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Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles and serum proteins Tian-Xu Zhang‡,1 , Guan-Yin Zhu‡,1 , Bo-Yao Lu1 , Chao-Liang Zhang1 & Qiang Peng*,1 1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China * Author for correspondence: Tel.: +86 28 85 501 484; Fax: +86 28 85 501 484; [email protected] ‡ Authors contributed equally
Aim: A comprehensive understanding of nanoparticle (NP)-protein interaction (protein corona formation) is required. So far, many factors influencing this interaction have been investigated, like size and ζ potential. However, NPs exposure concentration has always been ignored. Herein, we aim to disclose the correlation of NPs exposure concentration with protein adsorption. Materials & methods: Four polymeric NPs systems possessing similar sizes (230 ± 20 nm) but varied ζ potentials (−30 ∼ +40 mv) were prepared. Physicochemical properties and protein adsorption upon NP–protein interaction were characterized. Results: Protein adsorption capacity and adsorbed protein types were NPs concentration-dependent. Conclusion: Considering the critical impacts of protein adsorption on NPs delivery, our work could be an urgent warning about the possible risks of dosage adjustment of nanoformulations. First draft submitted: 24 July 2017; Accepted for publication: 5 September 2017; Published online: 11 October 2017 Keywords • adsorption • drug delivery • nanomaterials • nanomedicine • PHBHHx • protein corona • theranostics
Polymeric nanoparticles (NPs)-based drug delivery systems have shown great potentials in pharmaceutical and biomedical fields due to their biocompatibility, biodegradability and controlled release property [1–5]. However, the delivery of NPs is highly limited by their interaction with biomacromolecules at the nano–bio interfaces [6–8]. It has been well recognized that NPs will be rapidly covered by serum proteins upon into blood stream forming the so-called protein corona [9–13]. The protein adsorption, on the one hand, can lead to increases in particle size and changes in NPs surface properties [14–17]. More importantly, it significantly influences the biological impacts of NPs, leading to their rapid clearance from blood stream and loss of targeting capacity [18–22]. On the other hand, protein adsorption can also change the conformation and even biological activity of associated proteins [23–25]. The NP–protein interaction as well as the subsequent biological events is a kind of immune response of host to foreign particles, which can be considered as the direct reason for toxicity and uncontrollable in vivo fate of NPs [26,27]. Due to the importance of NP–protein interaction as described above, a lot of studies have reported the effects of physicochemical nature of NPs on such interaction, like particle size, shape, ζ potential and surface hydrophobicity [28–30]. In addition, the effects of temperature, local temperature and protein concentration have also been well documented [31–34]. However, the impacts of NP exposure concentration, which is closely related to the administration dose, are always ignored. This situation is not surprising since dosage adjustment of an ordinary formulation is just a common behavior in clinic and does not cause unpredictable results. However, there is no evidence that dosage change of nanoformulations would lead to the same results as ordinary formulations do. In consideration of the rapid development of nanoformulations for modern medication, where dosage adjustment is unavoidable, a comprehensive understanding of the correlation between NPs concentration and protein adsorption is required. In this present work, we aim to disclose the effects of NPs exposure concentration on protein adsorption via systematically investigating the interaction between serum protein and four NPs systems with different ζ potentials. Moreover, we aim to emphasize the importance of NPs concentration in the interaction at nano–bio interfaces and call attentions to the potential risks of dosage adjustment of nanoformulations.
C 2017 Future Medicine Ltd 10.2217/nnm-2017-0238
Nanomedicine (Lond.) (Epub ahead of print)
ISSN 1743-5889
Research Article
Zhang, Zhu, Lu, Zhang & Peng
Table 1. Detailed parameters for fabricating nanoparticles with different potential. NPs
PHBHHx (mg)
OA (mg)
F68 (%, w/v)
DOC-Na (%, w/v)
Sonication time (s)
Sonication power (%)
NP1
20
0
0.1
0.1
20
20
NP2
20
0
0.5
0.5
15
20
NP3
20
0.15
0.5
0
30
60
NP4
20
0.30
0.5
0
30
60
DOC-Na: Sodium deoxycholate; F68: Poloxamer188; NP: Nanoparticle; OA: Octadecylamine.
Materials & methods Materials Poloxamer188 (F68) was kindly provided by BASF (China) Co Ltd (Shanghai, China). Poly(3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx, Mw: 270 kD) containing 15 mol% 3-hydroxyhexanoate (3HHx) was kindly donated by Lukang Group (Jining, China). Octadecylamine was purchased from Sigma (MO, USA). Sodium deoxycholate (DOC-Na) was supplied by Amresco (OH, USA). Fetal bovine serum (FBS) was purchased from HyClone (UT, USA). BCA kit was obtained from Beyotime Institute of Biotechnology (Nantong, China). All other chemical reagents used in this study were of analytical grade or better. Preparation of PHBHHx NPs
Poly-(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), a biocompatible and biodegradable biopolymer, was used as the main carrier material to prepare NPs [31,32]. The PHBHHx NPs with different ζ potentials were prepared based on a previous report with some modifications [33]. The detailed preparation parameters are listed in Table 1. For negatively charged NPs, 20 mg PHBHHx was dissolved in chloroform, to which an aqueous solution containing different amount of F68 and DOC-Na was added at a volume ratio of 1:20 (aqueous: organic). The mixture was immediately sonicated, followed with rotation evaporation at room temperature for 20 min. For positive NPs, PHBHHx and octadecylamine were co-dissolved in chloroform, to which 0.5% F68 solution was added at the volume ratio of 1:20. The subsequent procedures were the same as above. Incubation of NPs with FBS
The above four NPs suspensions, with concentrations of 125, 250, 500 and 1000 μg/ml were added to 5% FBS (w/v), followed with vortex and incubation in a shaker (37◦ C, 100 rpm). The solvent used to dilute NPs and FBS is PBS (pH 7.4). At predetermined time intervals (10, 20, 30, 60 min), the formed NP-FBS complex was taken out for further tests and measurements. Measurement of size & ζ potential
The mean hydrodynamic size and ζ potential of NPs and NP–FBS complex were examined by dynamic light scattering and electrophoretic light scattering, respectively (Zetasizer Nano ZS90, Malvern Instruments, Ltd, UK) [14]. The measuring temperature was 25◦ C and the size was presented by intensity distribution. The size distribution was presented by polydispersity index (PDI). Scanning electronic microscopy
The morphology of NPs and NP–FBS complex was observed via scanning electronic microscopy (SEM). The specimen preparation for NPs was different from NP-FBS. Briefly, the NPs suspension was diluted with distilled water by 100-folds and one drop of the diluted suspension was placed on a clean glass sheet. After air-drying, the specimen was coated with gold before SEM (INSPECT F, FEI, The Netherlands). For NP–FBS, the NP–FBS suspension after incubation for 10 min was centrifuged (10 krcf, 5 min) and the supernatant containing free proteins was discarded. The pellets were washed with 0.1 ml distilled water for three times and then mixed with 0.1 ml 5% (v/v) glutaraldehyde. A few drops of the mixture were placed on a clean glass sheet and stored at 4◦ C overnight. Subsequently, the glutaraldehyde solution was removed and the specimen was washed with PBS (pH 7.4) for three times, followed with gradient dehydration using ethanol solutions. The dried specimen was coated with gold for SEM.
10.2217/nnm-2017-0238
Nanomedicine (Lond.) (Epub ahead of print)
future science group
Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
Table 2. Mean size, polydispersity index and potential of original nanoparticles. NPs
Size (nm)
PDI
ZP (mv)
NP1
213.9 ± 2.1
0.117 ± 0.014
-28.1 ± 2.3
NP2
221.2 ± 3.8
0.182 ± 0.004
-12.3 ± 0.2
NP3
245.9 ± 0.7
0.149 ± 0.015
23.5 ± 3.1
NP4
225.2 ± 9.0
0.124 ± 0.023
39.6 ± 5.3
Data are presented as mean ± sd (n = 3). NP: Nanoparticle; PDI: Polydispersity; SD: Standard deviation; ZP: potential.
Quantification of adsorbed FBS proteins
The proteins adsorbed on NPs surface were quantified using an indirect method according to a previous report [10]. Briefly, at fixed incubation time, NP-FBS was separated by centrifugation (10 krcf, 5 min). The supernatant was diluted by fivefold with distilled water and stored at -20◦ C till further content determination of nonadsorbed by BCA assay. The total protein content in FBS was also determined as above. Thus, the adsorbed protein amount was calculated by the difference between total and nonadsorbed protein amount. The protein adsorption capacity of NPs was calculated as dividing the adsorbed amount by the weight of NPs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The proteins adsorbed on NPs were characterized by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [10]. Briefly, at fixed incubation time, the formed NP–FBS was collected by centrifugation (10 krcf, 5 min) and washed with distilled water for three times. The resultant NP–FBS pellets were resuspended in loading buffer and boiled for 3 min, followed with centrifugation at 10 krcf for 3 min. An aliquot of 10 μl of supernatant was used for 12% SDS-PAGE. The separated protein bands were stained by Coomassie Brilliant Blue. Statistical analysis
All the data in this work were obtained by performing each experiment in triplicate and presented as mean ± SD (standard deviation). The one-way analysis of variance was used to analyze the statistical difference between groups. The difference was considered to be statistically significant when the p-value was lower than 0.05. Results & discussion Characterization of original NPs with different ζ potentials The hydrodynamic size, PDI and ζ potential of original NPs were shown in Table 2. As expected, the four NPs systems had similar particle size in a narrow range from ∼210 to ∼240 nm (NP1 was smallest and NP3 was largest) but varied ζ potentials ranging from ∼-30 to 40 mv. Moreover, via method modification, the ζ potential difference between any two NPs systems in this work was more than 10 mv, which is better than the previous report [33]. In addition, all NPs had a small PDI, indicating a narrow size distribution. These results support us to investigate the impacts of NPs concentration on protein adsorption in different NPs systems and thus make the conclusion of this work more universal. As we know, F68, DOC-Na and OA are neutral, anionic and cationic surfactant, respectively, and have been used to stabilize NPs system and modulate ζ potentials [38–40]. Therefore, the difference in ζ potential among the four NP systems has resulted from the different contents of these surfactants. Interestingly, there is a regular correlation between ζ potential and size. As shown in Table 2, the increase in the absolute value of ζ potential for both negative NPs (NP1 and NP2) and positive NPs (NP3 and NP4) resulted in a decrease in NPs size. It is well known that ζ potential plays important roles in stabilizing NPs-based colloidal systems via electrostatic repulsive force. Also, ζ potential is a regulator for particle size of NPs made of the same material. Increasing the absolute value of ζ potential (from NP2 to NP1 or from NP3 to NP4) leads to an increase in electrostatic repulsive force among particles and finally results in the decrease of particle size [34]. The morphology of original NPs examined by SEM is shown in Figure 1. All NPs are spherical in shape and small in size with a clean surface. In addition, the particles of each NP are well dispersed and almost no aggregation can be found. Size & ζ potential change of NPs upon interaction with FBS
The nonspecific interaction of NPs with serum proteins would change the physicochemical properties of NPs and result in a different in vivo fate [19]. We have demonstrated in our previous works that size increase and
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10.2217/nnm-2017-0238
Research Article
Zhang, Zhu, Lu, Zhang & Peng
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Figure 1. Scanning electronic microscopy images of four nanoparticles which have similar size but varied ζ potential. (A) NP1, (B) NP2, (C) NP3 and (D) NP4. Scale bar: 3 μm. NP: Nanoparticle; SEM: Scanning electronic microscopy.
ζ potential change are two typical physicochemical changes of NPs upon protein adsorption [10,14]. Herein, we further investigated the effect of NPs concentration on such changes in different NPs systems. As shown in Figure 2, the size increase after formation of NP–FBS complex is also found in this work for each NPs system. Furthermore, such increase started from the first sampling time (10 min postincubation, Figure 2A), indicating NP–protein interaction occurred very fast. Although NP–protein interaction would certainly lead to size increase, such change is not NPs concentration-dependent. In other words, the increase in NPs concentration does not always lead to the increase in size. It was assumed that NP–protein interaction is a dynamic process of protein adsorption/desorption, which would result in a fluctuation of NP-FBS size. Theoretically, size increase of NPs upon incubation with proteins may be attributed by both protein adsorption and protein-induced particle
10.2217/nnm-2017-0238
Nanomedicine (Lond.) (Epub ahead of print)
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 2. Hydrodynamic size of four original nanoparticles and their counterpart nanoparticle–fetal bovine serum complex formed upon incubating nanoparticles with fetal bovine serum for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. The NPs exposure concentration was 125, 250, 500 or 1000 μg/ml, and the FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
aggregation. Generally, particle aggregation would lead to fold increase in size. In this work, however, the largest size increase is lower than 40 nm. Hence, the size increase may be due largely to protein adsorption rather than particle aggregation. Compared with size change, ζ potential change was more significant and complicated (Figure 3). For the higher negatively charged NP1, the negative ζ potential decreased from -30 mv to ∼-20 mv after incubation with FBS. For the lower negatively charged NP2, however, the negative ζ potential increased from -12 mv to ∼-20 mv upon incubation with FBS. More interestingly, an obvious conversion of ζ potential from positive to negative value was found at NP3 and NP4 upon interaction with FBS. Furthermore, the ζ potential of NP3-FBS and NP4-FBS was almost the same as that of NP1-FBS and NP2-FBS when NPs concentration was ≤500 μg/ml (i.e., -20 mv). The similar results were also reported by other groups with different nanosystems [42–44]. These data imply that the serum proteins at concentration of 5% FBS can cover almost all the surface of NPs when NPs concentration is ≤500 μg/ml, and thus the ζ potential of these NPs-FBS is only derived from the proteins located on particle surface. Furthermore, these proteins should be negatively charged in total. This can explain why all NPs-FBS showed the same negative ζ potential despite the significantly different ζ potential of original NPs. This situation was changed for NP2, NP3 and NP4 when NPs concentration increased to 1000 μg/ml. At the highest exposure concentration, the ζ potential was −16 mv for NP2-FBS, -12 mv for NP3-FBS and -11 mv for NP4-FBS. These data indicate that the serum proteins at concentration of 5% FBS cannot cover all the surface of NPs when NPs concentration was 1000 μg/ml. Accordingly, the ζ potential of these NPs-FBS is the combination of associated proteins and uncovered part of original NPs. As such, the surface charge of original NPs (especially positively
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Zhang, Zhu, Lu, Zhang & Peng
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Figure 3. The ζ potential of four original nanoparticles and their counterpart nanoparticle–fetal bovine serum complex formed upon incubating nanoparticles with fetal bovine serum for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. The NPs exposure concentration was 125, 250, 500 or 1000 μg/ml, and the FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
charged NP3 and NP4) can neutralize the contribution of adsorbed proteins to the ζ potential of NPs-FBS. This can explain why the ζ potential increased to -16 mv for NP2-FBS and remarkably increased to -12 and -11 mv for NP3-FBS and NP4-FBS. In addition, the different composition of the protein corona at varied NP concentration and ζ potential, which will be shown in the SDS-PAGE section later, may also contribute to the above results. We can also see from Figure 3 that incubation time has little influence on ζ potential change. As shown above, NPs exposure concentration has uncertain effects on size increase (Figure 2) but has significant influence on ζ potential change (especially at the highest concentration, Figure 3) when interacting with FBS. In return, the size increase and ζ potential change can serve as a kind of evidence for the rapid and nonspecific interaction between NPs and serum proteins. Morphology examination of NP-FBS
In addition to size and ζ potential, association with serum proteins would also affect the morphology of NPs. We used SEM to examine the morphology of NP–FBS complex and further confirm the interaction between NPs and serum proteins. Unlike the relatively clean surface of the original NPs (Figure 1), the particles were found to be embedded in protein molecules after incubation with FBS (Figure 4). Meanwhile, compared with the well-dispersed original NPs, all the NPs-FBS seemed aggregated apparently. But this ‘apparent aggregation’ is not the real aggregation which cannot be studied by SEM, because these NPs-FBS were also well-dispersed in the state of suspension (PDI < 0.3). It was assumed that this ‘apparent aggregation’ of NPs-FBS observed from Figure 4 may be generated during the process of specimen treatment for SEM, especially the drying process, which led to
10.2217/nnm-2017-0238
Nanomedicine (Lond.) (Epub ahead of print)
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 4. Scanning electronic microscopy images of nanoparticle–fetal bovine serum complex. (A) NP1-FBS, (B) NP2-FBS, (C) NP3-FBS and (D) NP4-FBS. NPs concentration was 500 μg/ml. Scale bar: 3 μm. FBS: Fetal bovine serum; NP: Nanoparticle; SEM: Scanning electronic microscopy.
a strong protein–protein interaction and fusion. In summary, these SEM images are a kind of visualized and solid evidence for NP–protein interaction. Protein adsorption kinetics
We have demonstrated above the changes of NPs in size, ζ potential and morphology caused by NP–protein interaction. Undoubtedly, these changes are the result of protein adsorption onto the surface of NPs. Here we further quantified the protein adsorption and investigated the effects of NPs concentration on protein adsorption kinetics. The protein adsorption kinetics was presented by absolute amount of adsorbed protein-time curve (Figure 5). The adsorbed protein was detected from the first sampling time (10 min postincubation), further indicating the
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Absorbed protein amount (μg)
Zhang, Zhu, Lu, Zhang & Peng
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Figure 5. Protein adsorption kinetics of (A) NP1 (ZP -28.1 mv), (B) NP2 (ZP -12.3 mv), (C) NP3 (ZP 23.5 mv) and (D) NP4 (ZP 39.6 mv) upon incubating with fetal bovine serum (37◦ C, 100 rpm) at varied exposure concentrations of nanoparticles. The FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
rapid interaction between NPs and serum proteins. Almost all the protein adsorption kinetic profiles are fluctuant with time, suggesting that the NP–protein interaction is a dynamic process of protein adsorption and desorption and adsorption does not always dominate the process. Interestingly, more protein adsorption does not certainly mean more increase in hydrodynamic size. For example, the protein adsorbed amount of NP1 (125 μg/ml) at 10 min is much more than that of NP1 at 1000 μg/ml (Figure 5A), but the hydrodynamic size of NP1-FBS (125 μg/ml) is smaller (Figure 2A). This is because the associated proteins may be hardly bonded (hard corona, less contribution to size increase) or softly bonded (soft corona, more contribution to size increase) [35,36]. We can also see from Figure 5 that an increase in NPs concentration does not certainly mean an increase in the quantity of adsorbed proteins. For instance, at 10 min postincubation of NP1 with FBS, the adsorbed protein amount became less when NP1 concentration increased from 125 to 250 μg/ml but became more when the concentration increased from 250 to 500 μg/ml (Figure 5A). In contrast to NPs concentration, ζ potential seems to play a more important role in determining protein adsorption amount. Between negatively charged NPs of the same exposure concentration (NP1 and NP2, Figure 5AB), more proteins were adsorbed on the lower negatively charged NP2 (ZP -12.3 mv) than the higher negatively charged NP1 (ZP -28.1 mv). However, between positively charged NPs (NP3 and NP4, Figure 5C & D), more proteins were adsorbed on the higher positively charged NP4 (ZP 39.6 mv) than the lower positively charged NPs (ZP 23.5 mv). Only one condition can explain these results, that is the compositive charge of those adsorbed serum proteins being negative. As such, these proteins would have a lower electrostatic repulsive force for NP2 than NP1 and have a stronger electrostatic attraction force for NP4
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 6. Protein adsorption capacity of NP1 (ZP -28.1 mv), NP2 (ZP -12.3 mv), NP3 (ZP 23.5 mv) and NP4 (ZP 39.6 mv) after incubating with fetal bovine serum (37◦ C, 100 rpm) for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. Data are presented as mean ± SD (n = 3). Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001. FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
than NP3. In addition, the negative charge of adsorbed proteins can also explain why the ζ potential of all NPs-FBS is negative (Figure 3). Protein adsorption capacity
Protein adsorption capacity, defined as the weight of protein (mg) adsorbed on each milligram of NPs, is totally different from the concept of protein adsorption kinetics. Briefly, adsorption kinetics shows the total amount of proteins adsorbed on NPs at varied concentration, while adsorption capacity represents the protein adsorption ability of each milligram of NPs at varied concentrations. As shown in Figure 6, the protein adsorption capacity of NPs shows the same trend as protein adsorption kinetics (Figure 5) when comparing the NPs of the same kind of charge. Briefly, between negatively charged NP1 and NP2, the lower negatively charged NP2 shows the higher protein adsorption capacity, and between positively charged NP3 and NP4, the higher positively charged NP4 has the higher capacity. Additionally and more importantly, Figure 6 delivers a kind of key information that is the protein adsorption capacity increases significantly with decreasing NPs concentration, and this is a universal rule for any NPs at any incubation time. This result is not difficult to understand. When FBS concentration is constant, the decrease in NPs concentration (i.e., reduction in particle number) will surely lead to more protein molecules available for each particle and thus results in a higher protein adsorption potential (Figure 7). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The type of proteins adsorbed on NPs surface is of wide interests and can be affected by various physicochemical properties of NPs. Here, we used 12% SDS-PAGE to separate the adsorbed serum proteins. The impacts of NPs concentration on the type of adsorbed proteins were examined by incubating NPs (1000 and 250 μg/ml) with
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Low NPs concentration
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High NPs concentration
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Figure 7. Schematic of nanoparticles exposure concentration effect on protein adsorption. (A) More protein molecules adsorbed on each particle at low NPs concentration; (B) less protein molecules adsorbed on each particle at high NPs concentration. NP: Nanoparticle.
Figure 8. Twelve percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the proteins adsorbed on nanoparticles upon incubating with fetal bovine serum (37◦ C, 100 rpm) for 20 min. Lanes: 1 and 2, NPs-FBS with NPs concentration of 1000 (Lane 1) and 250 μg/ml (Lane 2), respectively. Arrows a–f indicate different protein bands. FBS: Fetal bovine serum; NP: Nanoparticle.
FBS. In order to intuitively present the effect of NPs concentration on protein adsorption capacity at the same time, fourfold volume of 250 μg/ml NP–protein were taken for SDS-PAGE (equivalent to 1000 μg/ml NPs). As shown in Figure 8, protein bands a–d are the main adsorbed proteins and can be found at each specimen. Interestingly, there is a regular difference in bands darkness between low and high concentration of the same NPs. The protein bands of lower concentration of NPs are always darker than that of higher concentration of NPs. This result indicates that NPs with a lower concentration have a higher protein adsorption capacity, which is consistent with the quantification result shown in Figure 6. However, bands e and f can only be found in the NPs-FBS formed by lower concentration of NPs. This result suggests that the exposure concentration of NPs can affect the type of associated proteins. We also found the effects of NPs ζ potential on adsorbed proteins type (Supplementary Figure 1). Protein bands of each NPs-FBS can be observed but no band is found for original NPs, further confirming NP–protein interaction. We can also see from Supplementary Figure 1 that a, b, c and d are the main protein bands of NPs-FBS with a little difference among them. NP2-FBS possesses the most abundant protein band d, suggesting that band d may mainly contain the weakly charged proteins that have a relatively high affinity to the lowest charged NP2. NP4-FBS possesses the most abundant protein band b, suggesting that band b may mainly consist of highly negatively charged proteins that have a relatively strong affinity to the highest positively charged NP4. These results indicate that ζ potential is a key factor influencing NP–protein interaction because proteins are also charged in solution and electrostatic force is one of the most important forces involved in the interaction [29].
10.2217/nnm-2017-0238
Nanomedicine (Lond.) (Epub ahead of print)
future science group
Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
We have demonstrated above the effects of NPs concentration on protein adsorption capacity and adsorbed protein type. To the best of our knowledge, this concentration-dependent protein adsorption of polymeric NPs was never reported before. But indeed, it is of great significance for clinical use of NPs-based nanoformulations. As we know, dosage adjustment of ordinary formulations is a common clinical strategy to optimize therapy and it does not cause unpredictable effects as long as the postadjustment dose falls into the therapeutic window. However, this normal situation would be different for nanoformulations since changes in NPs concentration would lead to substantial alteration in both protein adsorption capacity and adsorbed protein type. In consideration of the significant impacts of protein adsorption on delivery fate of NPs, like pharmacokinetics, biodistribution and pharmacodynamics [19,36], dosage adjustment of nanoformulations may cause unpredictable or uncontrollable effects due to the altered protein adsorption pattern. Therefore, this work is a serious warning about the possible risks of dosage adjustment of nanoformulations in practical use. On the basis of the findings, personalized therapy and thorough investigations on the pharmaceutical effects of each applied dose may be required in development and clinical use of nanoformulations. In addition to administration dose, the type of disease-caused personalized protein corona is another factor requiring personalized therapy. It has also been reported that corona decoration strongly depends on the type of disease, leading to the formation of personalized protein corona [47–49]. Conclusion In recent years, the negative effects of NP–protein interaction on NP-based drug delivery have drawn increasing attentions. A comprehensive understanding of such interaction is required for developing quality controllable, high-efficiency and low-toxicity nanoformulations. In this work, we showed the changes of polymeric NPs in size, ζ potential and morphology upon interaction with serum proteins. Importantly, we reported the important roles of NPs exposure concentration in NP–protein interaction. In consideration of the important impact of protein adsorption on the in vivo fate of NPs, our findings are quite meaningful for the design, study and practical application of NPs-based drug delivery systems. In addition to the basic physicochemical properties, the efficacy and toxicity of each applied dose of nanoformulations have to be thoroughly investigated during preclinical/clinical studies and closely monitored in clinical use. In a word, personalized therapy would be required in using nanoformulations. Financial & competing interests disclosure This work was supported by National Natural Science Foundation of China (No. 81402860) and the Excellent Young Scientist Foundation of Sichuan University to Q Peng (no. 2016SCU04A02). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Summary points r Four PHBHHx nanoparticle (NP) systems with similar sizes (230 ± 20 nm) but varied ζ potentials (ZPs) (-30 ∼ +40 mv) were successfully prepared. r Size, ZP and morphology of NPs changed upon protein adsorption.
r NP2 (ZP -12.3 mv) adsorbed more absolute protein amount than NP1 (ZP -28.1 mv) and NP4 (ZP 39.6 mv) adsorbed more than NP3 (ZP 23.5 mv). r Protein adsorption capacity increased when reducing NPs exposure concentration. r Type of adsorbed protein changed when NPs concentration was different.
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Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles and serum proteins Tian-Xu Zhang‡,1 , Guan-Yin Zhu‡,1 , Bo-Yao Lu1 , Chao-Liang Zhang1 & Qiang Peng*,1 1 State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China * Author for correspondence: Tel.: +86 28 85 501 484; Fax: +86 28 85 501 484; [email protected] ‡ Authors contributed equally
Aim: A comprehensive understanding of nanoparticle (NP)-protein interaction (protein corona formation) is required. So far, many factors influencing this interaction have been investigated, like size and ζ potential. However, NPs exposure concentration has always been ignored. Herein, we aim to disclose the correlation of NPs exposure concentration with protein adsorption. Materials & methods: Four polymeric NPs systems possessing similar sizes (230 ± 20 nm) but varied ζ potentials (−30 ∼ +40 mv) were prepared. Physicochemical properties and protein adsorption upon NP–protein interaction were characterized. Results: Protein adsorption capacity and adsorbed protein types were NPs concentration-dependent. Conclusion: Considering the critical impacts of protein adsorption on NPs delivery, our work could be an urgent warning about the possible risks of dosage adjustment of nanoformulations. First draft submitted: 24 July 2017; Accepted for publication: 5 September 2017; Published online: 11 October 2017 Keywords • adsorption • drug delivery • nanomaterials • nanomedicine • PHBHHx • protein corona • theranostics
Polymeric nanoparticles (NPs)-based drug delivery systems have shown great potentials in pharmaceutical and biomedical fields due to their biocompatibility, biodegradability and controlled release property [1–5]. However, the delivery of NPs is highly limited by their interaction with biomacromolecules at the nano–bio interfaces [6–8]. It has been well recognized that NPs will be rapidly covered by serum proteins upon into blood stream forming the so-called protein corona [9–13]. The protein adsorption, on the one hand, can lead to increases in particle size and changes in NPs surface properties [14–17]. More importantly, it significantly influences the biological impacts of NPs, leading to their rapid clearance from blood stream and loss of targeting capacity [18–22]. On the other hand, protein adsorption can also change the conformation and even biological activity of associated proteins [23–25]. The NP–protein interaction as well as the subsequent biological events is a kind of immune response of host to foreign particles, which can be considered as the direct reason for toxicity and uncontrollable in vivo fate of NPs [26,27]. Due to the importance of NP–protein interaction as described above, a lot of studies have reported the effects of physicochemical nature of NPs on such interaction, like particle size, shape, ζ potential and surface hydrophobicity [28–30]. In addition, the effects of temperature, local temperature and protein concentration have also been well documented [31–34]. However, the impacts of NP exposure concentration, which is closely related to the administration dose, are always ignored. This situation is not surprising since dosage adjustment of an ordinary formulation is just a common behavior in clinic and does not cause unpredictable results. However, there is no evidence that dosage change of nanoformulations would lead to the same results as ordinary formulations do. In consideration of the rapid development of nanoformulations for modern medication, where dosage adjustment is unavoidable, a comprehensive understanding of the correlation between NPs concentration and protein adsorption is required. In this present work, we aim to disclose the effects of NPs exposure concentration on protein adsorption via systematically investigating the interaction between serum protein and four NPs systems with different ζ potentials. Moreover, we aim to emphasize the importance of NPs concentration in the interaction at nano–bio interfaces and call attentions to the potential risks of dosage adjustment of nanoformulations.
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Zhang, Zhu, Lu, Zhang & Peng
Table 1. Detailed parameters for fabricating nanoparticles with different potential. NPs
PHBHHx (mg)
OA (mg)
F68 (%, w/v)
DOC-Na (%, w/v)
Sonication time (s)
Sonication power (%)
NP1
20
0
0.1
0.1
20
20
NP2
20
0
0.5
0.5
15
20
NP3
20
0.15
0.5
0
30
60
NP4
20
0.30
0.5
0
30
60
DOC-Na: Sodium deoxycholate; F68: Poloxamer188; NP: Nanoparticle; OA: Octadecylamine.
Materials & methods Materials Poloxamer188 (F68) was kindly provided by BASF (China) Co Ltd (Shanghai, China). Poly(3-hydroxybutyrateco-3-hydroxyhexanoate) (PHBHHx, Mw: 270 kD) containing 15 mol% 3-hydroxyhexanoate (3HHx) was kindly donated by Lukang Group (Jining, China). Octadecylamine was purchased from Sigma (MO, USA). Sodium deoxycholate (DOC-Na) was supplied by Amresco (OH, USA). Fetal bovine serum (FBS) was purchased from HyClone (UT, USA). BCA kit was obtained from Beyotime Institute of Biotechnology (Nantong, China). All other chemical reagents used in this study were of analytical grade or better. Preparation of PHBHHx NPs
Poly-(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx), a biocompatible and biodegradable biopolymer, was used as the main carrier material to prepare NPs [31,32]. The PHBHHx NPs with different ζ potentials were prepared based on a previous report with some modifications [33]. The detailed preparation parameters are listed in Table 1. For negatively charged NPs, 20 mg PHBHHx was dissolved in chloroform, to which an aqueous solution containing different amount of F68 and DOC-Na was added at a volume ratio of 1:20 (aqueous: organic). The mixture was immediately sonicated, followed with rotation evaporation at room temperature for 20 min. For positive NPs, PHBHHx and octadecylamine were co-dissolved in chloroform, to which 0.5% F68 solution was added at the volume ratio of 1:20. The subsequent procedures were the same as above. Incubation of NPs with FBS
The above four NPs suspensions, with concentrations of 125, 250, 500 and 1000 μg/ml were added to 5% FBS (w/v), followed with vortex and incubation in a shaker (37◦ C, 100 rpm). The solvent used to dilute NPs and FBS is PBS (pH 7.4). At predetermined time intervals (10, 20, 30, 60 min), the formed NP-FBS complex was taken out for further tests and measurements. Measurement of size & ζ potential
The mean hydrodynamic size and ζ potential of NPs and NP–FBS complex were examined by dynamic light scattering and electrophoretic light scattering, respectively (Zetasizer Nano ZS90, Malvern Instruments, Ltd, UK) [14]. The measuring temperature was 25◦ C and the size was presented by intensity distribution. The size distribution was presented by polydispersity index (PDI). Scanning electronic microscopy
The morphology of NPs and NP–FBS complex was observed via scanning electronic microscopy (SEM). The specimen preparation for NPs was different from NP-FBS. Briefly, the NPs suspension was diluted with distilled water by 100-folds and one drop of the diluted suspension was placed on a clean glass sheet. After air-drying, the specimen was coated with gold before SEM (INSPECT F, FEI, The Netherlands). For NP–FBS, the NP–FBS suspension after incubation for 10 min was centrifuged (10 krcf, 5 min) and the supernatant containing free proteins was discarded. The pellets were washed with 0.1 ml distilled water for three times and then mixed with 0.1 ml 5% (v/v) glutaraldehyde. A few drops of the mixture were placed on a clean glass sheet and stored at 4◦ C overnight. Subsequently, the glutaraldehyde solution was removed and the specimen was washed with PBS (pH 7.4) for three times, followed with gradient dehydration using ethanol solutions. The dried specimen was coated with gold for SEM.
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Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
Table 2. Mean size, polydispersity index and potential of original nanoparticles. NPs
Size (nm)
PDI
ZP (mv)
NP1
213.9 ± 2.1
0.117 ± 0.014
-28.1 ± 2.3
NP2
221.2 ± 3.8
0.182 ± 0.004
-12.3 ± 0.2
NP3
245.9 ± 0.7
0.149 ± 0.015
23.5 ± 3.1
NP4
225.2 ± 9.0
0.124 ± 0.023
39.6 ± 5.3
Data are presented as mean ± sd (n = 3). NP: Nanoparticle; PDI: Polydispersity; SD: Standard deviation; ZP: potential.
Quantification of adsorbed FBS proteins
The proteins adsorbed on NPs surface were quantified using an indirect method according to a previous report [10]. Briefly, at fixed incubation time, NP-FBS was separated by centrifugation (10 krcf, 5 min). The supernatant was diluted by fivefold with distilled water and stored at -20◦ C till further content determination of nonadsorbed by BCA assay. The total protein content in FBS was also determined as above. Thus, the adsorbed protein amount was calculated by the difference between total and nonadsorbed protein amount. The protein adsorption capacity of NPs was calculated as dividing the adsorbed amount by the weight of NPs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The proteins adsorbed on NPs were characterized by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [10]. Briefly, at fixed incubation time, the formed NP–FBS was collected by centrifugation (10 krcf, 5 min) and washed with distilled water for three times. The resultant NP–FBS pellets were resuspended in loading buffer and boiled for 3 min, followed with centrifugation at 10 krcf for 3 min. An aliquot of 10 μl of supernatant was used for 12% SDS-PAGE. The separated protein bands were stained by Coomassie Brilliant Blue. Statistical analysis
All the data in this work were obtained by performing each experiment in triplicate and presented as mean ± SD (standard deviation). The one-way analysis of variance was used to analyze the statistical difference between groups. The difference was considered to be statistically significant when the p-value was lower than 0.05. Results & discussion Characterization of original NPs with different ζ potentials The hydrodynamic size, PDI and ζ potential of original NPs were shown in Table 2. As expected, the four NPs systems had similar particle size in a narrow range from ∼210 to ∼240 nm (NP1 was smallest and NP3 was largest) but varied ζ potentials ranging from ∼-30 to 40 mv. Moreover, via method modification, the ζ potential difference between any two NPs systems in this work was more than 10 mv, which is better than the previous report [33]. In addition, all NPs had a small PDI, indicating a narrow size distribution. These results support us to investigate the impacts of NPs concentration on protein adsorption in different NPs systems and thus make the conclusion of this work more universal. As we know, F68, DOC-Na and OA are neutral, anionic and cationic surfactant, respectively, and have been used to stabilize NPs system and modulate ζ potentials [38–40]. Therefore, the difference in ζ potential among the four NP systems has resulted from the different contents of these surfactants. Interestingly, there is a regular correlation between ζ potential and size. As shown in Table 2, the increase in the absolute value of ζ potential for both negative NPs (NP1 and NP2) and positive NPs (NP3 and NP4) resulted in a decrease in NPs size. It is well known that ζ potential plays important roles in stabilizing NPs-based colloidal systems via electrostatic repulsive force. Also, ζ potential is a regulator for particle size of NPs made of the same material. Increasing the absolute value of ζ potential (from NP2 to NP1 or from NP3 to NP4) leads to an increase in electrostatic repulsive force among particles and finally results in the decrease of particle size [34]. The morphology of original NPs examined by SEM is shown in Figure 1. All NPs are spherical in shape and small in size with a clean surface. In addition, the particles of each NP are well dispersed and almost no aggregation can be found. Size & ζ potential change of NPs upon interaction with FBS
The nonspecific interaction of NPs with serum proteins would change the physicochemical properties of NPs and result in a different in vivo fate [19]. We have demonstrated in our previous works that size increase and
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HV Mag 11/18/2016 WD Mode 10:49:25 AM 20.00 kv 40 000 x 10.4 mm SE
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HV Mag 11/24/2016 WD Mode 9:42:39 AM 20.00 kv 40 000 x 10.0 mm SE
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HV Mag 11/25/2016 WD Mode 10:20:58 AM 20.00 kv 40 000 x 11.6 mm SE
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Figure 1. Scanning electronic microscopy images of four nanoparticles which have similar size but varied ζ potential. (A) NP1, (B) NP2, (C) NP3 and (D) NP4. Scale bar: 3 μm. NP: Nanoparticle; SEM: Scanning electronic microscopy.
ζ potential change are two typical physicochemical changes of NPs upon protein adsorption [10,14]. Herein, we further investigated the effect of NPs concentration on such changes in different NPs systems. As shown in Figure 2, the size increase after formation of NP–FBS complex is also found in this work for each NPs system. Furthermore, such increase started from the first sampling time (10 min postincubation, Figure 2A), indicating NP–protein interaction occurred very fast. Although NP–protein interaction would certainly lead to size increase, such change is not NPs concentration-dependent. In other words, the increase in NPs concentration does not always lead to the increase in size. It was assumed that NP–protein interaction is a dynamic process of protein adsorption/desorption, which would result in a fluctuation of NP-FBS size. Theoretically, size increase of NPs upon incubation with proteins may be attributed by both protein adsorption and protein-induced particle
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Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 2. Hydrodynamic size of four original nanoparticles and their counterpart nanoparticle–fetal bovine serum complex formed upon incubating nanoparticles with fetal bovine serum for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. The NPs exposure concentration was 125, 250, 500 or 1000 μg/ml, and the FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
aggregation. Generally, particle aggregation would lead to fold increase in size. In this work, however, the largest size increase is lower than 40 nm. Hence, the size increase may be due largely to protein adsorption rather than particle aggregation. Compared with size change, ζ potential change was more significant and complicated (Figure 3). For the higher negatively charged NP1, the negative ζ potential decreased from -30 mv to ∼-20 mv after incubation with FBS. For the lower negatively charged NP2, however, the negative ζ potential increased from -12 mv to ∼-20 mv upon incubation with FBS. More interestingly, an obvious conversion of ζ potential from positive to negative value was found at NP3 and NP4 upon interaction with FBS. Furthermore, the ζ potential of NP3-FBS and NP4-FBS was almost the same as that of NP1-FBS and NP2-FBS when NPs concentration was ≤500 μg/ml (i.e., -20 mv). The similar results were also reported by other groups with different nanosystems [42–44]. These data imply that the serum proteins at concentration of 5% FBS can cover almost all the surface of NPs when NPs concentration is ≤500 μg/ml, and thus the ζ potential of these NPs-FBS is only derived from the proteins located on particle surface. Furthermore, these proteins should be negatively charged in total. This can explain why all NPs-FBS showed the same negative ζ potential despite the significantly different ζ potential of original NPs. This situation was changed for NP2, NP3 and NP4 when NPs concentration increased to 1000 μg/ml. At the highest exposure concentration, the ζ potential was −16 mv for NP2-FBS, -12 mv for NP3-FBS and -11 mv for NP4-FBS. These data indicate that the serum proteins at concentration of 5% FBS cannot cover all the surface of NPs when NPs concentration was 1000 μg/ml. Accordingly, the ζ potential of these NPs-FBS is the combination of associated proteins and uncovered part of original NPs. As such, the surface charge of original NPs (especially positively
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Figure 3. The ζ potential of four original nanoparticles and their counterpart nanoparticle–fetal bovine serum complex formed upon incubating nanoparticles with fetal bovine serum for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. The NPs exposure concentration was 125, 250, 500 or 1000 μg/ml, and the FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
charged NP3 and NP4) can neutralize the contribution of adsorbed proteins to the ζ potential of NPs-FBS. This can explain why the ζ potential increased to -16 mv for NP2-FBS and remarkably increased to -12 and -11 mv for NP3-FBS and NP4-FBS. In addition, the different composition of the protein corona at varied NP concentration and ζ potential, which will be shown in the SDS-PAGE section later, may also contribute to the above results. We can also see from Figure 3 that incubation time has little influence on ζ potential change. As shown above, NPs exposure concentration has uncertain effects on size increase (Figure 2) but has significant influence on ζ potential change (especially at the highest concentration, Figure 3) when interacting with FBS. In return, the size increase and ζ potential change can serve as a kind of evidence for the rapid and nonspecific interaction between NPs and serum proteins. Morphology examination of NP-FBS
In addition to size and ζ potential, association with serum proteins would also affect the morphology of NPs. We used SEM to examine the morphology of NP–FBS complex and further confirm the interaction between NPs and serum proteins. Unlike the relatively clean surface of the original NPs (Figure 1), the particles were found to be embedded in protein molecules after incubation with FBS (Figure 4). Meanwhile, compared with the well-dispersed original NPs, all the NPs-FBS seemed aggregated apparently. But this ‘apparent aggregation’ is not the real aggregation which cannot be studied by SEM, because these NPs-FBS were also well-dispersed in the state of suspension (PDI < 0.3). It was assumed that this ‘apparent aggregation’ of NPs-FBS observed from Figure 4 may be generated during the process of specimen treatment for SEM, especially the drying process, which led to
10.2217/nnm-2017-0238
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 4. Scanning electronic microscopy images of nanoparticle–fetal bovine serum complex. (A) NP1-FBS, (B) NP2-FBS, (C) NP3-FBS and (D) NP4-FBS. NPs concentration was 500 μg/ml. Scale bar: 3 μm. FBS: Fetal bovine serum; NP: Nanoparticle; SEM: Scanning electronic microscopy.
a strong protein–protein interaction and fusion. In summary, these SEM images are a kind of visualized and solid evidence for NP–protein interaction. Protein adsorption kinetics
We have demonstrated above the changes of NPs in size, ζ potential and morphology caused by NP–protein interaction. Undoubtedly, these changes are the result of protein adsorption onto the surface of NPs. Here we further quantified the protein adsorption and investigated the effects of NPs concentration on protein adsorption kinetics. The protein adsorption kinetics was presented by absolute amount of adsorbed protein-time curve (Figure 5). The adsorbed protein was detected from the first sampling time (10 min postincubation), further indicating the
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Research Article
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Figure 5. Protein adsorption kinetics of (A) NP1 (ZP -28.1 mv), (B) NP2 (ZP -12.3 mv), (C) NP3 (ZP 23.5 mv) and (D) NP4 (ZP 39.6 mv) upon incubating with fetal bovine serum (37◦ C, 100 rpm) at varied exposure concentrations of nanoparticles. The FBS concentration was fixed at 5% (v/v). Data are presented as mean ± SD (n = 3). FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
rapid interaction between NPs and serum proteins. Almost all the protein adsorption kinetic profiles are fluctuant with time, suggesting that the NP–protein interaction is a dynamic process of protein adsorption and desorption and adsorption does not always dominate the process. Interestingly, more protein adsorption does not certainly mean more increase in hydrodynamic size. For example, the protein adsorbed amount of NP1 (125 μg/ml) at 10 min is much more than that of NP1 at 1000 μg/ml (Figure 5A), but the hydrodynamic size of NP1-FBS (125 μg/ml) is smaller (Figure 2A). This is because the associated proteins may be hardly bonded (hard corona, less contribution to size increase) or softly bonded (soft corona, more contribution to size increase) [35,36]. We can also see from Figure 5 that an increase in NPs concentration does not certainly mean an increase in the quantity of adsorbed proteins. For instance, at 10 min postincubation of NP1 with FBS, the adsorbed protein amount became less when NP1 concentration increased from 125 to 250 μg/ml but became more when the concentration increased from 250 to 500 μg/ml (Figure 5A). In contrast to NPs concentration, ζ potential seems to play a more important role in determining protein adsorption amount. Between negatively charged NPs of the same exposure concentration (NP1 and NP2, Figure 5AB), more proteins were adsorbed on the lower negatively charged NP2 (ZP -12.3 mv) than the higher negatively charged NP1 (ZP -28.1 mv). However, between positively charged NPs (NP3 and NP4, Figure 5C & D), more proteins were adsorbed on the higher positively charged NP4 (ZP 39.6 mv) than the lower positively charged NPs (ZP 23.5 mv). Only one condition can explain these results, that is the compositive charge of those adsorbed serum proteins being negative. As such, these proteins would have a lower electrostatic repulsive force for NP2 than NP1 and have a stronger electrostatic attraction force for NP4
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
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Figure 6. Protein adsorption capacity of NP1 (ZP -28.1 mv), NP2 (ZP -12.3 mv), NP3 (ZP 23.5 mv) and NP4 (ZP 39.6 mv) after incubating with fetal bovine serum (37◦ C, 100 rpm) for (A) 10 min, (B) 20 min, (C) 30 min and (D) 60 min. Data are presented as mean ± SD (n = 3). Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001. FBS: Fetal bovine serum; NP: Nanoparticle; SD: Standard deviation.
than NP3. In addition, the negative charge of adsorbed proteins can also explain why the ζ potential of all NPs-FBS is negative (Figure 3). Protein adsorption capacity
Protein adsorption capacity, defined as the weight of protein (mg) adsorbed on each milligram of NPs, is totally different from the concept of protein adsorption kinetics. Briefly, adsorption kinetics shows the total amount of proteins adsorbed on NPs at varied concentration, while adsorption capacity represents the protein adsorption ability of each milligram of NPs at varied concentrations. As shown in Figure 6, the protein adsorption capacity of NPs shows the same trend as protein adsorption kinetics (Figure 5) when comparing the NPs of the same kind of charge. Briefly, between negatively charged NP1 and NP2, the lower negatively charged NP2 shows the higher protein adsorption capacity, and between positively charged NP3 and NP4, the higher positively charged NP4 has the higher capacity. Additionally and more importantly, Figure 6 delivers a kind of key information that is the protein adsorption capacity increases significantly with decreasing NPs concentration, and this is a universal rule for any NPs at any incubation time. This result is not difficult to understand. When FBS concentration is constant, the decrease in NPs concentration (i.e., reduction in particle number) will surely lead to more protein molecules available for each particle and thus results in a higher protein adsorption potential (Figure 7). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The type of proteins adsorbed on NPs surface is of wide interests and can be affected by various physicochemical properties of NPs. Here, we used 12% SDS-PAGE to separate the adsorbed serum proteins. The impacts of NPs concentration on the type of adsorbed proteins were examined by incubating NPs (1000 and 250 μg/ml) with
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Zhang, Zhu, Lu, Zhang & Peng
Low NPs concentration
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Figure 7. Schematic of nanoparticles exposure concentration effect on protein adsorption. (A) More protein molecules adsorbed on each particle at low NPs concentration; (B) less protein molecules adsorbed on each particle at high NPs concentration. NP: Nanoparticle.
Figure 8. Twelve percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining of the proteins adsorbed on nanoparticles upon incubating with fetal bovine serum (37◦ C, 100 rpm) for 20 min. Lanes: 1 and 2, NPs-FBS with NPs concentration of 1000 (Lane 1) and 250 μg/ml (Lane 2), respectively. Arrows a–f indicate different protein bands. FBS: Fetal bovine serum; NP: Nanoparticle.
FBS. In order to intuitively present the effect of NPs concentration on protein adsorption capacity at the same time, fourfold volume of 250 μg/ml NP–protein were taken for SDS-PAGE (equivalent to 1000 μg/ml NPs). As shown in Figure 8, protein bands a–d are the main adsorbed proteins and can be found at each specimen. Interestingly, there is a regular difference in bands darkness between low and high concentration of the same NPs. The protein bands of lower concentration of NPs are always darker than that of higher concentration of NPs. This result indicates that NPs with a lower concentration have a higher protein adsorption capacity, which is consistent with the quantification result shown in Figure 6. However, bands e and f can only be found in the NPs-FBS formed by lower concentration of NPs. This result suggests that the exposure concentration of NPs can affect the type of associated proteins. We also found the effects of NPs ζ potential on adsorbed proteins type (Supplementary Figure 1). Protein bands of each NPs-FBS can be observed but no band is found for original NPs, further confirming NP–protein interaction. We can also see from Supplementary Figure 1 that a, b, c and d are the main protein bands of NPs-FBS with a little difference among them. NP2-FBS possesses the most abundant protein band d, suggesting that band d may mainly contain the weakly charged proteins that have a relatively high affinity to the lowest charged NP2. NP4-FBS possesses the most abundant protein band b, suggesting that band b may mainly consist of highly negatively charged proteins that have a relatively strong affinity to the highest positively charged NP4. These results indicate that ζ potential is a key factor influencing NP–protein interaction because proteins are also charged in solution and electrostatic force is one of the most important forces involved in the interaction [29].
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Research Article
Concentration-dependent protein adsorption at the nano–bio interfaces of polymeric nanoparticles & serum proteins
We have demonstrated above the effects of NPs concentration on protein adsorption capacity and adsorbed protein type. To the best of our knowledge, this concentration-dependent protein adsorption of polymeric NPs was never reported before. But indeed, it is of great significance for clinical use of NPs-based nanoformulations. As we know, dosage adjustment of ordinary formulations is a common clinical strategy to optimize therapy and it does not cause unpredictable effects as long as the postadjustment dose falls into the therapeutic window. However, this normal situation would be different for nanoformulations since changes in NPs concentration would lead to substantial alteration in both protein adsorption capacity and adsorbed protein type. In consideration of the significant impacts of protein adsorption on delivery fate of NPs, like pharmacokinetics, biodistribution and pharmacodynamics [19,36], dosage adjustment of nanoformulations may cause unpredictable or uncontrollable effects due to the altered protein adsorption pattern. Therefore, this work is a serious warning about the possible risks of dosage adjustment of nanoformulations in practical use. On the basis of the findings, personalized therapy and thorough investigations on the pharmaceutical effects of each applied dose may be required in development and clinical use of nanoformulations. In addition to administration dose, the type of disease-caused personalized protein corona is another factor requiring personalized therapy. It has also been reported that corona decoration strongly depends on the type of disease, leading to the formation of personalized protein corona [47–49]. Conclusion In recent years, the negative effects of NP–protein interaction on NP-based drug delivery have drawn increasing attentions. A comprehensive understanding of such interaction is required for developing quality controllable, high-efficiency and low-toxicity nanoformulations. In this work, we showed the changes of polymeric NPs in size, ζ potential and morphology upon interaction with serum proteins. Importantly, we reported the important roles of NPs exposure concentration in NP–protein interaction. In consideration of the important impact of protein adsorption on the in vivo fate of NPs, our findings are quite meaningful for the design, study and practical application of NPs-based drug delivery systems. In addition to the basic physicochemical properties, the efficacy and toxicity of each applied dose of nanoformulations have to be thoroughly investigated during preclinical/clinical studies and closely monitored in clinical use. In a word, personalized therapy would be required in using nanoformulations. Financial & competing interests disclosure This work was supported by National Natural Science Foundation of China (No. 81402860) and the Excellent Young Scientist Foundation of Sichuan University to Q Peng (no. 2016SCU04A02). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Summary points r Four PHBHHx nanoparticle (NP) systems with similar sizes (230 ± 20 nm) but varied ζ potentials (ZPs) (-30 ∼ +40 mv) were successfully prepared. r Size, ZP and morphology of NPs changed upon protein adsorption.
r NP2 (ZP -12.3 mv) adsorbed more absolute protein amount than NP1 (ZP -28.1 mv) and NP4 (ZP 39.6 mv) adsorbed more than NP3 (ZP 23.5 mv). r Protein adsorption capacity increased when reducing NPs exposure concentration. r Type of adsorbed protein changed when NPs concentration was different.
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