PLGA has been a major homopolypeptide model in the field of conformational transitions in proteins since the 1950s [1]–[7]. In neutral or basic environment, repulsive culombic interactions between charged Glu side chains favor random coil conformation of the PLGA main chain. With reprotonation of side chains at low pH, a rapid coil-to-helix transition takes place [6]. However, α-helical conformation is accessible only to PLGA chains of lengths above certain critical value – acidification of short disordered oligomers of α-L-glutamic acid converts them directly into aggregated β-sheets [8]–[9], as is also the case of long-chain α-helical PLGA subjected to high temperature (e.g.[10]). The ability to adopt, depending on pH and temperature, different conformations has made PLGA an insightful model for biophysical studies on protein folding. On the other hand, the reactivity and uniformity of Glu side chains triggered interest in using PLGA and its derivatives as biodegradable drug delivery systems (e.g. [11]–[12]), components of tumor targeting gene carriers (e.g. [13]), pH-switchable multi-topology vesicles [14], and multilayer films (in complexes with polylysine) for enhancement of cell adhesion (e.g. [15]). Apart from these biomedical applications of PLGA and its derivatives, the polypeptide, along with a number of other polymerized α-amino acids, has found an important explanatory function in the context of amyloid-related disorders such as Alzheimer's or Parkinson's diseases. These maladies distinguish themselves by in vivo deposition of amyloid fibrils – abnormal linear β-sheet-rich aggregates of misfolded proteins or peptides [16]–[17]. In 2002, Fändrich and Dobson demonstrated that several homopolypetides including PLGA form amyloid-like fibrils [18]. This finding led them to propose that the capacity to form such fibrils is not restricted to a handful of disease-associated proteins but rather is a generic property of polypeptides, and is primarily driven by main-chain interactions. The authors reported infrared spectra of PLGA fibrils formed through incubation at low pD (4.08) and 65°C. In the conformation-sensitive amide I′ band vibrational region, the spectra revealed a pair of peaks at 1616 (strong) and 1683 cm⁻¹ (weak) typically assigned to antiparallel intermolecular β-sheet. While these fingerprint features are commonly found in spectra of aggregated polypeptides they contrast with the infrared characteristics of similarly prepared β-aggregates described by Itoh and colleagues [19] who showed that PLGA may adopt two different antiparallel β-sheet structures: one with the spectral features similar to those described by Fändrich and Dobson (termed β₁) and another (β₂) with the amide I′ band position characteristically redshifted below 1600 cm⁻¹. X-ray diffraction analysis pointed to different packing modes in β₁ and β₂ aggregates [19]. While Itoh et al. did not study the β₂ architecture in the context of amyloid fibrils this has become subject of recent investigations carried out by our group, as well as by Keiderling′s, Kubelka's and Bouř's groups [20]–[24]. Detection of β₂-structures through infrared absorption coincides with formation of twisted supermolecular assemblies of PLGA fibrils [21]. In all our previous investigations employing high quality linear PLGA chains [20]–[21] only β₂-aggregates were observed, although the aggregation conditions were essentially the same or very similar to those used by Fändrich and Dobson. In our earlier studies on acid-and-temperature-induced conformational transitions in PLGA, β₁-aggregates were detected only as metastable transient forms occurring upon co–aggregation of PLGA and PDGA (poly-D-glutamic acid) [22]. This has led us to speculate that the amyloidogenic self-assembly of poly-Glu chains unperturbed by defects, or by polydispersity of the polymer, should produce higher order structures with β₂-like infrared characteristics. This idea also resonates with the recent study from the Keiderling's group demonstrating that synthetic (L-Glu)₁₀ oligopeptide forms β₂-fibrils [23].

The main goal of this work is to address the β₁-vs.- β₂ discrepancy by studying effects of increasing concentration of intentionally induced defects in PLGA on its tendency to form the two different types of aggregates. Covalent modification of Glu side chains through carbodiimide (EDC) - mediated formation of amide bonds with amines is often used for derivatization of PLGA (e.g. [25]–[26]). While the EDC-mediated chemistry is easy to control in aqueous environment in which PLGA is typically dissolved, NBA was selected on the basis of reasonable compromise between sufficient solubility in water and low violatility. In presented study, we have investigated NBA-derivatized and polylysine-doped PLGA aggregates with Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

The EDC-mediated modification of PLGA's Glu side chains with NBA (Figure S1) has been carried out at the fixed PLGA:NBA ratio and at varying concentrations of added EDC (Materials and Methods). PLGA does not react with NBA in the absence of EDC, and neither the amine itself nor EDC itself affect formation of β₂-aggregates (see control experiments in Figure S2). Thus in the presence of stoichiometric excess of NBA relative to Glu side chains, the number of carboxyl groups susceptible to reaction with amine is effectively controlled by the concentration of EDC. As the reaction leads to more hydrophobic and no longer ionizable side chains, it affects properties of PLGA even before polypeptide is converted into β-aggregates.

Figure 1 shows far-UV CD spectra of fresh PLGA samples progressively modified with NBA. The spectra were collected after initial alkalization of samples to pH 8.3 (A) and also after subsequent acidification to pH 4.9. (B). At any pH above 6, unmodified PLGA acquires random coil conformation (e.g. [27]), which is also the case to NBA-modified samples at EDC:Glu ratios 0.5 and lower – reflected by the characteristic negative at 197 nm and slightly positive at 217 nm spectral curvature visible in Figure 1A. However, the most profoundly modified sample (1.5 EDC:Glu ratio) shows double 208/222 nm minima which are distinctive for α-helical conformation – normally not accessible to PLGA at this high pH. This is a likely consequence of the fact that the extensive modification of Glu side chains causes simultaneous desensitization of PLGA to pH – the limited ionization of the polypeptide no longer prevents folding. CD spectra were again measured after pH of each sample was lowered to 4.9 (Figure 1B). All samples except for the most modified one (for which the spectrum is essentially flat) reveal well-defined α-helical signals. Because the acidification led to partial precipitation of the ‘1.5 EDC’ sample – reflected by the increase of light scattering at 350 nm (inset in Figure 1B), the lack of interpretable CD signals in the corresponding spectrum should be attributed only to poor optical quality of the sample.

Preliminary characterization of modified PLGA samples captures certain differences relative to unmodified PLGA which are rather intuitive. The thus obtained and characterized samples were used as precursors for preparation of β-aggregates. This was carried out by further lowering pD to 4.3 followed by prolonged incubation at 65°C. Importantly, IR spectra of NBA, EDC, and products of EDC hydrolysis either do not overlap the amide I′ band region, or are easy to subtract from polypeptide spectra (Figure S3). This coupled to D₂O being used as the reaction allowed us to probe the modification process in situ even before the final acidification and incubation step (when β₁/β₂ aggregates precipitate and become very easy to separate from the solution). FT-IR spectra of increasingly modified PLGA samples before and after the aggregation step are juxtaposed in Figure 2. In agreement with the CD data, at the close-to-neutral pD of the reaction, slightly modified PLGA samples retain random coil conformation with the corresponding amide I′ band at 1645 cm⁻¹ and prominent 1560 cm⁻¹ band assigned to antisymmetric stretching vibrations of Glu –COO– groups [28]. Only at 0.5 EDC:Glu ratio the latter band decreases in intensity (as the ionizable carboxyl groups are replaced with peptide bonds) while the amide I′ band splits into two components at 1646 and 1623 cm⁻¹. At the highest EDC concentration, the amide I′ band becomes further shifted down to 1619 cm⁻¹ with an overlapping broad component at approximately 1650 cm⁻¹ and another tiny and poorly resolved peak at 1690 cm⁻¹. The simultaneous appearance of strong spectral component below 1620 cm⁻¹ and weaker one above 1680 cm⁻¹ is routinely assigned to excitonic splitting taking place in an intramolecular antiparallel β-sheets (type β₁) – B(π,0) and B(0,π) modes, respectively [29]. Certainly, absorption from newly-created amide bonds in NBA-modified PLGA side chains will overlap these main-chain signals. In this respect, it should be mentioned that the amide I′ band of N-methylacetamide dissolved in D₂O is centered just above 1621 cm⁻¹ [33]. However, for the case studied in this work, reference data on isolated secondary amides is of limited use as the actual spectral contribution from the side chains may strongly depend on their local self-association behavior (and surroundings within the aggregate).

The key result of this study is presented in Figure 2B which shows FT-IR spectra of modified PLGA samples after acidification and prolonged incubation at 65°C. The most singular aspect of the IR characteristics of β₂-structure of PLGA is the pair of amide I′ band component peaks: a small one at ca. 1638 cm⁻¹ and the large one shifted to 1596 cm⁻¹. At this low pD Glu side chains undergo deuteration: -COOD band appears above 1700 cm⁻¹ at the expense of the 1560 cm^-1 band corresponding to –COO– stretches. Only in β₂-type aggregates of PLGA the –COOD band is characteristically split into two peaks at approximately 1731 and 1719 cm⁻¹ [20]–[21]. The extreme redshift of the amide I′ band in β₂–PLGA was attributed to reduced strength of amide C = O bonds upon formation of unusual three-center hydrogen bonds involving bifurcated main chain carbonyl acceptors and main chain ND (NH), as well as side chain –COOD (–COOH) donors [20]. According to the data in Figure 2B these spectral features are strictly maintained for aggregates of partly modified PLGA chains at the two lowest EDC concentrations (0.005 and 0.015 EDC:Glu ratios). However, a more extensive modification of PLGA at the even higher EDC concentration (EDC:Glu ratio  =  0.05), results in the emergence of peaks at 1618 and 1683 cm⁻¹ and broadening of the –COOD band. More remarkable spectral changes are seen for aggregates of PLGA chains modified in the presence of ‘0.15 EDC’. The pair of 1618/1683 peaks corresponding to β₁-type dominates the amide I′ region while the –COOD bandshape is deformed and reduced in intensity. Interestingly, this degree of modification of side chains does not affect so dramatically PLGA before the aggregation (spectra in panels A and B) implying a higher sensitivity of β₂-architecture to such defects. The β₂-components do not appear in the spectra of aggregated chains modified at the two highest EDC concentrations (‘0.5’ and ‘1.5’). Several factors are likely to contribute to the strong broadening of the amide I′ band including local conformational disorder, polydispersion of β₁-aggregates, overlapping absorption of side chains′ peptide bonds and different degree of solvent-exposure of surface-, and buried main chain amide bonds. The most thoroughly modified PLGA chains assemble into neat β₁-structure reflected by the pair of 1616 and 1683 cm⁻¹ peaks and the corresponding spectra appear to be least affected by the acidification/aggregation procedure of all examined PLGA samples.

Because random modifications of homopolypeptide side chains could, in principle, lead to complete disorder on all levels of the structural organization of aggregates, we have analyzed morphologies of β₁/β₂ samples. TEM (after negative staining - upper row) and SEM (bottom row) images of selected aggregates are shown in Figure 3. Clearly, the increasing degree of modification of side chains leads to rapid decrease in number of long helically-twisted aggregates, which are prominent only in SEM image of unmodified β₂-PLGA. Even the mildest modification in the presence of ‘0.005’ EDC appears to limit the long-range order of these assemblies. Interestingly, on the lower level of assembly corresponding to single PLGA fibrils (visible in TEM), the structural order appears to be preserved also in the case of ‘1.5’-EDC-modified aggregates.

The spectral data presented in Figure 2B strongly supports the idea that β₂-fibrils are the proper end product of defect-free amyloidogenic self-assembly of PLGA chains [20]–[22]. Formation of fibrils with β₂-type infrared features requires the occurrence of additional stabilizing interactions: bifurcated hydrogen bonds involving Glu carboxyl groups and main chain carbonyl groups. This may explain the earlier observations that in mixtures of PLGA and PDGA chains, β₂-fibrils appear to be more thermodynamically stable than β₁-aggregates [22]. Certainly, the dense packing and strict steric requirements for three-center hydrogen bonds narrowly define the structure of β₂-fibrils. On the other hand, aggregates with the β₁-like spectral features are more likely to constitute a broad ensemble of agglomerates unable to establish such bonding patterns for one reason (kinetic traps caused by co-aggregation of PLGA and PDGA chains [22]) or another (e.g. random covalent modifications of side chains used in this study). The spectral and SEM data shown in Figures 2 and 3 reflect high sensitivity of β₂-fibrils to structural defects. Even substoichiometric modifications of side chains result in barriers against formation of three-centered hydrogen bonds not only at the modification site, but most likely (through local mechanical strain and spatial separation of sheets – as explained in Figure 4) also in direct vicinity. It should be stressed that there are many possible perturbation scenarios for the self-assembly of β₂-fibrils. Here, we have employed covalent modification of side-chains with an arbitrarily selected amine. Arguably different amines could prove more effective in inducing structural defects in β₂-fibrils, as also could be the case of sporadic branching of main chains [30]. Non-covalent binding and inclusion of molecules sterically incompatible with this type of PLGA self-assembly could prove yet another way of disrupting the β₂-architecture. We have tested this hypothesis using PLL and PDL as model disruptors, since polylysine is known to form stable β-pleated electrostatic complex with PLGA [31]–[32]. Figure 5 shows FT-IR spectra of PLGA doped with either PLL or PDL at different molar ratios (expressed as Glu:Lys side chains). The β₁-type spectral features set in at approximately 11-fold excess of PLGA and begin to dominate over β₂ when this excess is lowered to 3.8∶1 – clearly, addition of polylysine prevents formation of PLGA β₂-fibrils at substoichiometric levels. The minor spectral shifts of PLGA-PLL relative to PLGA-PDL complexes are due to locally different β-sheet geometries and have been described before [32].

Aggregation and amyloidogenic self-assembly of linear and undamaged PLGA chains appears to lead exclusively to β₂-type fibrils. The process is very sensitive to structural perturbations which may consist either in covalent modifications of side chains or entrapment of foreign molecules leading to aggregates with the regular amide I/I′ band traits associated with intermolecular beta sheets. One could speculate that the outcome of at least some of published studies on PLGA aggregates revealing only β₁-type spectra (formed in the apparent absence of such purposefully induced perturbations) might have been influenced by contaminations or unsuccessful synthesis (ineffective removal of protecting groups from side chains or branching of main chains). Interestingly, out of several homopolypeptides with side chains capable of hydrogen bonding and therefore potentially favoring formation of β₂-like aggregates (along with poly-L-lysine, poly-L-threonine, poly-L-aspartic acid) such forms were only observed for PLGA. To what extent this was influenced by chemical imperfection of synthetic homopolypeptides remains unclear. This also underlies urgency to revisit several decades-old works describing limits to structural transitions and superstructural self-assembly of homopolypetides.

Unperturbed aggregation of defect-free PLGA chains ultimately leads to superstructures of amyloid-like fibrils with unusual infrared characteristics in the amide I′ band region, so-called β₂-fibrils. With increasing number of structural defects introduced either by covalent modification of Glu side chains or through non-covalent co-binding of foreign peptides PLGA aggregates progressively depart from the β₂-type towards less-ordered forms with β₁-type spectral features that are commonly associated with intermolecular antiparallel β-sheet. Our study highlights the importance of thorough physicochemical characterization of polymerized-α-L-amino acids used in basic and applied research.