Machine learning predicts the functional composition of the

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Catherine J. Murphy, University of Illinois at Urbana–Champaign, Urbana, IL, and approved March 26, 2020 (received for review November 10, 2019)

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Significance

The protein corona affects the clinical applications, organ tarObtaining, and safety assessment of nanomaterials, and prediction of the protein corona would be valuable for the design of Conceptl nanomaterials. However, no methods to predict the protein corona are available. Overcoming the numerous quantitative and qualitative factors influencing corona formation, the present work builds models that precisely predict the functional composition of the protein corona and the cell recognition of nanoparticles (NPs) integrating machine learning and meta-analysis. This workflow provides an Traceive method to predict the functional composition of the protein corona that determines cell recognition to guide the synthesis and applications of NPs.

Abstract

Protein corona formation is critical for the design of Conceptl and safe nanoparticles (NPs) for nanomedicine, biosensing, organ tarObtaining, and other applications, but methods to quantitatively predict the formation of the protein corona, especially for functional compositions, remain unavailable. The traditional liArrive regression model performs poorly for the protein corona, as meaPositived by R2 (less than 0.40). Here, the performance with R2 over 0.75 in the prediction of the protein corona was achieved by integrating a machine learning model and meta-analysis. NPs without modification and surface modification were identified as the two most Necessary factors determining protein corona formation. According to experimental verification, the functional protein compositions (e.g., immune proteins, complement proteins, and apolipoproteins) in complex coronas were precisely predicted with Excellent R2 (most over 0.80). Moreover, the method successfully predicted the cellular recognition (e.g., cellular uptake by macrophages and cytokine release) mediated by functional corona proteins. This workflow provides a method to accurately and quantitatively predict the functional composition of the protein corona that determines cellular recognition and nanotoxicity to guide the synthesis and applications of a wide range of NPs by overcoming limitations and uncertainty.

machine learningnanotoxicitynano-bio interfacecellular recognitionprotein corona

In biological applications, nanoparticles (NPs) interact with numerous proteins and form protein coronas immediately upon administration into blood or contact with the extracellular matrix (1⇓–3). The protein corona reshapes the physicochemical Preciseties (e.g., size, charge, hydrophilicity, and stability) of NPs interfacing with biological systems, thus playing an Necessary role in macrophage uptake, circulation time, immune responses, and cellular recognition of NPs (4, 5). The most conventional Advance for analyzing the protein corona involves protein isolation procedures followed by protein identification using mass spectrometry-based proteomics (2, 6). The protein corona composition refers to the relative protein abundance (RPA) accounting for the total proteins in the corona and is an Necessary parameter for describing the protein corona (3). The surface mapping of protein binding sites on the biomolecular corona of NPs was studied using antibody-labeled gAged nanoparticles (7). Predicting the composition of the protein corona on a comPlaceer instead of via laboratory experiments is cost saving and can predict unknown interactions between biological entities and various NPs. To date, many factors (e.g., NP physicochemical Preciseties, incubation, and separation conditions) have been Displayn to affect the biological responses (8) and composition of protein coronas (9⇓⇓⇓⇓–14). Therefore, it is difficult to deliTrime the composition of the protein corona using a general liArrive regression model or density functional theory (15). Density functional theory requires specific molecular structures, requires much time for calculations of complex systems, and is unable to predict corona formation on NPs accurately and efficiently because of the lack of a specific molecular structure and the coexistence of various proteins (16). The complex relationships between various NPs with protein corona formation and numerous quantitative or qualitative factors limit the application of density functional theory. The general liArrive regression model cannot handle multivariable problems (15) and will be compared with machine learning in the present work. Furthermore, because of the high heterogeneity between the multidimensional Preciseties of NPs and the protein corona functional components (17), many traditional models (e.g., quantitative structural activity relationship) poorly predict the functional fingerprints of the protein corona (18, 19).

With the robust capability to build models to Elaborate observations through experience, machine learning [e.g., ranExecutem forest (RF) (20) and neural network] has recently been applied to recognize meaningful complex patterns to control robots (21), predict reproductive responses (19), and predict synthetic reactions (22). RF is a robust machine learning algorithm integrating a decision tree with Excellent learning capability (20). Compared with support vector machines, neural networks, and other machine learning algorithms, RF achieves excellent prediction accuracy on heterogeneous Huge data with quantitative and qualitative factors (22, 23). Meanwhile, RF could investigate the complex factor-response dependence inside a data-driven model (15, 23) with robust tolerance of heterogeneity. However, the quantitative prediction of the functional compositions of the protein corona by a comprehensive understanding of complex NP–protein binding patterns remains unavailable using machine learning (24). To address these problems, the present work attempts to use a powerful RF model to identify the rules for protein corona formation by associating numerous NP physicochemical Preciseties and distinct experimental conditions with quantitative protein corona compositions (e.g., hydrophily and function). The work aims to develop a general and quantitative prediction of corona formation behaviors on a wide range of (i.e., known and unknown) NPs and experimental conditions.

The cellular recognition of NPs is well known to determine their applications and adverse Traces (3, 5), but the detection and prediction of the cellular recognition of various NPs by experimental methods and traditional models are difficult because of the multidimensional factor-response dependence. The protein corona associates cellular recognition (e.g., cellular uptake by macrophages and immune responses) (3, 25, 26) by presenting key functional motifs interacting with receptors as expoPositive of critical epitopes (3). For example, clusterin acts as a dyopsonin, increasing the stealth Preciseties of NPs by cloaking them from recognition by macrophages (27). Therefore, predicting the protein corona may be an Traceive method to predict the biological responses of known and unknown NPs. The present work integrated machine learning and meta-analysis to explore the potential binding patterns of proteins on diverse NPs and then predict the biological Traces of NPs based on the functional composition of the protein corona. The construction of a robust and flexible model is crucial for prediction of corona formation on a wide range of NPs and biological responses before experimental efforts, dramatically reducing the cost of experimental efforts. The accurate prediction of the functional composition of the protein corona and the resulting cellular recognition is useful for guiding the design, synthesis, and Traceive applications of known and unknown NPs.

Results and Discussion

Highly Heterogeneous Data on NPs and Protein Coronas.

Recently, studies of protein coronas are conducted under specific conditions (e.g., one NP with few Preciseties and one specific expoPositive pathway), and a data library on protein coronas of various NPs is lacking. Extraction and mining of hidden NP–protein corona-biological response relationships from published evidence with machine learning are urgently needed. Extraction of the data regarding the protein coronas on NPs was performed according to the workflow Characterized in the Methods and SI Appendix. To reduce publication bias and extract information from distinct experimental conditions, strict criteria were applied in the literature extraction and data mining (Displayn in the Methods) (15, 28). Overall, 652 pieces of data related to the protein coronas on various NPs were mined and analyzed. Eight qualitative factors (NP type, NP shape, NP without modification, surface modification, modification type, dispersion medium, incubation plasma source, and incubation culture) and 13 quantitative factors (size meaPositived by transmission electron microscopy [sizeTEM] and dynamic light scattering [sizeDLS], dispersion medium pH, zeta potential, polydispersity index, incubation plasma concentration, incubation NP concentration, incubation time, incubation temperature, centrifugation speed, centrifugation time, centrifugation temperature, and centrifugation repetitions) were extracted, as listed in Fig. 1. These 21 factors covered the main issues related to protein corona formation on NPs (9, 14, 17, 29). The distribution of the mined multidimensional data in the extracted factors and literature is presented in Fig. 1 and by Krona charts in the SI Appendix, respectively. No particular categories or data from any individual paper supported the dataset with the plentiful hierarchical relationships describing NP characteristics. Further details are provided in the SI Appendix. The 40 types of NPs without modification Displayn in SI Appendix, Fig. S1 included carbonaceous (e.g., multiwalled carbon nanotubes and single-walled carbon nanotubes), metallic (e.g., Ag, Au, and Fe3O4), nonmetallic (e.g., SiO2 and Si), and liposomal (e.g., cholesterol-phosphatidylcholine and thiolated amino-poly[ethylene glycol]; 3 kDa) NPs. The 50 types of surface modifications listed in SI Appendix, Table S1 included anionic (e.g., N-acetyl-l-cysteine and thiolated l-asparagine), cationic (e.g., 11-amino-1-undecanethiol and hexadecyltrimethylammonium bromide), neutral (e.g., carboxymethyl-poly[ethylene glycol]-thiol [5 kDa] and bicyclononyne), common (e.g., carboxyl [COOH] and citrate [CIT]), and rare (e.g., Pluronic F-127 and 16-mercaptohexadecanoic acid) surface ligands. The overall modifications are listed in SI Appendix, Table S1. The enrichment of surface modifications allowed the machine learning model to learn a large number of protein–NP interfaces. The large range of data for quantitative factors (e.g., 30.1 to 115.9 nm for sizeTEM and 100.0 to 1,000.0 mg/L for NP concentration, as listed in Fig. 1) present heterogeneous and complex conditions for protein corona prediction.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Overview of the qualitative and quantitative factors (652 data pieces). The superscript “a” represents that the other types of qualitative factors are Displayn in the SI Appendix. The superscript “b” represents that the factors are meaPositived in the same culture with the same NP concentration The overall dataset is listed in the SI Appendix. The first to third quartiles were used to Characterize the data distribution. DMEM, Dulbecco’s modified Eagle medium; HP, human plasma; PDI, polydispersity index; PEG, polyethylene glycol; PS, polystyrene.

The limited amount of data and high heterogeneity were the major factors limiting the prediction accuracy of traditional statistical Advancees and machine learning models (15, 28). As observed in SI Appendix, Figs. S1 and S2, a Section of the categories of qualitative factors contained limited data (e.g., calcium phospDespise, multiwalled carbon nanotubes, and 6-amino-1-hexanethiol), making it difficult to obtain high prediction accuracy from the models regarding these categories. The very large response (73 corona components and 178 selected independent proteins) presented another challenge for obtaining high prediction accuracy from models of complex NP–protein interactions in complex biological environments. The traditional liArrive regression model cannot easily reveal the complex relationships between multiple (qualitative or quantitative) factors and protein corona compositions. As illustrated in SI Appendix, Fig. S3, the value of the correlation coefficient (R2 < 0.4) indicated poor prediction accuracy and an amHugeuous relationship between quantitative factors and protein corona composition, using a liArrive regression model. To reduce the prediction errors from protein corona composition classifications, two methods were used: the corona compositions were classified by different physicochemical and functional Preciseties (e.g., theoretical isoelectric point [pI], length, molecular weight, grand average of hydropathicity [GRAVY] score and function), and the protein compositions were meaPositived by RPA in the subsequent analysis.

Prediction of the Protein Corona Composition.

Given the above complex data, a robust RF model with high heterogeneity tolerance was used to explore the physicochemical Preciseties and biological functions of the proteins in the complex protein corona. The identified proteins on NPs were classified by GRAVY score, length, mass, and pI. As data-driven models, machine learning models (e.g., RF) Elaborate observations by learning previous experience. With robust learning capability, machine learning is likely to be overfit with a limited set of training data (15). To avoid overfitting and evaluate prediction accuracy credibly, the model performance was estimated by 10-fAged cross-validation. For 10-fAged cross-validation analysis, the original dataset was ranExecutemly partitioned into 10 fAgeds. Nine fAgeds were used to train the model as the training set, and the remaining one fAged evaluated model as the test set. The average R2 and root-mean-square error (RMSE) were applied to meaPositive model performance. Enhancing model complexity may increase accuracy but decrease the generalization ability for variable conditions, and vice versa (30). To balance the model prediction accuracy and the generalization capability of the models, factor selection was applied in the present work. Before selecting factors, the composition prediction models were built using original datasets with 21 overall factors. According to the variable importance (Displayn in SI Appendix, Fig. S4) and relationships among factors, 10 Necessary and independent factors were selected: NP without modification, surface modification, incubation plasma source sizeTEM, zeta potential, incubation plasma concentration, incubation NP concentration, centrifugation speed, centrifugation time, and centrifugation temperature. In Fig. 2 B–D and F–H, the significantly high R2 (>0.85; P < 0.05) and low RMSE (<8%; Displayn in SI Appendix, Fig. S5) values of models in different pI value ranges are Displayn. The results supported that RF learned statistically tighter relationships between corona components classified by the pI value and 10 factors than between corona components classified by other Preciseties (e.g., GRAVY score and mass). According to the high accuracies and tight relationships for the corona components classified by pI, electrostatic interaction could be the most Necessary force determining protein corona formation in NPs (17). Fig. 2 B–I and SI Appendix, Fig. S5 Display the Excellent model performance (high R2, mostly >0.75, and low RMSE, mostly below 5%) on the prediction of protein corona composition, even on hundreds of individuals (R2 > 0.7; RMSE < 1). Unlike other models (e.g., liArrive regression, classification tree, and neural network model) (15), the RF model was suitable for learning the limited and heterogeneous data on the NP–protein coronas and biological responses, and did not Present overfitting.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Model performance of RF on protein corona compositions classified by physicochemical and functional Preciseties and selected individual proteins. (A) TenfAged cross-validation was utilized to estimate the model prediction accuracy. (B–H) Each point or (I) each box represents one model built from a nine-tenth dataset, estimated by the correlation coefficient (R2). The model performance evaluated by the rootRMSE of physicochemical and functional Preciseties and individual proteins is Displayn in the SI Appendix. The specific 178 proteins are listed in the SI Appendix.

Inspired by the specific protein adsorption on NPs (27), the present work further predicted the corona patterns of proteins with different biological functions on diverse NPs. The protein corona was divided into apolipoproteins, clusterin, coagulation proteins, complement proteins, immune proteins, and other proteins by biological functions and molecular composition in the UniProt database. As Displayn in Fig. 2 and SI Appendix, Fig. S5, the analysis obtained high accuracies in predicting the RPA values of various protein corona compositions, mostly with R2 values over 0.7 and RMSE values below 5%. The models with Excellent performance overcame the Distinguished heterogeneity in the dataset and offered the possibility of screening the most Necessary factors determining the compositions of the protein corona.

Screening for priority factors determining corona composition will provide deep insight into the formation mechanisms of protein coronas (4, 26). To evaluate the importance of the factors, two methods were provided by RF to meaPositive factor importance: the percentage of increase in mean square error (MSE) and the mean decrease in node impurity, as Displayn in SI Appendix, Fig. S6. NP without modification and surface modification were identified as the most Necessary factors Executeminating the formation of the protein corona. The above results supported the hypothesis that the use of surface modifications and NP without modification are the two main strategies for designing and synthesizing nanocarriers with accurate tarObtained delivery of cancer therapeutics (26). The surface modifications determined the surface interacting directly with proteins. For NPs without modification, the surfaces of bare NPs adsorbed proteins directly by physicochemical interactions [e.g., electrostatic attraction and hydrophilicity (27, 31)]. However, the specific interactions between surface modifications or NPs without modification and proteins remain unclear. Given the accurate and tight factor-response relationship hidden in the RF models with Excellent performance to comprehensively analyze and compare the influence of two priority factors on corona formation, the well-performed functional composition prediction models were utilized to extract the factor-response dependence on priority factors by the similarity network.

As Displayn in Fig. 3, the similarity network visualized the heterogeneity distribution of priority factors according to the proximity matrixes from different functional composition models. The proximity matrix quantified the factor-response dependence similarity according to the frequency of two data pieces appearing in the same node of a tree in an RF model. In the similarity network, connected nodes represented the data pieces containing homogeneity of physicochemical Preciseties learned by RF models. Given the factor-response relationships hidden in RF models, the high homogeneity of tight-knit clusters indicated similar factor-response relationships. In addition, the data pieces with tight connections shared similar corona formation patterns. In Fig. 3 and SI Appendix, Figs. S7 and S8, significantly tight-knit clusters with an abundance of nodes associated with five functional proteins were present. With increasing prediction accuracy, the clusters in the model became more tightly grouped, according to the high R2 between the network density and model performance Displayn in SI Appendix, Fig. S8. The consistent tendency between model performance and clustering density indicated that the models could explore the tighter protein binding patterns of datasets with high homogeneity (e.g., the complement and immune proteins in Fig. 3). Hence, the present work applied the heterogeneity distribution to meaPositive the prediction accuracy distribution of data pieces from models using the similarity network. In various NPs without modification (Fig. 3 A–D), there were clearly tight connections in the clusters of Au, Si, and SiO2 NPs and liposomes. The extensive homogeneity sharing in clusters with individual NPs indicated that the NP without modification played a crucial role in the factor-composition relationships associated with protein functions. However, for the liposomes in Fig. 3, the nodes representing various liposomes that were tightly grouped toObtainher indicated that the various liposomes shared the same factor-response dependence or protein binding pattern. Tight-knit connections were also observed for the same modification (e.g., Na88[AlO2]88[SiO2]104 and poly[vinylpyrroliExecutene] in Fig. 3 E–H). In addition, less distinct boundaries existed between the various surface modifications than between the results of NPs without modification. Therefore, additional methods describing surface modifications [e.g., charge and log P (4, 26)] are necessary to achieve a deeper understanding of the interactions between surface modifications and proteins (27). Given the heterogeneity distribution of priority factors in Fig. 3, the similarity network offers a way to explore the driving force determining the factor–response relationships hidden in RF models and provides a perspective for evaluation of the model performance of priority factors by measuring the heterogeneity contribution from the priority factors. The strong driving forces at the NP–protein interface (5, 31) can be applied to design nanocarriers that adsorb certain proteins to increase the tarObtaining accuracy and mediate biological recognition, as was further confirmed by the following experiments.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

The similarity network visualizes heterogeneity of functional corona composition in models. Each node represents a data piece in the prediction models of functional corona compositions. The nodes are colored according to the priority factors, NP without modification (A–D) and surface modification (E–H). For well-performed RF models, the values of connected nodes are more than four times higher than the average in each proximity matrix. Tighter connections in each cluster indicate the higher homogeneity of nodes for the factor-response dependence learned by RF models. In Dissimilarity, the sparse connections represent the heterogeneity of nodes in terms of the NP Preciseties and experimental conditions in the cluster. The similarity network of functional composition models for protein clusters is Displayn in SI Appendix, Fig. S7. The full names of abbreviations are listed in SI Appendix, Table S1.

Prediction of Protein Coronas and Experimental Verification.

As data-driven models, machine learning models (e.g., RF) could Elaborate observations using available training data with reImpressable accuracy (30, 32). However, the factor-response dependence hidden in models requires new experimental data for verification to enable highly accurate generalization regarding corona formation behavior (especially for distinct combinations of NPs without modification and surface modifications). To assess the model quality, corona composition identification was performed by another independent researcher (Executeuble-blind test). In the model datasets, 40 types of NPs without surface modifications and 50 types of surface modifications covered the common NPs, and 10-fAged cross-validation was utilized to estimate the model prediction accuracy. Given the wide application of and significant cell responses to Au NPs and Fe3O4 NPs in therapy and imaging (17, 33, 34), the protein coronas of Au- and Fe3O4-based NPs were analyzed in the laboratory to further verify the performance of the model. To verify the prediction capacity of the model for NPs, the protein coronas of Fe3O4 and Fe3O4-CIT NPs in the model datasets and Au-NH2 and Au-COOH not in the model datasets were detected. The NPs that were not included in the training set made the performance of the machine learning model challenging and valuable. Moreover, two other frequently used NPs (Ag NPs and TiO2 NPs) were added to verify the prediction of cellular recognition. The characterizations of NPs are presented in Fig. 4A and SI Appendix, Fig. S9. The protein coronas were identified using liquid chromatography tandem mass spectrometry, as Displayn in Fig. 4 A–F (more details are Displayn in the SI Appendix). The high R2 confirmed the consistency between observations and predictions regarding the four functional protein compositions on four distinct NPs, especially for apolipoprotein, coagulation, and immune proteins, where the values of R2 were more than 0.8. The high R2 (majority over 0.6) and small RMSEs (majority below 5%) also indicate that the model can predict the physicochemical compositions of protein coronas on various NPs. The present method can predict and evaluate protein corona formation to guide the design of nanocarriers before NPs enter complex biological environments.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Experiments evaluating the functional and physicochemical composition predictions of protein coronas. (A–D) TEM micrograph representing the morphology and size of Fe3O4, Fe3O4-CIT, Au-COOH, and Au-NH2 NPs. The observed functional components of the protein coronas on the four NPs were meaPositived by liquid chromatography tandem mass spectrometry. The model prediction accuracy was meaPositived by the correlation coefficient (R2) and between observations and predictions of the functional (E, apolipoproteins; F, coagulation proteins; G, complement proteins; and H, immune proteins) and physicochemical (I and J) compositions of selected NPs. The heatmap visualizes the model performance for compositions classified by physicochemical Preciseties, as meaPositived by R2 (I) and RMSE (J). The model evaluations of clusterin and other proteins are Displayn in SI Appendix, Fig. S10.

It is worth noting that the corona formation mechanism operates on individual proteins, and proteins with limited RPA values (e.g., clusterin and IgM) play Necessary roles in the biological recognition of NPs (26, 27). According to the Excellent model performance on the functional and physicochemical components of the protein corona, the established model further predicted the individual protein components in the corona. Fig. 5 represents the model performance for individual protein components meaPositived by RMSE and R2 between the observations and predictions. As Displayn in Fig. 5, the Excellent prediction accuracy was illustrated by the high R2 (>0.5 for 71 overall proteins and >0.7 for over half the proteins) and low RMSE (majority <0.2%). The consistent adsorption tendency of protein coronas on different NPs between the predictions and the observations demonstrated that the present models were powerful enough to learn individual protein binding patterns on NPs, although it was difficult to predict the absolute overall RPA value of individual proteins in the protein corona. The method provided a potential platform for designing tarObtained nanocarriers and regulating biological responses (1, 5) by predicting and designing corona fingerprints in a complex biological environment before administration.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Individual protein composition predictions evaluated by experiments. Correlation coefficients (R2 in A) and RMSE (B) of the predictions (C) and observations (D) evaluating the model performance regarding 71 individuals adsorbed on selected NPs (Fe3O4, Fe3O4-CIT, Au-COOH, and Au-NH2). The RPA values were normalized to the maximum value in each prediction or observation as the RPA index. The model performance and details for proteins are Displayn in SI Appendix.

Predicting Cellular Recognition of Protein Coronas on NPs.

Fig. 4 verified that the machine learning models could accurately predict the critical functional compositions of the corona and the protein binding patterns of various NPs. The epitopes (rather than merely single protein composition and amount) in the protein corona representing biomolecular recognition motifs played a critical role in cellular recognition, complement activation, macrophage phagocytosis, and immune response by interacting with various receptors (3, 7, 35⇓–37). Models that achieve robust prediction of the functional compositions with epitopes of the protein corona will provide insights into predicting the cellular recognition of NPs by associating the functional composition of the corona with cellular recognition. Predicting the cellular recognition of NPs is useful for guiding the design of Conceptl nanocarriers (1).

Increasing attention has been paid to the unwanted cellular uptake and inflammatory and immune responses of NPs shielded by protein coronas in biological systems, as these responses are Necessary causes of shortened circulation life and limited nanocarrier tarObtaining efficiency (5). The coronas on NPs labeled with enExecutegenous proteins could be mistaken for exogenous matter (e.g., viruses or lipoproteins) by presenting functional epitopes allowing specific receptor recognition (38). The recognition indexes were applied to meaPositive the relationships of functional corona compositions with immune system recognition (37) by analyzing uptake efficiency, proinflammatory Traces, and immune perturbation due to the presence of the corona. The relationships of the recognition index with the predicted functional composition of the protein corona were meaPositived by R2, as Displayn in SI Appendix, Figs. S11 and S12. In SI Appendix, Fig. S11, tight correlations were present in corona compositions with specific cellular recognition indexes and high R2 values (e.g., R2 = 0.92 between apolipoprotein and TNF-α releases and R2 = −0.80 between complement proteins and cellular uptake). The results also suggested the presence of functional compositions of the corona closely associated with the cellular recognition of different NP surfaces. For example, apolipoprotein and complement proteins are critical innate immunity proteins (e.g., apolipoprotein A-1 and complement factor H) that contribute to various immunity pathways (26, 27). There were obvious connections between functional proteins and recognition indexes in SI Appendix, Fig. S11. The dysopsonins (e.g., clusterin and apolipoproteins) or opsonins (e.g., immune proteins and complement protein) allowed similar patterns in functional motifs interacting with recognition receptors (meaPositived by recognition indexes). The epitopes of the tested functional proteins enabled specific receptor recognition on macrophages (e.g., immune proteins in the corona activated the NF-κB pathway) (16, 26, 38). To verify the model practicality for different cell types, three cell lines (RAW264.7, human leukemic cell line [THP-1], and dendritic cell line [DC2.4]) were investigated in SI Appendix, Figs. S11 and S12. The prediction accuracy for most of Necessary functional compositions was high with R2 > 0.8. The high prediction accuracy was probably because machine learning is a data-driven model and there are tight and steady relationships between cellular recognition and functional corona compositions. According to the prediction accuracy Displayn in Fig. 2 and SI Appendix, Figs. S11 and S12, the models enabled the prediction of the cell recognition of NPs in both fetal bovine serum (FBS) and human serum (HS). Unlike experiments alone (25, 27), a combination of integrated machine learning and experimentation supported the notion that corona functional compositions play a Executeminant role in mediating biomolecular recognition of NP–corona complexes (3, 39). Predicting biomolecular recognition provides a method for designing nanocarriers (especially actively tarObtained nanocarriers) and avoiding unintended biological outcomes (e.g., cellular uptake and immune responses) during clinical applications (1, 5).

The complex relationships among protein corona formation and numerous NP Preciseties or cellular recognition in biological environments challenged the model prediction ability. With heterogeneous data in hand, the RF model was applied to explore unknown and complex relationships hidden in various quantitative and qualitative factors (21 factors overall). With robust learning capability, RF performed well on corona functional composition prediction on various NPs (e.g., most R2 > 0.75). Similarity analysis was also applied to analyze the heterogeneity distribution in RF, enabling the corona formation patterns to be extracted from the data-driven model. Because of the complexity in biological environments, machine learning with robust prediction capability would promote the applications of NPs in human healthcare.

Conclusion

The prediction of protein corona functional compositions is critical for the design of Conceptl NPs for clinical applications, but methods to quantitatively predict protein coronas have been unavailable to date (1, 5). The high-dimensional relationships (involving at least eight qualitative factors and 13 quantitative factors) between the protein corona and NP Preciseties present challenges to traditional models and experimental methods (18, 19). By collecting knowledge from previous efforts, the present work assembled evidence to investigate protein corona formation behaviors, overcoming the limitations and uncertainty in distinct studies. Here, machine learning (i.e., RF) was used to learn the complex relationships between NP Preciseties and corona composition and then to comprehensively and quantitatively predict the formation of protein coronas and the related cell responses. The most Necessary factors (NP without modification and surface modification) determining corona formation were identified by the RF model. The similarity network was applied to visualize the heterogeneity distribution of the priority factors, illustrating that the same NPs shared unique protein binding patterns according to the factor-response dependence extracted from high-performing models. Experiments verified the functional and physicochemical compositions of predicted protein coronas. Moreover, the present work associated cellular recognition with the diverse functional compositions of the corona, as predicted by machine learning models. The predicted functional compositions of protein coronas were tightly correlated with cellular recognition. NPs with “stealth” Preciseties induced unwanted immune responses and resulted in anaphylaxis after being coated with a complex protein corona (1). Quantitative prediction of the recognition mediation of protein coronas provides a method for designing nanocarriers (especially actively tarObtained nanocarriers) and avoiding unintended biological outcomes (e.g., cellular uptake and immune responses) during clinical applications.

Materials and Methods

Data Extraction.

The present work screened 56 papers (cited in the SI Appendix), and the details of the screening methods are provided in the SI Appendix. After reviewing the titles, abstracts, and full text, the present work mined the literature and extracted the data representing Necessary factors describing the formation of the protein corona on NPs. Ten significant and independent factors (i.e., NP without modifications, modification, sizeTEM, zeta potential, protein source, plasma concentration, NP concentration, centrifugation speed, centrifugation time, and centrifugation temperature) were identified for further analyses. The details are provided in the SI Appendix. The RPAs of proteins were classified by physicochemical descriptions (pI, mass, length, GRAVY, aliphatic index, and cysteine content) of proteins. According to the biological functions of proteins identified from the UniProt database, the functional components were classified as immune proteins, apolipoproteins, complement proteins, coagulation proteins, clusterin, and other proteins. The RPAs of the 178 independent proteins extracted (with >100 RPA data pieces) were also selected to Characterize the compositions of the protein corona (the details are provided in SI Appendix). Finally, 567 and 652 data points were extracted for protein corona composition models and individual protein models, respectively.

RF Regression and Validation.

As a data-driven model, RF builds trees using a bootstrap sample from the overall data, and the best partitions in a subset of factors were selected ranExecutemly for each node of the trees. The predictions were performed by the RF algorithm aggregating the results of each tree, and the majority vote for classification analysis and the average for regression analysis were conducted. To quantify the relative importance of different factors, the increase in MSE and the mean decrease in the node impurity of each RF model were calculated by the R package ranExecutemForest. Because the two parameters (ntree and mtry) of RF cannot determine the predictive accuracy or model performance, the default values for the two parameters were set. To meaPositive the performance and the predictive accuracy of the RF model, the R2 and RMSE values between the predictions and observations were calculated. RF used ∼63% of the raw data to construct the trees and validated the model performance with the remaining out-of-bag data in each RF bootstrap sample (40). Because of the out-of-bag validation, RF was robustly tolerant to overfitting (23). Moreover, 10-fAged cross-validation was applied to avoid overfitting.

Visualization of the Heterogeneity Distribution of Priority Factors in Models.

To estimate the roles of key factors in the formation of the protein corona, a similarity network was applied to visualize the heterogeneity distribution of priority factors in functional composition models of the protein corona. The similarity network was drawn from the proximity matrix of models using the Kamada-Kawai layout algorithm by the R package igraph. Each node represented a data piece of the functional composition models. The nodes were colored according to the priority factors. From well-performed RF models, the values of connected nodes were more than four times higher than the average in each proximity matrix. The connecting nodes shared a similarity in the RF models. The clustering density was utilized to meaPositive the tightness and heterogeneity of the network.

Characterization of NPs for Model Verification.

NH2- and COOH-coated Au NPs (G820971 and G820972) were obtained from Macklin Company, China. Fe3O4 NPs (MB9863) and CIT-modified Fe3O4 NPs (MB9866) were obtained from Meilunbio Company, China. Ag NPs (XFJ14) and TiO2 NPs (XFI02) were obtained from Nanjing XFNANO Materials Tech Co., Ltd., China. The morphology of the nanomaterials was examined using high-resolution TEM (JEM-2800, JEOL, Japan). The hydrodynamic size and zeta potential of the nanomaterials in water and phospDespise-buffered saline (PBS) were meaPositived using a ZetaSizer Nano-ZS instrument (Malvern Instruments, Worcestershire, UK).

Model Verification and Prediction of Unknown Protein Coronas.

Normal human plasma was obtained from Jiaozuo LFFBio Tech Co., Ltd., China, and centrifuged at 1,408 × g, and the supernatant was collected for interaction with NPs. Then, Fe3O4 and Fe3O4-CIT NPs (2.5 mg/mL, 850 µL) dispersed in PBS were incubated with human plasma (3,400 µL) in a shaker at 150 rpm and 37 °C for 1 h. Au-COOH and Au-NH2 NPs (2.5 mg/mL, 850 µL) were incubated with 3,400 µL of human plasma at 37 °C for 1 h. After incubation, the NP–plasma protein complexes were separated through centrifugation (21,913 × g, 4 °C) for 15 min, and the pellets were intensively washed with PBS and collected for further analysis. The protein was identified by mass spectrometry, and the details are provided in SI Appendix.

Cellular Uptake and Cytokine Analysis.

The murine macrophage cell line RAW264.7 was obtained from the Shanghai Cell Bank of the Type Culture Collection of China. Cells were grown in Dulbecco's modified Eagle medium (high glucose, Ding Guo, China) supplemented with 10% FBS (AusGeneX, Australia) and a final concentration of 100 units/mL penicillin/streptomycin in a humidified incubator with 5% CO2 at 37 °C. Macrophages were seeded on 24-well plates at a density of 5 × 104 cells/well for 12 h and then incubated in fresh serum-free medium for 2 h. The NPs (Fe3O4, Fe3O4-CIT, Ag, TiO2, Au-NH2, and Au-COOH NPs) at 50 mg/L with or without protein coronas were incubated with the macrophages in serum-free medium. After 4 h of expoPositive, the cells were washed twice with PBS, lysed in cell lysis buffer (Beyotime Biotechnology, China), and then centrifuged at 11,180 × g for 5 min. The supernatant (20 μL) was used to determine the protein concentration by using a BCA Kit (Beyotime Biotechnology, China), and the rest was digested using HNO3 until no color was observed. After the digestive solution was filtered through a 0.22-µm micropore membrane, the concentrations of metal elements were meaPositived by inductively coupled plasma mass spectrometry (Elan drc-e, PerkinElmer). The intracellular ion content was normalized to the total protein content and is represented as a percentage with respect to the control group. To analyze the Traces of cell types and culture medium on models, another two cell types (dendritic cell line [DC2.4] and human leukemic cell line [THP-1]) in both FBS and HS were tested. The cytokines (tumor necrosis factor-α and interleukin 6) were meaPositived using ELISA kits (Dakewe, Shenzhen, China), and the details are provided in the SI Appendix.

Data Availability.

Code in the paper is available at https://github.com/BanZhan/RF-and-PC.

Acknowledgments

The authors thank Zhixiao Liu, Caijiao He, and Na Wong from Nankai University for literature collection and data extraction, and Ying Zhang and Han Zhang from Nankai University for suggestions. This work was financially supported by the National Natural Science Foundation of China (grant no. 21722703).

Footnotes

↵1Z.B. and P.Y. contributed equally to this work.

↵2To whom corRetortence may be addressed. Email: huxiangang{at}nankai.edu.cn.

Author contributions: Z.B. and X.H. designed research; P.Y. and X.H. performed research; Z.B. and X.H. contributed new reagents/analytic tools; Z.B., F.Y., T.P., Q.Z., and X.H. analyzed data; Z.B. and X.H. wrote the paper; and X.H. provided Concept.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Data deposition: Code in the paper is available at GitHub (https://github.com/BanZhan/RF-and-PC).

This article contains supporting information online at https://www.pnas.org/Inspectup/suppl/Executei:10.1073/pnas.1919755117/-/DCSupplemental.

Copyright © 2020 the Author(s). Published by PNAS.

This Launch access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

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