(352f) Field Flow Fractionation-Based Analysis for Evaluating Slight Aggregation of Surface-Functionalized Nanoparticles That Can Impact Their Biological Interaction

Surface modification of nanoparticles (NPs) has been widely studied for biomedical applications, e.g., imaging, drug delivery, therapy, and sensing. Various functional molecules such as antibodies and DNAs have been modified to NPs for mediating their biological interaction. During these surface modifications, aggregation of NPs frequently occurs, leading to loss of their functions. Therefore, special efforts have been made to prevent the aggregation of NPs, where evaluation of dispersion stability is particularly important.

Recent studies have revealed that physicochemical property of NPs, including size, shape, and surface chemistry, significantly affect their biological interactions¹. Because aggregation of NPs dramatically changes their physicochemical properties, their interaction with biological environment, such as cellular uptake, was reported to be altered depending on the degree of aggregation.^2,3 From these studies, an emerging concern is that although NPs seem to be monodispersed, slightly aggregated particles contained in the dispersion, such as NP dimers and trimers, could significantly impact their biological fate. However, this effect has never been addressed due to the lack of an exact evaluation method. Slight aggregation possibly occurs during all the conventional surface modifications, therefore a method that can detect it needs to be developed.

One of the common evaluation methods for aggregation is Dynamic Light Scattering (DLS). DLS is a method for estimating the hydrodynamic size of NPs from fluctuations in light scattering from particles, which has the advantage of simplicity. However, because curve fitting is performed assuming spherical particles and certain particle size distributions, it is difficult to detect slight aggregation. Furthermore, since DLS measures a hydrodynamic size, the measured particle size increases due to the surface modifications, making it difficult to distinguish it from slight aggregation.

In this study, we developed a new evaluation system for analyzing slight aggregation of NPs based on Field-Flow Fractionation (FFF). Centrifugal FFF (CF3) is a method for size classification of NPs using a balance between diffusion and centrifugal force⁴. Since density is a dominant factor in centrifugal force, NPs with high density, such as metal or semiconductor NPs, are expected to be classified based on the size of their solid contents with negligible influence of the surface chemistries. Therefore, it is expected that this characteristics of CF3 would make it feasible to detect the slight aggregation of NPs. Using a sample prepared by a conventional method that could cause slight aggregation as a model, the difference on cellular uptake between slightly aggregated and monodispersed sample was investigated by CF3-based evaluation. The impact of the slight aggregation on the biological interaction of NPs will be examined via cellular uptake assay.

Experimental

Preparation of slightly aggregated NPs

Fluorophore-conjugated antibody (Ab) was modified with NHS- and SH- terminated polyethylene glycol (PEG) (MW=5000) and then modified to gold nanoparticle (AuNP) surface. During Ab modification to AuNP, SH-terminated shorter PEG (MW=2000) was also added to stabilize them. For purification of Ab-modified AuNPs, centrifugation have been conventionally used, although it could potentially cause aggregation due to their forced packing. In this study, slightly aggregated sample was prepared on purpose using relatively strong centrifugal force (15,000×g, 3 times). Synthesis of Ab-modified AuNP was confirmed by FT-IR spectroscopy. Size distribution of slightly aggregated and monodispersed Ab-modified AuNPs were evaluated by both DLS and CF3 analysis, followed by their comparison. Morphologies of each fraction obtained from CF3 analysis were also observed by TEM.

Cellular uptake study

To examine the difference in cellular uptake between slightly aggregated and monodispersed samples, anti-EGFR Ab was modified to AuNP by the same method with above. Then, we incubated anti-EGFR Ab-modified AuNPs with A431 cells for 6 hours, followed by washing with PBS. After digesting the cells, the amount of AuNPs internalized into the cells was evaluated using ICP-OES.

Results and Discussion

Ab modification on AuNPs

In both slightly aggregated and monodispersed samples, same peaks including broad bands on 3300 cm^-1, which correspond to amide bonds of Ab, were observed by FT-IR spectroscopy. These results suggested successful modifications of Ab on AuNP surface.

Size evaluation via CF3 to distinguish slight aggregation

Size distribution of slightly aggregated and monodispersed Ab-modified AuNPs was evaluated by both DLS and CF3. DLS analysis showed that compared with bare AuNP, hydrodynamic sizes of Ab-modified AuNP were increased in both slightly aggregates and monodispersed samples. However, the difference between them was not obvious enough to identify the slight aggregation. On the other hand, in CF3 analysis, the main elution peak of monodispersed Ab-modified AuNP sample was same with that of bare AuNP. However, elution peak of slightly aggregated Ab-modified AuNP showed distinct multiple peaks, corresponding to AuNP dimers and trimers as evidenced by TEM observation.

The observed difference between DLS and CF3 analysis would be attributed to the different evaluation mechanism. Because DLS detects hydrodynamic size of NP, measured particle sizes increase due to not only slight aggregation of NPs but also surface modification to NPs, making it difficult to distinguish them. On the other hand, because CF3 classifies NPs basically based on the mass of NPs, even if the surface was modified with Ab and PEG, the total mass changes were small enough to be considered as negligible because of the high density of AuNPs themselves. Therefore, CF3 could classify monodispersed and slightly aggregated Ab-modified AuNPs based on the number of NPs bound together. These results demonstrated that CF3 could detect slight aggregation of Ab-modified AuNPs, which was not detectable by conventional DLS analysis.

Effect of slight aggregation on cellular uptake

So far, we proposed a new evaluation system for slight NP aggregation using CF3. Herein, we show how problematic the presence of slight NP aggregation could before biomedical application. To evaluate the difference between slightly aggregated and monodispersed Ab-modified AuNPs in their biological interaction, the cellular uptake amount by A431 cells was compared. The cellular uptake level was 1.8-times higher for slightly aggregated sample than monodispersed sample (p < 0.001). This trend was consistent with previous reports on receptor-mediated endocytosis in this size range, around 50 nm; larger particles have more Abs on surface and thus bind more frequently to the receptor than smaller one⁵. Since a mere presence of slight aggregation could change the amount of cellular uptake as shown above, attention and removal of these slight aggregation would be required to obtain more accurate and reproducible results. These results demonstrated the impact of slight aggregation of NPs on their biological interaction and the importance of its evaluation via CF3.

Conclusions

CF3 was able to detect slight aggregation of Ab-modified AuNPs prepared via a generally used method, which was difficult to be detected by DLS. It was demonstrated that the slight aggregation of Ab-modified AuNP significantly affect their cellular uptake behavior. For more accurate NP-based biomedical applications, evaluation via CF3 would be important.

Acknowledgement

This work was joint research with SHIMADZU CORPORATION and supported by JST PRESTO.

References

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