Preparation of the NPs
Firstly, PLGA and PLGA-PEG NPs were prepared using commercially available biodegradable polymers [41]. While the hydrophobic PLGA polymer possesses carboxyl end groups, the PLGA-PEG diblock copolymer is uncharged. The hydrophilic PEG units covalently attached to the PLGA block dispose in a “brush” conformation at the NPs’ surface leading to protein-repellent surface properties [50]. Both PLGA and PLGA-PEG NPs were prepared by using an emulsifier, PVA, that forms a neutral, hydrophilic shell on the surface of the NPs, as it has been previously described [51]. For over 25 years, the versatile NPs of PLGA and PLGA-PEG have been utilized to encapsulate and deliver in a controlled manner a wide range of hydrophilic and hydrophobic drugs [52].
Secondly, biodegradable MIL-100(Fe) nanoMOFs were synthesized by a “green” (organic solvent-free) microwave-assisted hydrothermal method generating an open 3D porous crystalline architecture. The nanoMOFs possess mesoporous windows (5.6 and 8.6 Å) and internal cages of around 24 and 29 Å in diameter [28]. Discovered more recently than the polymeric PLGA and PLGA-PEG NPs, they have the advantage of being able to incorporate a variety of drugs without the use of organic solvents reaching payloads up to 20–70 wt%[28] and were well tolerated in vivo [53].
Aiming to develop biomedical applications of nanoMOFs, some of us have set up strategies to modify the nanoMOFs’ surfaces with PEG chains by mild, one-step procedures devoid of reactants and organic solvents[30] [31]. The coating was based on cyclodextrin (CD) derivatives that are large enough to avoid their penetration within the nanoMOF’s pores, and not to interfere with and displace the loaded drugs [31]. The CD scaffolds displaying grafted PEG moieties (average MW ~ 2000 g/mole) spontaneously adsorbed onto the nanoMOFs, where they are firmly anchored by coordination with the available metal sites at the surface [30]. Advantageously, the procedure was carried out in a one-step procedure in water and without using any toxic additives and led to stable coatings without altering the nanoMOFs’ crystalline structure.
Morphology and characteristics of NPs
PLGA and nanoMOF NPs, PEGylated or not, were successfully prepared with similar sizes to allow a comparative study of their nano-bio interactions as a function of both their core and surface properties Fig. 1A-D). Whereas the PLGA NPs appeared round-shaped, the nanoMOFs were crystalline with facetted structures in agreement with previous studies[43] [30]. In all cases, the average diameters ranged from 80 to 370 nm. These sizes were in agreement with DLS measurements, which showed that the PLGA and nanoMOFs NPs had mean hydrodynamic diameters of 310 ± 15 and 234 ± 16 nm, respectively (Table 1, SI). The polydispersity indexes (PDIs) were respectively 0.1 and 0.2, indicating that the NPs were monodisperse. PLGA-PEG and nanoMOF-PEG NPs had larger diameters of 418 ± 30, and 286 ± 20 nm, respectively, possibly because of the presence of a PEG shell and differences in molecular weights.
The Zeta potential for PLGA and PLGA-PEG NPs was around 0 mV, which was attributed to their neutral PVA shell, as previously reported [51]. This protective steric layer shields the negative charges of the carboxyl end group of PLGA. The nanoMOFs, coated or not, had Zeta potentials of -17 ± 3 mV, in good agreement with previously published data[43] [30].
Nano-bio interactions
Kinetics of protein adsorption
The kinetics of HSA adsorption onto the two types of NPs, hybrid and polymeric, with different core and shells, were compared. The NPs were incubated with HSA solutions and incubated up to 24 h. The unbounded HSA was quantified using a BCA colorimetric assay at different time intervals (Fig. 2).
For nanoMOFs, HSA adsorption occurred as soon as they were in contact with HSA (amount of adsorbed proteins 24 ± 2 µg/mg, 5min). Then, HSA adsorption gradually increased in a time-dependent manner up to around 55 µg/mg until a plateau is reached at the 8th hour of incubation (Fig. 2). In contrast, the amount of adsorbed HSA was very low in the case of nanoMOF-PEG (less than 7 ± 2 µg/mg) regardless the incubation time. This suggests that the affinity of HSA for the nanoMOF surface decreased while they gained “stealth” properties through PEGylation.
Surprisingly, neither the PLGA nor the PLGA-PEG NPs did adsorb any significant protein amounts. Even within three days of incubation, the adsorbed amounts were undetectable by the BCA assay. This was the case also when increasing the HSA-NP contact area (i.e. decreasing the HSA:NP ratio to 1:10 and 1:100). Taking into account the surface area developed by the NPs, it was calculated that less than 0.15 mg/m2 HSA was adsorbed. Therefore, it was concluded that the affinity of HSA for both PLGA and PLGA-PEG NPs was very low as compared to bare and PEGylated nanoMOFs.
The striking differences in protein adsorption between the two types of analyzed NPs could be explained based on their composition and physicochemical properties such as surface charge and hydrophilicity. PLGA NPs possess a neutral and hydrophilic surface in reason of their PVA coatings [38]. In contrast, the bare surface of the nanoMOFs is negatively charged and displays numerous accessible iron sites prone to bind large molecules such as proteins [54]. Whatever the NPs (PLGA or nanoMOF) the PEG shells protected them against protein adsorption.
Finally, the kinetic studies performed here provided the incubation conditions to be carried on for the following PC investigations in phosphate buffer. In the case of nanoMOFs, two hours were sufficient to form a large PC without NP degradation [55]. However, nanoMOFs progressively degraded in phosphate buffer after longer incubation times [55] [56]. In contrast, PLGA formulations were stable in phosphate buffer, so they were studied over three days in this medium.
NP morphology upon contact with protein
NP morphologies were investigated after incubation with HSA by TEM (Fig. 1E-H). The micrographs revealed that both PLGA and PLGA-PEG NPs maintained their uniform and spherical shapes and the images were similar to the prepared NPs (Fig. 1A-B). Both PLGA and PLGA-PEG NPs preserved their morphology after contact with HSA for 3 days with no evidence of PC formation (Fig. 1E-F), in line with the HSA kinetic findings (Fig. 2), which demonstrated no HSA adsorption onto the polymeric NPs.
On the other hand, TEM images showed that the facetted morphology of nanoMOFs and nanoMOF-PEG were preserved after interaction with HSA (Fig. 1G-H). Noticeably, only uncoated nanoMOFs after incubation with HSA presented a blurry aspect (Fig. 1G), not observed with PEG-coated ones (Fig. 1H). Given the high HSA adsorption onto these NPs (Fig. 2), it’s plausible to hypothesize that this blurry aspect is due to the formation of a PC.
CE was further used to investigate the corona formation on the nanoMOFs. This versatile technique also brings insights into the possible alterations of the unbound HSA after contact with the different NPs studied here.
CZE
CZE is a versatile method allowing investigation of HSA behavior on NPs by both quantitative and qualitative approaches [57] [58] [59] [60] [61]. For this study, PVA-coated capillary has been employed considering its excellent ability to separate different forms of HSA: native, glycated, and cysteinylated [44].
Bar-Or and his coworkers highlighted the heterogeneity of albumin by liquid chromatography coupled with mass spectrometry [62]. Several HSA isoforms have been found, among them native HSA, cysteinylated HSA, and glycated forms [60]. Glycated HSA is an additional clinical marker to monitor blood glycemia, and cysteinylated HSA is a key plasma marker of oxidative stress [63]. In the above-mentioned studies [44], the analysis of the protein peak area will provide quantitative information on protein adsorption. Therefore, potential modifications on the relative amounts of the different HSA forms can be evaluated qualitatively to gain more detail on HSA interactions with NP surfaces [59]. Therefore, CZE was a method of choice here to investigate nano-bio interactions combining both quantitative and qualitative aspects.
CE experiments were performed firstly on HSA solutions (without NPs) to investigate the stability of the protein in the experimental conditions employed here for adsorption studies. Thus, the HSA solutions were incubated up to three days at RT or 37°C and were subjected to the centrifugation conditions employed to sediment the NPs (without any NPs in the medium). The CZE electropherograms were then compared to those of fresh HSA solutions (Fig. SI1). The electrophoretic profiles of the HSA solutions remained unaltered whatever the experimental conditions, pointing out the stability of HSA under the experimental conditions used here to study its interaction with the NPs. Indeed, when reference HSA is incubated without any NPs, three reproducible peaks at 12.4, 12.5 and 12.7 min were obtained. These data are in agreement with previous studies using the same type of capillaries which showed the three HSA peaks assigned to Glycated, native and Cysteinylated forms, respectively [44].
Therefore, the NP series (nano MOF and PLGA) were then incubated with HSA and their supernatants were collected to investigate the aforementioned two aspects: i) quantitative by analyzing the peak areas of unbound HSA and ii) qualitative by analyzing potential peaks modification for the three HSA forms (native, cysteinylated, and Glycated). Figure 3 presents the CE-UV profiles of HSA recovered from NP supernatants after the incubation studies.
CE profiles of unbound HSA obtained after incubation with PLGA and PLGA-PEG NPs at an HSA: NP ratio of 1:3 (Fig. 3A, blue and orange lines) were almost identical to the HSA reference (not incubated with NPs). However, an increase in the NP concentration from 3 to 10 mg/mL (ratio 1 :10) resulted in a significant decrease of the peak at 12.7 min corresponding to the Cysteinylated HSA. Moreover, a new peak appeared at 11.8 min for this ratio. Interestingly, the total area of all the HSA peaks remained similar to the reference, with differences of less than 5%. It is worth mentioning that identical CZE profiles were obtained when PLGA and PLGA-PEG NPs were incubated with HSA for either one (Fig. SI2) or three days (Fig. 3A) whatever the ratio of HSA: NPs. In contrast, when the incubation time was limited to 2h, there were no significant modifications in the HSA profile even at the HSA: NP ratio of 1:10 (Fig. SI3). This shows the dependence of the qualitative aspect of nano-bio interactions on both concentration and incubation time.
Thus, we speculate that the shift of Cysteinylated HSA might correspond to a structural alteration of the protein, although it did not adsorb onto the NPs’ surfaces (Fig. 2). These findings suggest that HSA in contact with PLGA-based NPs might undergo structural modifications despite their low affinity towards them. As shown here, these modifications could be observed whatever the incubation time and NP concentration (i.e. developed surface areas). It was then interesting to investigate if structural changes occur with HSA upon incubation with nanoMOFs, where important amounts (30 ± 2 µg/mg) were adsorbed within 2h (Fig. 2). For nanoMOFs with higher affinity for HSA, no modification of the HSA profile (ie no change of the retention times) was observed on the unbound HSA species after incubation (Fig. 3B). However, the 11 ± 3% decrease in the peak area after incubation with the nanoMOF is an indication that around 37 ± 9 µg of the HSA was adsorbed per mg of nanoMOF, which is in a good agreement with the BCA protein assay (30 ± 2 µg/mg adsorption, Fig. 2). Moreover, the shielding effect gained through PEGylation was evident as the area of unbound HSA remained unchanged after incubation with nanoMOF-PEG (less than 5% changes) compared to reference HSA. To conclude, to the best of our knowledge, no study has yet reported potential conformational modification for HSA forms upon incubation with NPs. However, more detailed investigations would be needed to explain the observations concerning the Cysteinylated forms after incubation with PLGA NPs.
In complement to the CZE studies, LC-MS investigations were carried on in an attempt to resolve the HSA forms in the NP supernatants.
LC-MS investigations
Total ion count (TIC) chromatograms provided in Fig. 4 represent the ion intensities displayed as a sum of the individual HSA fragments as a function of incubation time with the PLGA NPs and nanoMOFs, PEGylated or not. TIC chromatograms of unbound HSA after incubation with or without NPs revealed similar shapes and same retention time (75.2 min) in all the studied cases (Fig. 4). When HSA was incubated with PLGA and PLGA-PEG NPs, an additional small peak appeared at 74.7 min. It corresponds to free PVA emulsifier in the NP suspensions, and it induced no effect on the main peak (Fig. 4B-C).
Values derived from the areas under the intensity curves give insights about the abundance of the protein thus relative protein quantity between samples. As indicated next to the chromatograms in Fig. 4A-B, areas under the curve values did not vary between incubated HSA reference and HSA in contact with PLGA (< 14% deviations in the areas). Therefore, it is in agreement with the quantitative BCA assay findings, which showed that HSA was not adsorbed on the PLGA NPs. In contrast, a dramatic change was recorded when HSA was in contact with nanoMOFs (non-PEGylated): 50% of reduction occurred in comparison to incubated HSA reference (Fig. 4D-E). This observation aligns with the previously mentioned findings, which suggest a decrease in unbound HSA in the supernatant, thereby indicating the formation of a PC. Interestingly, PEGylated NPs, namely PLGA-PEG and nanoMOF-PEG, demonstrated a clear resistance to protein adsorption. This was evidenced by the minimal differences of 12% and 4% respectively, when compared to the HSA reference. (Fig. 4E-F).
SR-CD investigations
To complete these studies, SR-CD was a method of choice to investigate the changes in the secondary structure of the HSA protein due to its interaction with the NPs. The CD provides access to the relative content of the secondary structure components of HSA (alpha-helix, beta-sheet, beta-turns, and random coil). While the extended spectral range was reached from 175 to 250 nm using synchrotron radiation in the DISCO beamline, the entire protein peak at 192 nm was recorded which is of interest regarding the singlet electron transitions in the HSA peptide backbone (Fig. 5A-B). As presented in Fig. 5, HSA exhibited a typical spectrum of a predominantly α-helical protein with two minimums at 224 and 208 nm and a maximum at 192 nm. These data were in agreement with the literature [50]. The peak at 224 nm was attributed to the nπ* transition from the lone pair of oxygen to a π* anti-bonding orbital. The peak observed at 208 nm was due to the ππ* electronic transitions of the peptide bond, whereas a positive band at 192 nm was due to the transition of non-bonding π orbital to the π* orbital [64]. The region around 180 nm is known as a charge transfer region. Unlike other transitions, the transitions in this region occur intramolecularly from one peptide bond to another [65].
The amount of α-helical secondary structure of HSA was obtained by deconvolution of the CD spectra. Fresh HSA reference used here presented a 70% helix content which was in agreement with the literature [66]. This helicity content was preserved 2h after incubation (70%), whereas it dropped to 61% after three days of incubation (Fig. 5A). The decrease in the HSA helicity upon incubation has been shown in various studies based on CD spectroscopy, confirming the results found here [67] [68].
The interactions of bare and PEGylated NPs with HSA were analyzed by SR-CD directly on the NP suspensions as the NPs did not interfere with the experiments (Fig. 5). A special attention was given to HSA secondary structure differences between the reference (HSA incubated without NPs) and HSA in the presence of the four types on NPs. Firstly, the contact of HSA with the PLGA NPs for 3 days (1:10, HSA:PLGA NP) resulted in a dramatic loss in α-helix content (from 61 to 27%) with visible signs of modifications that affected the bands at 224 nm (nπ* parallel transitions), 208 nm (ππ* parallel transitions) as well as at 192 nm (ππ* perpendicular transitions) (Fig. 5A). A significant change observed around 180 nm indicated that HSA was subjected to intramolecular charge-charge transitions. On the other hand, the increase recorded on HSA’s parallel β-strand (from 0 to 4%) and antiparallel β-strand (from 0 to 12%) signified the increase in β-sheet populations. Possibly, a minor denaturation or reorganization with the formation of β-strands took place during collisions at the NPs’ surfaces, without leading to adsorption as it was previously established (Fig. 2). Noticeably, when lower quantities of PLGA and PLGA-PEG NPs were in contact with the same amount of HSA, the α-helix structure of the protein remained unaltered confirming the concentration dependence of the nano-bio interactions (Fig. SI4).
HSA incubated (2h) with nanoMOFs was then analyzed and compared with incubated (2h) HSA reference (Fig. 5B). After contact with NPs, a loss in α-helix content of HSA was observed, from 70–52%. This decrease was remarkable taking into account the short incubation time of only 2h. Further analysis of the SR-CD data showed only a slight increase in antiparallel β-strands (from 0 to 2%) and in beta-turns (from 9 to 14%). Besides, the percentage of other secondary structure components (unordered) increased from 21 to 32%.
Conversely, the CD spectra in the case of HSA incubated with PLGA-PEG NPs and nanoMOF-PEG were similar to their reference HSA spectra (Fig. 5A-B). The α-helix content was preserved with less than 5% differences (within the detection limit of the method) as compared to control HSA.
In a nutshell, these results suggest that the PEGylated surfaces preserve the α-helix structure of the protein, whereas uncoated surfaces disturb it regardless of whether adsorption has taken place or not.
To go a step further, protein unfolding was analyzed through thermal denaturation studies that give insights into the effect of NPs on protein’s thermal stability. Far-UV SRCD spectra were collected over a wide temperature range, from 26 to 80°C. Figure 6A shows a typical spectrum of HSA unfolding upon an increase in temperature. All the thermal unfolding spectra intersected at 203 nm which corresponds to the isosbestic point. The HSA thermal unfolding was then assessed after incubation with the NPs. The changes in the unfolding behavior of HSA due to interactions with the PLGA NPs are presented in Fig. 6B. The isosbestic point was shifted from 203 nm to 201 nm, which is a clear indication of a reduction in HSA’s stability.
In addition, changes in other spectral features were also noted. The spectra from the first scan appeared to be in a less compact state compared to the reference HSA. This was in line with the results shown in Fig. 5A where the same NP was shown to cause a decrease in the helicity of HSA. The unfolding behavior characterized by the peak at 208 nm (Fig. 6B) was strikingly different as compared to control HSA solutions. This could be attributed to NP-induced modifications on the HSA secondary structure, particularly on the ππ* parallel transitions. The disappearance of the band at 208 nm at high temperatures (> 70°C) can be taken as evidence for the modifications of the melting behavior of HSA resulting from structural changes due to their contact with the NPs.
On the opposite, the thermal behavior of HSA has not been affected by the incubation with PLGA-PEG NPs, as the characteristic bands were fully maintained (Fig. 6C). A more compact HSA structure was observed, suggesting that the protein has gained thermal stability. It is worth mentioning that HSA denaturation depends on its concentration and experimental conditions [21],[69]. Thus, in this study, given that all the experimental conditions were the same, qualitative comparisons could be made between HSA samples incubated or not with NPs. The key discovery was the alterations in HSA stability when it came into contact with PLGA NPs, despite the fact that they did not adsorb onto their surfaces. (Fig. 2).
Thermal denaturation profiles of HSA incubated with nanoMOF and nanoMOF-PEG were represented in Fig. 7. Results were similar to the ones of PLGA and PLGA-PEG NPs. HSA was in a more unfolded state after incubation with the nanoMOF, and in particular, its characteristic band at 208 nm was diminished when increasing the temperature (Fig. 7B). Moreover, HSA unfolding occurred, as the isosbestic point was sifted from 204 nm (HSA reference) to 202 nm (HSA in contact with the nanoMOFs. However, in the case of nanoMOF-PEG, there was no significant modifications neither on the characteristic band at 208 nm nor on the isosbestic point. The protein was maintained in a more folded state upon heating and remarkably preserved its compact form throughout the temperature increase. It can be therefore concluded that the PEG-based coating conferred thermal stabilization to the HSA molecules. This is also clearly shown in Fig. 8, presenting the melting curves of reference HSA, and HSA after contact with each NP. The protective effect of the PEG coatings on both PLGA and nanoMOFs is in line with the previous CZE findings [70][71].