Based on previous results of IONPs activity [16], in this study, it has been explored systematic and exhaustive proteomics characterization of different nano-constructions in order to identify differences and similarities between them, useful in further characterizations of biological activity or possible biomedical application. In Fig. 1, it is schematically depicted the IONPs studied as model in this report, displaying the associated nomenclature to facilitate the follow-up and outcomes observed from the protein corona characterization and other screening approaches.
Biological interactions and biocompatibility of nanodrugs conjugates based on multifunctional copolymer coated iron oxide NPs and platinum derivatives
Firstly, it is required the evaluation of the interaction with biomolecules contained on the biological fluids, which inherently bound to the surface of the nanodrug conjugate, known as protein corona. During last decade, several methodological strategies have been proposed to determine protein corona and further determination of differences in the intrinsic interaction between the NPs and the biological milieus, which can ultimately define their pharmacological activity [17, 22]. Among others described methodologies, high-throughput techniques are required because the wide molecular variety and multi-functionalities of the NPs.
As previously mentioned, the protein corona (interaction between NPs surface and the physiological environment) is playing a critical role in the pharmacokinetics and pharmacodynamics of any novel nanodrugs conjugates, as it is directly related with functional process (such as blood absorption and distribution, to cellular endocytosis and pharmacological response, among others…) and the different wide dynamic range of biomolecules on biological proximal fluid, which are in continuous dynamic equilibrium between soluble and adsorbed biomolecules [17, 18]. Consequently, several coating layers constitute the protein corona depending on the NPs surface and molecules diversity. Thus, NPs are coated with a dynamic layer conformed by the most abundant proteins that is named as “soft-corona”. Gradually, some of these proteins are replaced by low abundant proteins but higher affinity for the NP, resulting in a closer layer to the NP surface, called “hard-corona” [23, 24]. Considering that these relevant for modeling novel NPs, in this work, it has been designed and performed a cost-effective assay that allowed to systematically decode the composition of hard- and soft- protein corona (in different biological fluids: human plasma, rabbit plasma and fetal bovine serum (FBS)) by centrifugation process and further proteomics analysis. IONPs and IONPs conjugated with biliar-acid-CisPt derivative [16] (IONP-Pt) has been evaluated by this strategy (Fig. 2B).
In Fig. 3, it is shown a quantitative comparative analysis about the number of unique proteins detected in the protein corona of each biological fluid. The overlap analysis revealed notable differences across the analyzed protein coronas at different proximal fluids; being reported a high number of identified proteins in human plasma compared with other studied proximal fluids. In particular, human IONP-Pt protein corona was > 5 and > threefold larger than FBS and rabbit-derived corona, respectively. Overall, the affinity protein corona displayed larger differences when comparing soft and hard corona (Fig. 3A and B) than when evaluating the presence or absence of Pt derivative conjugated to NPs (Fig. 3A and C). Newly, this trend is particularly conspicuous at human-derived protein coronas (Fig. 3, bottom row) where the highest number of identified proteins allows to observe the most remarkable differences between soft- and hard-corona. In a general overview, the fraction of proteins common to both -soft & hard- is quite relevant because it is highlighted a constantly presence in the final nano-drug conjugate. Although it the largest presence of proteins was determined in the human plasma-derived corona, according to the expected abundance of proteins in this fluid, the rabbit plasma provided a greater number of common proteins between the two coatings (Fig. 3, middle row). FBS-derived analysis resulted in the smallest numbers on proteins comparing with the other studied species. (Fig. 3, top row). However, in this case, the influence of the presence of Pt seems remarkable because 67 proteins identified at FBS-derived hard-corona, which were also detected at soft IONP-Pt. This is suggesting that the presence of Pt is modifying the interaction affinity of those proteins (Fig. 3, top row). The functional enrichment of the detected proteins provide a view of the type of protein binding in this case, where mostly of them are related to blood functions (coagulation, would healing…). Knowing that these factors are in the protein contain of FBS, it is understandable that the presence of Pt displaces these proteins towards the soft-corona, as these proteins interact with more affinity with the metal [25, 26] and do not reach to interact with the IONP surface (Additional file 1: Table S1). Finally, human protein corona reveals the largest differences between identified proteins at hard and soft coronas (Fig. 3, bottom row). Human plasma, on the other hand, presents much more diversity regarding the protein content. Thus, the number of proteins is higher and with more diversity between the layers of the protein corona as expected. When the NPs are conjugated with the Pt derivative, IONP-Pt, the human hard corona incorporates additional 275 different proteins (representing 40.1% of total proteins) (Fig. 3, bottom row).
Number of identified proteins gives the prospect of a different dynamic behavior between species which is to be expected due to the diverse variety of biological content of plasmas; but also it is revealing the importance to evaluate the protein corona in all the proximal fluids involved in the development of nanodrug conjugates (from cell culture, animal model and preclinical). As well as, wide diversity on protein structures which directly influence in the interaction of protein corona onto the surface of NPs. This premise was also taken into account for the functional analysis of the protein as parts of protein corona (Fig. 4).
Cellular Component-Functional enrichment analysis reveals the distinct biological hallmarks associated to hard and soft corona in the plasma of species of interest (human, rabbit and bovine) (Fig. 4A). To characterize the biological hallmarks of the proteins found along the multiple protein coronas, two independent functional enrichment analyses of Gene Ontology-Cellular Component (CC) terms were performed separately for hard and soft protein coronas at each animal model (Fig. 4). Functional enrichment of Cellular Component-Gene Ontology annotations is a powerful tool to identify the most prominent macromolecular complexes represented in a protein set. In the same way, it brings relevant indications regarding the protein’s subcellular localization and thus, their biological roles.
As expected, due to the size of original protein set, the analyses of FBS and rabbit-derived protein coronas barely retrieved functional annotations (Fig. 4A). On this basis, the next analysis was restricted to human-derived protein corona. In order to summarize Functional Enrichment Analysis (FEA) results, the enriched CC annotations were classified into 13 groups representing the most relevant subcellular localization or processes. Finally, the CC-functional enrichment commonalities between soft and hard of IONP and IONP-Pt corona were assessed (Fig. 4B). The functional enrichment revealed notorious differences between hard and soft corona, both in total size and functional signatures (Fig. 4).
Interestingly, the functional annotations of identified proteins at soft corona are mainly related to macrocomplexes—commonly found at peripheral blood—as lipoproteins, platelet-coagulation C protein-related complexes or IgG and IgA immunoglobulin complexes as previously reported [tenzer2013] (Fig. 4B-left). Conversely proteins identified at hard corona and therefore, the NP stable interactors are drastically distinct (Fig. 4B-rigth). At hard corona proteome, identified proteins are involved in cell–cell contacts as integrins or cytoskeleton protein (ie. actin, microtubles, among others). Likewise, hard-corona proteins prompt to be particularly and significantly enriched in processes such as endocytosis, phagocytosis and intracellular trafficking (including COPI or clathrin-coated vesicles and endomembrane system complexes). Hence, these results are suggesting the activation of late endocytic-NP digestion processes because it is observed enriched proteins involved in endosome and lysosome-related proteins together with proteasome core and regulatory complexes. These observations correlate with the normal distribution of proteins in plasma, where proteins in highest concentration (such as the function “blood”) appears in soft-corona while the proteins in lower concentration conform the hard-corona. The influence of the platinum complex is also noteworthy, which promote proportional modification on the functions associated to soft-corona and not those relative modifications to the hard-corona as might be expected from the dynamics of the protein-corona formation. These changes in the different composition of the protein layers close the NPs as a consequence of the functionalization must be considered when evaluating and establishing the biocompatibility of each of the modifications that are established in the nanoconjugates.
Apart from general functional enrichment, here it is also relevant to explore the differences in distribution at cellular compartment between soft- and hard-protein corona. At Human Protein Atlas (HPA) according to the tissue of origin, the proteins observed are related with blood secreted proteins, extracellular matrix (ECM), and other tissues as lung, epithelium or endothelium. Also, it provides proteins involved in secretion pathway frequently found restrained at organelle lumen or membrane. By this, it is obtained a global view of the histological origin of the proteins related to the human-derived corona (Additional file 1: Fig. S1). While soft-corona, in both situations ± Pt derivative functionalization, is composed by large percentage of blood secreted proteins; hard-corona proteome barely maps into a global secretome universe (Additional file 1: Figs. S1 and S2). Around 80% of detected proteins at hard- corona have not been previously reported at HPA secretome; it was evaluated the most frequent tissue of origin by assessing their RNA expression levels at 33 tissue-specific RNA-Seq datasets (also retrieved at HPA database) (Additional file 1: Figs. S1 and S2, Table S2). For 410 and 570 proteins found at hard IONP and IONP-Pt corona, respectively, approx. 90% were identified in at least one of the 33 tissue-specific RNA-seq data sets. Disregarding differences in total number, the tissue of origin most over-represented, (hard-corona in both NP and functionalized NP), are bone marrow, tonsil, esophagus and colon. On the other side, as it may be expected that less frequent tissues of origin correspond to sexual tissues as endometrium, testis, prostate or fallopian tubes (Additional file 1: Figs. S1 and S2, Table S2).
In vitro cytotoxicity of multi-functional NPs
The cytotoxicity of this novel NPs, it is another critical aspect in the characterization of the novel multi-functional nanodrugs, as these novel NPs are containing a fluorochrome covalently coupled to the surface. The main goal was determining whether this functionalization with the fluorescence dye may affect the release of Pt from the initial structure, or the fluorophore coupling could affect to the biocompatibility of the IONP (Fig. 5). Then, it has been explored the cell viability of two tumoral cell lines (colorectal cancer (Caco-2) and T lymphoma (Jurkat)) as models and studied the effect at different concentrations (1–0.1-mg/mL) of IONP-Pt and the multi-functional NP with a fluorophore (IONP-Pt-Flu). In Fig. 5A, it is observed a similar cell viability in both studied human cell lines. In the case of IONP-Pt, cell viability resulted similar to previously reported studies which described the compound triggers cellular instability reflected with a large increase in cell viability [16]. Some slightly differences have been observed in both tumor cell lines. At first glance, it can be seen that the use of IONPs improves the activity of the platinum compound as it shows less cytotoxicity than the IONP conjugate. In general, it has also been shown that there is a slightly higher cytotoxicity in Caco-2 cells than in the Jurkat tumor cell line. In addition, in the Caco-2 tumor cells, it can also be seen that there are more similarities in the behavior of IONP without any influence from the degree of functionalization. About Jurkat tumor cell line, it can be observed that there are certain discrepancies in the behavior of the functionalized IONPs at different incubation times (Additional file 1: Fig. S3). This is due to the oxidative stress-inducing effect mediated by Pt-derivatives as a cytotoxic mechanism [27], and it was reported in previous studies of this kind of platinum-IONPs [16]. In a similar way, it is observed that the fluorochrome did not affect the biocompatibility of IONPs as the detected cytotoxicity did not significantly changes when cells were exposed under both conditions (Additional file 1: Fig. S2). To further characterization of biocompatibility of novel multifunctional NP, cell cycle and apoptosis were determined by flow cytometry (Fig. 5B). IONP-Pt and IONP-Pt-Flu were tested (0.1 mg/mL) at 24 h of incubation using a combination of annexin V and propidium iodide (PI) in order to outline the profile of drug-induced cell death. In addition, the cell cycle analysis will provide a view of possible effects at the different phases. Platinum derivatives induce a cycle arrest in the G2/M phase preventing the correct DNA replication due to their crosslinking effect and formation of adducts between the DNA base pairs [28]. As depicted in Fig. 5B, both tested cell lines show a different behavior in the cell cycle; however, it appears that in both cases the NPs induce an equal response. About apoptosis, it is slightly increased in both cell lines under the assay conditions, which are set up according to obtained results about the cell viability experiments (Fig. 5A). Bearing in mind the NP functionalization, no significant variation is observed between IONP-Pt and IONP-Pt-Flu in either of the two tested cell lines. Regarding the cell cycle analysis, an increment of cells in G2/M phase was observed in the Caco-2 cells with both studied NPs; and similarly, in Jurkat cells. Overall, these results perfectly match with expected mechanism of action for Pt derivatives. In summary, the preservation of the apoptotic response as well as minimal variations in the cell cycle suggest that the multifunctionalities of the IONP-Pt-Flu does not have an impact on drug release from the IONPs nor a variation in the pharmacological response.
Proteomics approach for deciphering the intracellular biology of multi-functional NPs
Further analysis, it is focused on the exploration of intracellular pathways by systematic proteomics characterization. Intracellular study of the activity carried out by the NPs is necessary to confirm that the Pt derivative has been successfully released and also to identify the expected and novel protein targets. Deciphering the perturbations in the signaling pathways linked to this compound are key to know if there are modifications in the release process of the pharmacological load of the NP. Also, they are useful to know the implications that the functionalization of the NP may cause in the intracellular signaling pathways on the cells of interest.
Proteomics is a precise and suitable technique to understand the behavior of cellular stimuli, providing a general and detailed overview of the proteins involved in signaling pathways, organelle activation or cellular communication processes; despite the potential of proteomics approaches, it is required to design experimental procedures focused on the analysis of targeted proteins or proteome landscape. Here, in this study, a technique based in protein labeling with non-canonical amino acid azidohomoalanine (AHA) provided the ability to specifically and selectively detected of de novo synthesized proteins uniquely related to intracellular pathways triggered by NPs. Tumoral cells lines (Caco-2 and Jurkat) were exposed to the functionalized NPs including in the cell culture medium AHA amino acid that was integrated into the nascent proteins, as described in materials and methods section. Further bio-orthogonal chemistry between the amino acid and a copper-catalyzed azide–alkyne ligation allowed isolation of tagged proteins (which are newly synthetized). A column previously prepared with a resin functionalized with an alkyne derivative allowed the specific capture of the labeled proteins. This methodology also allowed in situ trypsin digestion of the newly synthetized proteins, directly providing trypsin digested and purified peptides (Fig. 6).
At first glance, the global proteome is analyzed in order to distinguish differences across several studied tumoral cell lines and NPs of interest. Attending of the total number of proteins in each condition and cell lines, a significant quantitative difference can be observed in the total number of proteins depending on the cell line, being in general twice as many proteins detected in the colorectal cancer cell line (Caco-2) than in the hematological (T cell leukemia) tumor cells line (Jurkat) (Fig. 7A), (according to the functional enrichment of Gen Ontology-Biological Process (GO-BP)). This difference becomes even more remarkable when observing the effect triggered by the IONP-Pt, where in the Caco-2 cells a fourfold higher number of proteins were detected than in the Jurkat cells (Fig. 7B).
Considering this general analysis provided by the high-content proteomics characterization, a simplification of the functional enrichments has been carried out to facilitate the biological outcome from cell signaling pathways comparison. The functional enrichments were simplified into broader functional groups according to Lin’s semantic similarity > 0.8 using REVIGO method [29]. The simplified functional commonalities were assessed only for the proteins de novo synthesized at Caco-2 and Jurkat cells at different IONPs conditions and controls (Fig. 7C right). In this analysis, the differences between tumor cell lines are minimized. Nevertheless, this proteomic approach allows us to discern certain differences in the pharmacology of the tumor cell lines. Interestingly, differences between cell lines were found in the most functionalized NP (IONP-Pt-Flu), where a higher number of functions (more than double) were detected in the hematological tumor line compared to the Caco-2 cell line. This phenomenon is explained by the different functional specialization of the cells. The adaptive immune system is composed of highly-specialized systemic cells. The functional specialization is patent when considering the total number of proteins identified in the assays and the respective universe of functions annotated in GO repository for the proteomes of Jurkat and Caco-2 cells. We have corroborated that the initial functional annotations present almost the same hierarchical depth; thus, the semantic simplification did not bias the results in Additional file 1: Fig. S4. On this basis, we claim the difference observed in functional enrichment of the two cell lines reflects the difference in the level of functional specialization between Jurkat and Caco-2 cells.
The potential pharmacological features of these novel nanodrugs might be described by the selective and specific qualitative functional enrichment analysis of the newly synthetized proteins in studied conditions. Moreover, it was possible the one-step and simultaneous global analysis of intracellular effects related to the multi-functionalities of the IONPs. The analysis of the 15 most important biological functions in the Caco-2 control line are in accordance with the reported and expected for this type of solid tumor (Fig. 8, Additional file 1: Table S3). It has long been known that in colon cancer, there is an alteration of the anaphase-promoting complex (APC) involved in the control of the transition of the cycle to G2/M phase. This also has implications in the activation of the Wnt pathway [30]. Amyloid-beta clearance [31], regulation of folding and stability of proteins as wells as RNA metabolism are known to be disrupted in colorectal cancer [32, 33]. When cells were treated with IONP-Pt, the protein expression profile has changed according to the effect of Pt-derived compound conjugated with the IONP. The activation of cellular functions such as “DNA replication” (including proteins such as TOP1A) are consistent with the pharmacological effect of Pt as a DNA crosslinker. Also interesting is the activation of the “purine ribonucleotide metabolic process” function where the PARP is significantly observed which is a protein related to mechanisms of repairing DNA damage. Likewise, the “chaperone-mediated autophagy” function is related to the endoplasmic reticulum stress mediated by platinum compounds as it reveals the appearance of the protein HSP90. Regarding the multifunctional IONP, general cellular processes related to Rho GTPase family are observed. Other previous functions of the analysis are observed (both actin and axon are mediated by the Rho GTPase family). Also, FEA is not highlighting any other pathways that can be related to cytotoxicity directly related with the multifunctional properties of these IONPs. Thus, by these studies, it is confirmed, in Caco-2 cells, the release of Pt compound which makes feasible the specific molecular targeting, and it is also observed that the influence of the multi-functionality with respect to the pharmacological effect is not affecting a successful drug disposition.
The analysis in the hematological cell line yielded very similar conclusions about the behavior of multifunctional NPs and the pharmacological effect of the platinum derivative. The basal state of the analyzed Jurkat cells as a control condition resulted in the appearance of 15 most relevant functions related to activation of the immune system as well as alterations in cell replication. These functions are in accordance with the nature of cell (T lymphocyte) and the tumoral pathology (Fig.8, Additional file 1: Table S3). In cells exposed to IONP-Pt, the function related to non-homologous recombination of damage repair underlined, which is directly linked to the action of the platinum compound. According to the specific proteomic data obtained, in this cell line it is due to activation of histones as well as DNAPKcs kinase. The oxidative stress mediated by the Pt compound is reflected in the “ER-nucleus” function where EIF2S1 appeared. Also highlights again HSP90 protein in this group; being both proteins known as key factors in response to cellular stress. Similarly, these proteins are also part of the function “response to unfolded protein” which is also related to oxidative stress. Regarding the alkylating power of platinum derivatives, in this case also analysis provided “peptide crosslinker” as a leading function. Coupled with the DNA alkylation, this process represents a cytotoxic mechanism of platinum derivatives. As for the IONP-Pt-Flu, results linked to DNA damage were also obtained, such as the impairment of nucleotide synthesis where PARP appears detected. Likewise, the mini-chromosome maintenance (MCM) proteins family also appear within the “nuclear DNA replication” function, which are responsible for the formation of the nuclear replication complex. Likewise, HSP90 participates in the regulation of reactive oxygen species. This more specific analysis of the different conditions in the Jurkat cells has resulted in a correlation of functions in accordance with those proposed for the colorectal cancer assay (Fig. 8).
The main functions associated with the drug are maintained independently for both IONP-Pt and IONP-Pt-Flu. Therefore, a low number of significantly detected proteins is maintaining a high number of similarly functions, which mainly be related to redundant, pleiotropic, cooperative and synergically effect of these proteins on the immune system and its response. Bearing in mind the results from functional proteomics characterization, it is observed a correct drug delivery to the molecular target as far as the IONP is concerned and no significant influence of the fluorochrome is appreciated that could interfere in the pharmacokinetic process. In summary, the proteomic technique chosen to decipher the intracellular signaling of these novel multi-functionalized IONPs can be considered very suitable, since it has been able to differentiate the implications of each of the NP components. Likewise, the bioinformatic analysis performed on the data has been highly enriched. General and detailed analytical results have been tightly correlated between cell lines as well as coherent with the studied nanotechnological platform.
In vivo evaluation of NPs activity
To conclude with the integral characterization of the NPs and their biocompatibility, an initial in vivo approach has been carried out. IONPs and CisPt precursors were tested using rabbit as an animal model. In this case, a liver tumor by implantation of fragments of VX2 tumors (squamous cell cancer line) were developed to test the efficacy of platinum compounds and the corresponding nano-drugs conjugates. For this test, apart from BilPt (biliar platinum complex, see Fig. 1) and IONP-Pt, [Pt(Cl)2(NH3)] cisplatin compound (CisPtCl) and the corresponding IONPt-CisPtCl (positive controls), IONPs (negative control) were also included in the screening.
When the animals were intervened, the tumor mass was allowed to grow until 10 days later, then treatment with drugs was started. After 8 days of treatment initiation, the animals were sacrificed. The localization and follow-up of the tumor was carried out by periodic measurements with ultrasound scans, finally verifying the correspondence between the images and the tumor after excision of the tumor mass (Fig. 9).
Histological examination of the samples yielded interesting results about the compound studies and the corresponding nano- drugs conjugates (Fig. 9C and Additional file 1: Table S3). The saline solution used as vehicle (negative control) as well as the administration of the naked/plain IONPs (without functionalization) resulted in tumors of large volume and similar size (Fig. 9C). This demonstrates the function as drug carriers of these NPs, with no other cytotoxicity per se. The CisPtCl used as a positive control resulted in the appearance of a minimal tumor mass or the absence of tumor in the animal (Fig. 9C). Treatment with the standard CisPtCl was able to reduce tumor mass to a volume 40-fold less than the negative control (Fig. 9C). Similar reduction was observed when the BilPt was administered, indicating an outstanding efficacy of this product. When the standard CisPtCl was encapsulated in the IONPs, similar reductions were found with respect to the product alone, thus indicating the effectiveness of IONPs as suitable drug carriers. The administration of IONP-Pt did not achieve the values of the positive control nevertheless, it was still able to reduce the tumor mass to one third of the mass developed without treatment (Fig. 9C).