, the technology was used to encapsulate the enzyme myoglobin which remained active even when the complex was exposed to trypsin, a common protease [61]. As interest and utility of PICsomes increased, inclusion of homopolymers, selection of chiral polypeptides, alteration of PEG fraction, and crosslinking enabled control over the morphology and stability of the PICsomes [62–64]. As it stands now, robust PICsomes have been developed as circulating nanoreactors for therapeutic applications [65, 66] and may soon be in the clinic [67]. Likewise water soluble BICs formed by reacting PEG-b-poly(methacrylate) with divalent cations, can be chemically core-cross-linked and then remain stable as core cross-linked swollen nanogels after removal of the cations by a chelating agent [68].

Fig. 3.
figure 3

Strategies for protein protection. (a) The most mature technology is the pegylation of proteins for persistence in the blood or to improve bioavailability of immunogenic proteins. (b) Water soluble BICs can incorporate charged proteins to the interior to protect from interaction with opsonin proteins or the extracellular matrix. (c) PICsomes can encapsulate proteins while allowing small molecules access to enzymes or targeting release to specific environments.

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PIC micelles (water soluble BICs) and PICsomes are two of the leading technologies and the later chapters of this work focus on a PIC micelle system. In the discussion of protein-PICs we will reference numerous examples. However, we will diverge to focus on the physics of complexation and new developments in PIC complexes before returning to discussion of the applications and pitfalls of these technologies.

1.3 Physical Characteristics of Polyion Complexes

Similarities between BICs/PICs and PECs comprised of homopolymers were clear from the start [14]. However, understanding of the physics determining complexation was needed in order to understand the improved stability and utility and further develop the technology. Much like canonical PECs, an early BIC, PEG-b-poly(methacrylate) complexed with poly(N-ethyl-4-vinylpyridinium), was sensitive to disruption by altering ionic strength and pH [38]. However, most physiologically relevant conditions were accommodating to BIC formation [69]. The particles were disrupted by high temperature via desolvation of the PEG blocks indicating the importance of the exterior block to particle formation. Later studies showed that the interior was hydrated and reconfirmed temperature sensitivity [70, 71], characteristics shared with PECs. Additionally, BICs associate cooperatively, consistent with PECs [72].

Another hallmark of PECs, especially NSPECs, is the ability to mediate interpolyelectrolyte complex reactions where the GPE transfers from one HPE to another or vice versa (Fig. 2). Using a polyion shell micelle, where a polystyrene-polyelectrolyte block copolymer formed a complex with a hydrophobic core, Chelushkin, et al. demonstrated that interpolyelectrolyte reactions occurred where a polyelectrolyte partner was displaced by a second. Compared to interpolyelectrolyte complex reactions using linear homopolymers, the rate of the reaction was much lower, likely due to the high charge density in the PIC core and the high aggregation numbers of the complexes [73]. However, in a system using more traditional water soluble BIC, when a free HPE was introduced, the substitution reaction proceeds with rates such that NSPEC < BIC < core cross-linked BIC which indicates that the PEG shell does not inhibit penetration and substitution [74]. These two studies indicate the dynamics of the PIC systems which can be tuned by altering different parameters including ionic strength, pH, and component parts.

As mentioned above, in the case of polypeptide PICs, the associative blocks of PEG-PLK and PEG-b-poly(α/β aspartic acid) needed to be similar lengths [48]. A more thorough study by Harada and Kataoka revealed some important properties and explored the underlying mechanisms of association. The specificity of the associative block lengths was reconfirmed and attributed to a balancing of the conformation entropy of the core block stretching and the interfacial energy. Furthermore they also showed that the size of a PLK homopolymer and PEG-b-poly(α/β aspartic acid) complex was dependent on PEG length, but not on complementary sizes of the associative, core blocks [75].

The complicated nature of the PEG-PLK and PEG-b-poly(α/β aspartic acid) hints at the complexity of the thermodynamics driving the complexation of PIC micelles. Generally, the complexation of PECs can be attributed to electrostatic interactions being stabilized by a hydrogen bond network and hydrophobic effects [14]. BICs are a more complicated case where electrostatic interactions still mediate the initial association and complexation drives water out of the core [76]. The study of the water soluble BICs formed by cationic block copolymers with plasmid DNA revealed that the formation of the complexes, disproportionation and the direction of the polyion interchange reactions depend on the polycation charge density and the DNA linear vs. circular (supercoiled) topology [77]. Later work recognized the most important contributions come from entropy including counterion release and hydrophobic effects [78]. The system environment was also recognized as important, especially ionic strength or pH changes which minimize the effect of counterion release. Finally, work looking at the entropic contributions found that they outweighed the enthalpic contribution over physiologically relevant salt concentrations [79]. While these results recognize the importance of entropy, confounding results still arise from isothermal titration calorimetry (ITC) studies with proteins and polymers which are even more complicated systems [80, 81].

Understanding the critical components and phenomena which dictate BIC formation has enabled the development of technologies which control association, morphology, and architecture of BICs. BICs have been developed which release cargo or dissociate based on polymer architecture [82], temperature [62, 71], pH [83], sugar [83], type of ion [84, 85], and hydration [86]. Additionally, the introduction of metals have yielded magnetic nanoparticles which are effective drug carriers [87].

With this basic overview of some of the important concepts needed to understand the formation of BICs we will focus on several areas of research in developing new and novel ways to formulate PECs and BICs.

1.4 Intrinsically Disordered Proteins, Polyampholytes, and Zwitterions

Coacervation is a natural phenomenon observed in nature and simple systems were described over 70 years ago [7]. These systems were suggested as an early, pre-membrane, method of organizing the molecules necessary for life [8]. In a more modern context, coacervation is recognized as an important biological process which mediates many specific interactions [88]. Notably, one of the critical components to forming many intracellular coacervate structures is intrinsically disordered protein (IDP) [89]. These proteins have one or more domains which are disordered, that is, they do not contain recognizable secondary structure and are generally flexible and sample space. However, a hallmark of these domains is strong and specific binding, often with a specific partner. Furthermore, the observation of phase separation mediated by IDPs led to purposeful design of IDP polyelectrolyte systems [78, 88, 89].

Two recent reports demonstrate the ability of an IDP, based on the hepatitis C core protein, to associate with RNA and form a nucleocapsid-like particle which looks remarkably like a PEC and may be one depending on the definition used. Another, more classical system of polyampholyte polymers was used to model IDPs which self-associate. Specifically, they considered how “blocky” the polymers needed to be in order to mediate self-association and found that blocks of 8–12 charged subunits were necessary to observe cooperative binding and salt resistance [90]. Notably, similar findings were reported for PECs using homopolymers [31]. While the field of controllable IDPs for polyelectrolyte complexation is in a nascent state, there is much promise in using these materials for PECs and PICs for drug delivery in the future.

Zwitterionic polymers are similar to polyampholytic polymers in having both positive and negative charges in a single polymer (Fig. 5c). However zwitterionic polymers have a positive and negative charge on each subunit, first described by Ladenheim and Morawetz in 1957 [92]. More recently, these molecules have found utility as both the associative core block of block copolymers in BICs or as non-fouling blocks, that is, they can act as either functional block of a block copolymer [93, 94]. Self-associative, zwitterionic, tri-block copolymers have even been designed to form PECs [95]. Looking forward, these polymers will continue to be used for non-fouling surfaces, formation of PECs, and designing thermoresponsive complexes [94].

Fig. 4.
figure 4

Nanoformulation of BDNF and receptor triggered release. (a) Nanoformulated BDNF selectively releases BDNF to the intended receptor TrkB stimulating dissolution of the complex to constituent parts. (b) The primary regions of interaction with the polymer are located at the highly charged poles of BDNF, the same surface the TrkB interacts with, identified as Patch 1 and Patch 2. A computational analysis identified residues of high interaction frequency and mapped them on the protein surface. (c) Specific binding of TrkB and P75 successfully abstract BDNF from the BIC whereas nonspecific opsonin proteins do not compromise the integrity of Nano-BDNF as shown on a horizontal agarose gel assay. Parts (b) and (c) are replicated from Jiang, et al. [80].

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Fig. 5.
figure 5

Drastic increase in PEG usage has led to increased prevalence of anti-PEG antibodies emphasizing the need for alternative “stealth” or anti-fouling polymers. (a) Increased use of PEG in pharmaceutical applications and commercial products along with the diversification of PEG structures has correlated with a drastic increase in anti-PEG antibodies in the population of healthy blood donors. (Reproduced from Haddad, et al. [196]). (b) PEG structure along with that of poly(sarcosine) (PSR) and poly(2-methyl oxazoline) (PMeOx) all of which demonstrate stealth/antifouling effects due to their hydrophilic and flexible characteristics. (c) Two of the most commonly used monomers for polyzwitterion synthesis; 2-methacryloyloxy)ethyl 2-(trimethylammonio)ethyl phosphate (phosphorylcholine methacrylate, MPC) and N-(3-methacryloyloxy)ethyl-N,N-dimethylammonio acetate (trimethylglycine methacrylate, TGMA) [200].

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1.5 Rational Design of BIC Nanoparticles

From the above discussions, it is clear that our understanding of the physics and phenomena of PECs have advanced such that more rational design principles can be explored and utilized for developing stable and responsive BIC/PIC nanoparticles. One of the earliest examples of formulation requiring careful consideration of components was PEG-PLK and PEG-b-poly(α/β aspartic acid) PIC micelles [48] and the subsequent development of PICsomes [60, 61]. Taking inspiration from the plasma membrane ubiquitous in living systems, hydrophobic alkyl spacers were introduced to stabilize the vesicle wall of the PICsome leading to stable PICsomes which did not require crosslinking [96]. Later, inclusion of guanidinium groups stabilized the PICsome even further by strengthening the hydrogen bonding network [66]. These hydrophobically stabilized PICsomes are distant relatives of a very broad class of water soluble BICs formed by block polyelectrolytes and surfactants of opposite charge [97, 98]. These BICs can form multiple morphologies including various shaped micelles and vesicles depending on the block polyelectrolyte block length and the packing parameter of the charged surfactant [99, 100]. These BIC nanoparticles can be made environmentally responsive to pH, salt concentration temperature and chemical nature of low molecular mass counterions [101, 102]. Moreover, the transition between different morphologies in such BIC systems can be controlled by light that alter the conformation of the lipid tail groups Similarly to PICsomes, the BIC vesicles can be stabilized by crosslinking the surfactant molecules [103].

Chemical modification is not the only method of improving stability of PICs. Recent advances in polypeptide production has enabled fine control of the sequences and chirality present on the associative blocks of block copolymers [104, 105]. Additionally, development of facile polypeptide synthesis has further expanded the repertoire of available sequences and structures [106]. Notably, for polypeptide coacervate PECs, inclusion of a racemic mixture for one of the binding partners destabilizes the hydrogen bonding network and control the phase of the coacervate [107]. Furthermore, design of the sequence and thus the copolymer architecture, whether by blockiness of charged units [108] or inclusion of hydrophobic residues [109], drastically affects the electrostatic and entropic contributions to stability [110, 111]. It is also worth noting that with advances in computing, rational design of polymers has begun to greater understanding of how to design more stable or responsive PECs and BICs [112].

1.6 Bio-inspired Cationic Polymers

The last section of this part of the review will take us away from rational design to focus on bio-inspired polymers. In particular, we will consider bioadhesives inspired by marine animals. These PEC networks function in particularly harsh conditions which often overwhelm chemical adhesives. Since time immemorial, people who lived on the water were familiar with animals which adhere to surfaces extremely strongly or create structures which are remarkably resilient. With modern analysis, our understanding has evolved to know these are secreted proteins with specific sequences. During these exploratory studies one of the most notable features was that most of the binding partners were both cationic. Also, they contained high proportions of 3,4-dihydroxyphenylalanine (Dopa), a catecholic amino acid derivative of tyrosine. These unique amino acids mediate π–π binding and cation–π interactions which form strong bonds in water. The above is a summary of details found in Kord Forooshani, et al., in which can be found a detailed review of mussel foot proteins and bio-inspired bioadhesives [113]. Bioadhesives are of particular interest in wound healing so as to limit the need for mechanical sutures. Similarly, the “cement” made by sandcastle worms is a mixture of sand and a polyelectrolyte complex comprised of a polycationic peptide and a Dopa prevalent anionic peptide [114].

Initially, bioinspired adhesives were developed based on mussel foot proteins which could stably and strongly bind underwater [115]. Later, complex coacervates were formed using two like-charged (cationic) mussel inspired proteins to form a PEC which was spherical [116]. This type of adhesion has been of interest in non-fouling coating for underwater or medical device purposes [117].

1.7 Conclusions

PECs are present in a wide variety of applications in nature and our own understanding, despite over 50 years or work, is still increasing. Not only that, but our ability also to design and implement PECs continues to grow in a variety of application. For our purposes, we will focus on water soluble BICs from here on out as drug delivery vehicles, particularly for protein therapeutics. As our understanding of PECs continues to grow, we will hopefully see novel applications in material science, drug delivery, and other yet unknown functions.

2 PROTEIN THERAPEUTIC DELIVERY IN CONTEXT OF BIC

2.1 Protein Drugs

Proteins are polypeptide electrolytes which often fold to globular shapes with complicated surface chemistry to effect function and mediate interactions in the biological milieu. Most proteins considered for drug delivery have well defined structure or are short peptides which may or may not have complicated secondary structure [51, 118–120]. Per the scope of this review, protein-PECs and the role of proteins in coacervation has be realized since the advent of the field where gliadins were involved in the earliest recognition of coacervate phenomena [6]. As mentioned above, polyelectrolyte resins, and ion exchange columns preceded the development of protein PECs [10–12, 14].

Many proteins may be utilized for therapeutic purposes [120], however, delivering the protein drug or mediating its survival in serum may require modification or encapsulation [121, 122]. Exogenous proteins are particularly susceptible to immunological clearance and even endogenous proteins can be efficiently cleared [80, 120, 123]. One of the most successful strategies to reduce opsonization and preserve protein therapeutic efficacy is to modify with hydrophilic and flexible polymers, most notably PEG (Fig. 3a). Protein PEC hydrogels, PIC micelles, PICsomes, and multilayer BICs are also viable strategies [51, 122, 124, 125]. We will review examples of each below (Figs. 3b, 3c).

While certain principles can be applied across proteins, it is important to remember that proteins are extremely complex and even apparently similar proteins can behave differently in the context of the same polyelectrolyte homopolymers [126]. Still though, we can make some broad assertions. Electrostatic complexation creates hydrophobic patches such that hydrophobicity in the polymer can stabilize protein PECs or PICs/BICs [126]. The complexes are sensitive to salts and environmental manipulation can tune nanoparticle formation [127]. At low ionic strength, association can be entropically driven via counterion release [128]. Notably, these physical principles correlate to many of the principles set down in the early PEC work [14]. However, modern techniques and computation have enabled a deeper understanding and modeling of these complex systems [129].

As a final note on protein drug delivery, we must discuss the complexity of delivering a specific therapeutic to a specific target via a specific route of administration. For instance, oral delivery has many considerations that are not present in intravenous (IV) or intranasal to brain (INB) administration [80, 123, 130, 131]. We will not be discussing routes of administration in depth, but it is useful to keep in mind with the below applications. Additionally, we will discuss some methods by which administration is mediated or triggered using targeting moieties [132] or environmental sensitivity [80, 133].

2.2 Polyelectrolyte Complexes and PIC Micelles

Development of PECs inspired the early development of incorporating proteins into BICs. In 1977, bovine serum albumin (BSA), a protein with multiple charged patches as well as hydrophobic patches, was complexed with quaternized poly(4-vinylpyridines) [134]. Later, amphoteric polymers as well as cationic polymers were also complexed with BSA [135, 136]. BSA though, is not a particularly useful therapeutic protein. Thus, penicillin amidase, an enzyme, was covalently attached to a cationic polymer where it was active. However, activity was curtailed with addition of the anionic polymer which could impart a level of control over the enzyme, even in the presence of its substrate [137]. Later complexation of enzymes or other proteins with hydrogels was based on these early works [14].

Kokufuta et al., in concurrent efforts, demonstrated the complexation of hemoglobin with potassium poly(viny1 alcohol) sulfate and poly(diallyldimethylammonium chloride) [138]. The same group later demonstrated that human serum albumin could complex to strong polyacids and polybases depending on the pH of solution [139]. Kokufuta’s group also made significant contributions to the development of microencapsulated PEC biocatalysts, which fall outside the scope of this review [140].

Later work performed by P.L. Dubin and collaborators demonstrated the use of polyelectrolytes to precipitate several proteins and to form soluble protein-PECs [141–143]. They also demonstrated the complexation of alcohol dehydrogenase and trypsin with poly(diallyldimethyl-ammonium chloride) to form complexes with preserved enzymatic activity [144]. Dubin and others contributed significantly to our understanding of the physics of PECs, reconfirming some things and expanding our understanding of how buffer conditions affect the complexation of proteins and polyelectrolytes as well as polyelectrolyte interactions with charged micelles [145, 146].

Protein-polymer nanoparticles were the earliest application of noncovalently bound polymers to protect and preserve therapeutic proteins and peptides. The next step of protein-block copolymer nanoparticles was comprised of PEG-poly(lactic acid) and the tetanus toxoid, a hydrophobic peptide. Spherical particles of less than 150 nm were formed with a polydispersity index of less than 0.2. Most notably, when a gelatin stabilizer was included, particles continued to release cargo for a full month indicating a stealth effect whereby the particles were ignored by the immune system [147]. It is an easy leap to the application of ionic block copolymers for charged proteins.

Kataoka’s group preferred to work with lysozyme which deserves some discussion as a model system. Lysozyme has long been a popular model system for protein scientist due to its thorough characterization, stability, easily measured activity, and availability. In the BIC context, the relatively dense and uniform cationic charges on its surface made it somewhat simple to associate with anionic block copolymers. A BIC formulation whereby PEG-b-poly(α/β aspartic acid) and lysozyme were complexed demonstrated formation of spherical nanoparticles that were salt sensitive [148]. Also, the BIC occluded access to large substrates so that the protein was inactive until the complex was dissociated with salt [148]. Further characterization showed that for small substrates, the PEG corona could actually act as a reservoir and increase the catalysis rate for the encapsulated protein [149]. A similar BIC micelle was formulated with trypsin, a common protease, which improved its stability and demonstrated the utility of crosslinking using glutaraldehyde in the context of PIC micelles [150, 151]. Crosslinking and inclusion of hydrophobic elements were used to improve BIC formulation with lysozyme as well which improved salt and pH resistance [152, 153].

Stabilization was useful to a point but control over the size and morphology was another focus as well as where and how to release protein cargo. BICs are complex systems which exist over a range of conformations which can take days to relax into a preferred conformation [154]. Similarly, changes in the solution via ionic strength can alter complex behavior [155]. In one notable example, lipase was ejected from the core and thus became active [156]. Other experiments using homopolymers to displace lysozyme echoed the interpolyelectrolyte exchange reactions observed by V. Kabanov and colleagues with PEC [157].

While these model systems allowed fruitful exploration of protein BIC formulation, most were not therapeutically useful. Unfortunately, useful proteins tend to be less amenable to nanoformulation which can be overcome in various ways. The ensuing sections will explore ways in which proteins and BIC systems have been manipulated to produce therapeutically interesting formulations. The sections will be organized by application followed by an overview of some cutting-edge solutions.

2.3 Charge Conversion

One of the most notable features of lysozyme, the premier model protein, is its cationic exterior charge which facilitates formulation with anionic polymers. Most proteins are not so strongly and singly charged, often having a mix of patchy surfaces with cationic, anionic, neutral, and hydrophobic areas. One solution is to convert positive patches to negative so that the surface is only positive or neutral. Citraconic anhydride can be used to convert positive residues on the exterior of the protein to negative moieties [158]. Moreover, these moieties are unstable at pH 5.5 and break down to the original cationic amine. Endosomal pH can be as low as pH 5, thus enabling release of the BIC cargo which was demonstrated using small, fluorescent molecules [58, 158]. These technologies have been applied to antibodies aimed at intracellular targets [133]. In the future, other proteins with patchy surfaces may be used.

2.4 Enzyme Encapsulation

Many proteins of therapeutic interest are enzymes, a class of proteins which catalyze chemical reactions, usually of a specific substrate or class of substrates. Exogenous enzymes often cause an immunogenic response or can generally be unstable. Endogenous proteins may be cleared more quickly than desired or may be unstable. In short, there are many reasons to encapsulate or formulate enzymes for delivery.

The nanoformulation of enzymes addresses some major issues in protein drug administration [118]. Most notably, formulation can enable long circulation, improve stability, enable targeting, and even improve enzyme kinetics. However, care must be taken to avoid inactivation [137, 159]. These issues are generally easy to avoid with careful formulation [160].

Cu, Zn superoxide dismutase 1 (SOD1) is an extremely well characterized protein involved in reducing reactive oxygen species throughout the body. Reactive oxygen species levels are raised in inflamed tissues, neurodegenerative disease affected brain tissue, and cancerous lesions. SOD1 has been stabilized via genetic manipulation [161] but nanoformulation was still preferred for later administration. An initial BIC nanoformulation of SOD1 complexed with PEG-PEI has shown ability to attenuate angiotensin II-dependent pressor response and intra-neuronal signaling after intracarotid injection in a rabbit [162]. Subsequent development of BIC nanoformulation of SOD1 cross-linked with PEG-PLK produced small (<20 nm) and narrowly dispersed particles [163]. These SOD1 nanoparticles were amenable for intravenous injection, showing improved delivery to the central nervous system (CNS) and therapeutic activity in a rat model of stroke after a single injection [163–165]. A later reformulation using PEG-DET yielded a nanoparticles with reduced toxicity and low mononuclear phagocyte system accumulation [59]. Notably, this formulation demonstrated considerably improved outcomes in a mouse model of ischemic stroke. Finally, the development of cross-linked multilayer BICs entrapping SOD1 resulted in prototypical antioxidant modality showing promising therapeutic effects in treatment of acute spinal cord injury in a rat model [125]. Here, we see how several rounds of reformulation may lead to a much-improved final product.

The diversity of enzymes encapsulated by nanoformulations continues to grow and expand with the further development of novel forms and methods of encapsulation. Recently, organophosphate hydrolase, a protein of interest in preventing organophosphate intoxication, was modified with a 6-histidine tail and complexed with PEG-PLE, improving the bioavailability of the enzyme and protecting against lethal doses of organophosphate pesticides and even a chemical weapon [166]. A competing effort actually grew a zwitterionic polymer on the surface of the protein and yielded similarly successful results [167]. Also recently, a PICsome was developed carrying glucose oxidase for release in targeted cells where it would induce cell death by pyroptosis [168]. Another study demonstrated encapsulation of the therapeutic protein uricase and nonspecific uptake by macrophages [169].

As we consider novel technology using nanoparticles, the targeting and utility of macrophages becomes more topical. In a series of works, Batrakova and A. Kabanov have led an effort that began with enzyme encapsulation and evolved to macrophage mediated delivery. In the first phase, catalase was packaged with PEG-PEI and taken up by macrophages, these macrophages released active nanozymes over 24 hours and were able to deliver some of the dose to the brain in a mouse model [170]. A later study confirmed and quantified the results in a mouse model of Parkinson’s disease [171]. Most importantly the strategy improved the bioavailability of catalase at areas of inflammation of the disease model mouse brain [172]. Ultimately, it was found that macrophages mediated protein delivery via release of exosomes [173, 174]. Work within these systems has continued in the Batrakova laboratory but the developments leave the scope of this review.

2.5 Signaling Proteins

As with advancements in nanoparticles containing enzymes, the technologies for preservation of signaling macromolecules has benefited from PICs and other polymer-based technology. Signaling macromolecules present several distinct challenges in contrast to enzymes. Primarily, the signaling protein must access its receptor or binding partner [118]. Other therapeutic macromolecules face similar challenges such as nucleic acids, antibody therapies, and proteins which must act at specific sites. These alternative macromolecules fall outside the scope of discussion here, but the accompanying technologies have informed and improved developments for delivery of signaling molecules.

Careful PEGylation has been one of the leading technologies for signaling molecules. Leptin is a signaling molecule of particular interest in the treatment of obesity and has been called the “satiation signal.” Covalent modification of leptin with PEG-b-poly(propylene oxide)-b-PEG, a polymer known to promote crossing of the blood brain barrier (BBB), as a single polymer and multiple polymers was studied. A single polymer improved the bioavailability in the brain via the leptin transferase and lasted longer in the periphery. Modification with multiple polymers mediated transporter independent accumulation [175]. In order to make a more homogenous protein drug, specific modification to the N-terminus of the leptin protein by the same polymer was tested via delivery using an intranasal to brain (INB) route. The N-terminal modified leptin showed improved brain delivery and stimulation [176]. Finally, in an effort to avoid covalent modification, delivery via macrophage exosome was demonstrated as effective as well [177].

2.6 A Special Focus on the Nanoformulation of Brain Derived Neurotrophic Factor (BDNF)

In this section of the review, we will more thoroughly describe BDNF, its structure and function, its role in the body, prospective therapeutic effects, and existing formulation work. BDNF is a homodimeric protein of approximately 28 kilodaltons which is involved in various brain signaling pathways [178, 179]. The protein is found in the central nervous system as well as peripherally from where it can be transported across the BBB by a designated transporter [180]. The primary receptor is the tropomyosin receptor kinase B which stimulates the Ras-mitogen-activated protein kinase pathway, the phosphatidylinositol-3-kinase pathway, and the phospholipase Cγ pathway [179, 181–183]. A second, low affinity receptor also exists, the p75 receptor though the effects are less well defined [184]. These stimulatory pathways function in proper brain development, synaptic plasticity, neuropotentiation, axonal pathfinding, and neuron survival [179, 183]. While BDNF function is complicated, the effects of BDNF deprivation is clear in improper brain development and it is notably downregulated in a number of disease states [179]. Hence, BDNF has been suggested as a potential therapeutic option in a myriad of brain and neuronal diseases and disorders.

BDNF is notably downregulated in several psychiatric and neurodegenerative disorders. Additionally, application of BDNF to many of these disorders or diseases has yielded promising results. Giacobbo et al., has provided an expansive account of publications reviewing these disorders and BDNF’s role [179]. Notably, BDNF seems to have a possible therapeutic application across many diseases and has demonstrated utility in many animal models including Alzheimer’s, Parkinson’s, ischemic stroke, and several psychiatric diseases.

Water soluble BIC formulation of BDNF was motivated by the rapid clearance of administered BDNF from the serum as well as the rapid efflux from the brain [180]. BIC encapsulation is particularly attractive for two reasons, (1) BDNF is densely and positively charged [178], and (2) BICs offer a way in which to noncovalently preserve BDNF in the brain. BDNF is already known to associate with the lightly negatively charged branches of polysialic acid which comprise a major part of the brain’s extracellular matrix [185, 186]. The same characteristics that enable that association also mediate association with negatively charged polymers [80, 81, 123].

As is noted before, covalent modification is undesirous when considering formulation of most signaling proteins [160]. Simple vortex mixing of PEG-PLE and BDNF sufficed to yield a nanoparticle, termed Nano-BDNF which was approximately 225 nm in diameter with low dispersity. The particle resisted dissolution with small electrolytes and preferentially released BDNF to specific binding proteins which competed for similar areas of interaction, e.g. areas of dense, positive charge. Most importantly, this group of specific binders includes its target receptor, tropomyosin receptor kinase B, meaning that there is, effectively, receptor triggered release. Notably, Nano-BDNF PEG-PLE also improved delivery to the brain via intranasal to brain (INB) delivery and reduced efflux from the brain [80]. In terms of efficacy against disease models, Nano-BDNF delivered intravenously and INB improved outcomes in models of ischemic stroke and Parkinson’s disease respectively [80, 123]. Later work describing PEG-free reformulation is reviewed in Section 1.3.5.

2.7 Conclusion

The complexation and encapsulation of protein drugs is a promising field with much overlap with the encapsulation of other macromolecules. However, due to difficulties in passing the approval process, the promise of PIC/BIC and protein-PECs has not yet been achieved. However, the field is active, and it is the wide expectation that with increasing technological sophistication and manufacturing ability, more protein polymeric nanoparticles will soon reach the human trial stage.

3 PEG APPLICATIONS, DRAWBACKS, AND ALTERNATIVE TECHNOLOGIES

The human immune system is a true marvel of biology which every biomedical scientist should occasionally remember to appreciate, or, for drug delivery specialists, reel in horror at its complexity and efficacy. The body is naturally adept at clearing even endogenous macromolecules, the challenges for molecules or structures are even more innumerable if they are not native to the body. While some therapies, most notably vaccines, take advantage of the natural immune system, much of the work described above has focused on avoiding clearance. The most potent tool in the field of “stealth nanoparticles” and non-fouling surfaces has been PEG. A more thorough review on PEG for the interested reader might be found in the recent publication by Shi et al. [122].

3.1 PEG Function and Application

An early, if not the earliest, report of the immunological properties of PEG was presented by Abuchowski et al. in 1977 [187]. Covalent attachment of PEG to bovine serum albumin yielded a nearly complete evasion of the immune system and stymied the immune response [187]. A later study demonstrated that PEG was particularly anti-fouling due to its hydrophilic and flexible properties [188]. Densely PEGylated surfaces or particles exclude opsonins and other immune proteins via entropic exclusion which is mediated by those characteristics [122, 188]. This “stealth” effect is commonly utilized in covalently modified biotherapeutics and nanoparticle systems [118, 122, 160]. Indeed, further study has demonstrated that dense PEGylation can effect mucus and brain penetration, two environments which often stymy unprotected pharmaceutical candidates [189, 190]. Furthermore, protein PEGylation has been particularly effective in developing protein drugs present in the clinic [119]. The utility of PEG is demonstrated by its inclusion in numerous pharmaceutics in the clinic and it will continue to be used for the foreseeable purpose.

3.2 PEG Drawbacks

While the utility of PEG is not in question, there are some clear drawbacks and concerns which have been raised in recent years. Summarily, PEG is immunogenic and due to that property, repeat administration of PEGylated therapeutics can lower efficacy, immune reactions are possible, and other allergic reactions can be triggered [122]. Complicating these effects is the ubiquity of PEG in modern society (Fig. 5a). It is present in common consumer products, cosmetics, food, and many medicines. It is generally regarded as safe, and its use is not regulated beyond the general safety assessment consumer products undergo. It is also worth mention that PEG was present in the Moderna and Pfizer vaccines against the SARS-COVID-2 virus which were widely distributed [191].

The repeat administration of PEGylated therapeutics leads to the rapid clearance of the therapeutic agents, identified as the accelerated blood clearance effect [192]. The cause of this effect has been identified as the presence of anti-PEG antibodies present in the sera [193]. Anti-PEG antibodies have been identified as a cause of reduced mobility in mucus as well [194]. Recognition of these phenomena led to investigations of the prevalence of anti-PEG antibodies in the general population and over time [194, 195]. Incidence of antibodies in the general population was as high as 72% in contemporary populations [195]. Additionally, anti-PEG antibodies were found in blood samples from several decades before though at lower incidence level [195].

Anti-PEG antibodies may be part of the normal milieu of antibodies in the body and should not be identified as a negative trait, rather formulation scientists and medical doctors must be careful to identify individuals who may have negative allergic reactions [196]. Recent incidents have brought this issue to the public consciousness due to the government sponsored and distributed SARS-COVID-2 virus vaccines and their unfortunate politicization. Despite their nearly miraculous efficacy and minimal toxic effects, incidents of allergic reactions were identified and attributed to PEG sensitization in some cases [197]. Anaphylaxis was found to be correlated to presence of anti-PEG antibodies and the mechanism was identified through several routes including contact system activation by nucleic acids, complement activation of immune system, direct mast cell activation, and most relevant to us, the presence of anti-PEG antibodies [198]. As an interesting aside, incidents were higher in women and it was hypothesized this may be to higher exposure to PEG in general life [199].

Despite these possible drawbacks and the worrying prevalence of PEG in everyday life, these works should not be taken to insinuate that PEG is not a useful molecule. The utility of PEG is self-evident in the many approved drugs which contain PEG for the purposes described above. It is an incredibly useful molecule which will continue to be the gold standard noun-fouling molecule for the foreseeable future. The following sections will describe alternatives in development. It should be noted that, in most cases, the purpose is not to replace PEG, but rather to expand the repertoire of molecules which can achieve similar results.

3.3 Poly(sarcosine) (PSR) Polymers

Poly(N-substituted glycines) (Peptoids) are a class of molecules very similar to amino acids and polypeptoids are analogous to amino acid peptides. The simplest of these peptidomimetic molecules is poly(sarcosine) (PSR) which is analogous to the amino acid alanine [201] (Fig. 5b). Like PEG, it is flexible and hydrophilic, allowing entropic exclusion of opsonins and has been recognized as a prime candidate for PEG substitution [202, 203]. PSR has been identified as anti-fouling and appropriate for inclusion in nanoparticles as a “stealth” component [204, 205]. A recent study also shows that it can protect conjugated proteins from proteolysis [206]. In another study, an unexpected result was observed that PSR coated liposomes avoided the accelerated blood clearance effect [207]. These findings and others inspired part of work described in our recent publication on reformulation of Nano-BDNF [81].

3.4 Poly(oxazoline) (POx) Polymers

POx polymers are a versatile set of polymers composed of oxazoline monomers which can be modified in a number of ways to include hydrophobic moieties, charged groups, and flexible, hydrophilic monomers. Facile synthesis, via living cationic ring opening polymerization (LCROP), have made them a useful and diverse set of polymers for formulation science [208]. In particular poly(2-methyl-oxazoline) (PMeOx) has been identified as a strong candidate to replace PEG (Fig. 5b) [44, 209–211]. In 1994, liposomes coated with PMeOx demonstrated an immune evasion effect similar to PEG, this was later confirmed again by the same group [212, 213]. Conjugates with proteins have also shown immune evasion by shielding against opsonins [210]. POx polymers have been demonstrated as appropriate for non-fouling surfaces [214]. Indeed, numerous POx micelles have been developed with hydrophobic cores for delivery of small molecules demonstrating the diversity of possible polymers as well as the protective ability of hydrophilic and flexible POx polymers [81, 215–222].

3.5 Zwitterionic Polymers

Finally, we will describe the utility of zwitterionic polymers as alternatives to PEG. Zwitterionic polymers contain both a positive and negative charge on each monomer (Fig. 5c). These polymers have been of particular interest in non-fouling surfaces where several recent works have reported successful synthesis of non-adsorptive surface coatings [94, 200, 223, 224]. Another application reported is the protection of organophosphate hydrolase with polymers grown on the surface of the protein which successfully protected the immunogenic protein from clearance and preserved activity [167]. Notably, the application of zwitterionic polymers is a relatively new field where rapid advances have been made recently. Novel applications and inclusion in particles is an exciting area of research with many novel complexes being designed.

3.6 Reformulation of Nano-BDNF

Nano-BDNF PEG-PLE, described in Section 2.6, is a promising BIC which has demonstrated effect in the context of two animal models of disease and injury [80, 123]. However, PEG comprises the exterior block of the complex. Fay et al. describe the reformulation of Nano-BDNF with two polymers, PSR-PLE and PMeOx-poly(oxazolepropanoic acid)-PMeOx (PMeOx-PPpaOx-PMeOx), with the stated intent of diversifying the available pharmaceutic options [81]. They report that Nano-BDNF PSR-PLE and Nano-BDNF PMeOx-PPpaOx-PMeOx form similar particles which look and act nearly the same as Nano-BDNF PEG-PLE in a biophysical context. While the distribution of particles in vivo is yet unknown, successful expansion of Nano-BDNF options indicates that broadening pharmaceutical variations may prevent PEG resistance from becoming a population level problem.

3.7 Conclusions

PEG has been the gold standard, non-fouling polymer and is successfully used in many therapeutic applications currently. There are clear concerns with its overuse though. Allergic reactions and accelerated clearance have been reported, most likely due to specific recognition by the immune systems. Many alternatives have been proposed and shown some level of efficacy. However, their novelty has precluded extensive use in the clinic. It is worth noting though that more PEG-free formulations are expected in the future.

4 SUMMARY

The promise of PECs identified in during the 1970’s is slowly being realized with the development of PICs, modern PECs, hydrogels, and other similar technologies. Part of our effort here is to emphasize the historical nature of science where one work builds on the generations before and attempts to clarify part of the historical timeline. Furthermore, we can see how various interconnected efforts inform and support each other. Briefly, we will discuss each section and the conclusion which should be drawn.

The early work, especially by V. Kabanov and colleagues, established many of the defining principles which continue to inform later works. Understanding the mechanisms of association and cooperative nature of complexation continues to inform more recent developments. PEC technology led directly into the development of block copolymers and BIC/PIC micelles. The studies exploring the mechanisms of association were dependent on the description of PEC complexation. Understanding these principles has led to the development of new nanoparticles including PICsomes, and multilayer BICs. The recent development of rationally designed polymers and bioinspired polymers are understood through the lens of PECs and often contrast these novel chemistries to the earlier studies using uniform, charged polymers. Furthermore, the packaging of therapeutic cargos is best understood as polyelectrolyte complexation in many cases.

Many nanoparticles described are dependent on PEG which can help drive complexation in addition to providing a protective effect. Despite the success of PEG, the drawbacks are well documented. Additionally, ubiquitous use can eventually lead to population level problems with PEG resistance or anaphylactic reactions. Alternative polymers which can provide similar effects are needed to diversify and supplement the therapeutic arsenal. We have described several leading candidates for PEG-free alternatives as well as one example of a successful reformulation.