1 Introduction

Over the past few decades, notable new therapies and technologies have emerged in the field of nanotechnology, particularly for applications in medicine and pharmaceuticals. Nanotechnology pertains to the deliberate manipulation of materials at the nanoscale, a practice renowned for its ability to augment the existing properties of materials while concurrently introducing novel attributes. Nanomaterials, characterized by an average particle size of approximately 100 nm, comprise a diverse spectrum of substances, including both organic and inorganic nanoparticles, nanoparticulate ceramics, fullerenes, and carbon nanotubes (CNTs) [1].

Carbon nanotubes are among the most promising nanomaterials. Recent advancements have seen the development of biologically compatible CNTs, a significant achievement in nanotechnology that has attracted considerable attention in recent years [2]. The functionalization of CNTs through covalent and noncovalent methods has opened avenues for precise targeted drug delivery in the field of nanomedicine. Furthermore, CNT functionalization offers a range of advantages, including enhanced cellular internalization and improved stability within biological environments. These attributes hold significant promise for the biomedical and pharmaceutical research sectors and industries, particularly in terms of diagnostic and therapeutic delivery [3].

CNTs, as remarkable nanomaterials with a diverse range of applications, exhibit immense potential in the field of immunology and have been proposed as candidates for manufacturing nanovaccines since they can be bioconjugated with peptides and proteins: in this way, CNTs carry antigens, promoting an efficient immune response[4,5,6,7,8]. A robust immune response against tumors can be effectively triggered through a combination of therapeutic strategies that involve the use of tumor antigens in conjunction with immune modulators designed to counteract the immune suppression induced by the tumors [9]. Currently, nanomaterials combined with tumor-derived antigens used in immunotherapy have shown biocompatibility, permeability, and specificity. Furthermore, some nanomaterials are antigenic per se and can stimulate the immune system, acting as adjuvants[10].

The identification of overexpressed tumor antigens is essential for correctly establishing cancer vaccine immunotherapies. Of particular interest is the fucosylation pattern, a type of protein glycosylation that plays a key role in the development of OvCa. The expression of fucosylated proteins such as haptoglobin has been observed [11, 12]. In addition, increased expression of fucosyltransferases in transformed ovarian tissues and cell lines has also been described by Escrevente et al. [13]. Fucosyltransferase-4 (FUT4) is an enzyme indispensable for type II Lewis antigen synthesis, and its overexpression has been reported in leukemias and colon, breast, pancreas, lung, and gastric cancers [14,15,16,17,18,19]. Furthermore, FUT4 has been found to be related to epithelial-mesenchymal transition, proliferation, and metastasis during cancer development [17, 20, 21]. Thus, FUT4 is proposed as a possible therapeutic target to explore its use as an antigen in generating immunotherapies against ovarian cancer. Among gynecological cancers, OvCa is the seventh most common and has the highest mortality rate. Although OvCa is less prevalent than breast cancer, it is three times more lethal [22, 23]. Tumor-associated macrophages (TAMs) are important innate immune system cells with various physiological roles in the OvCa microenvironment. Macrophages can become either traditionally activated M1 macrophages or alternatively activated M2 macrophages in response to various stimuli. TAMs are regarded as M2-like in malignancies and support almost all characteristics of cancer by creating a significant quantity of growth factors, extracellular matrix remodeling molecules, and cytokines that promote cancer development, migration, and angiogenesis, whereas M1 macrophages have been reported to act as antitumoral cells that enhance the immune response to cancer cells, promoting tumor antigen presentation to T cells [24].

We hypothesize that the bioconjugation of carbon nanotubes with tumor peptides will potentiate and generate an antitumor immune response and, as part of this, activate and polarize macrophages to a proinflammatory M1-type profile, which would have an antitumor role in cancer.

Thus, this study aimed to synthesize and characterize CNTs bioconjugated with FUT4-derived peptides and to evaluate their cytotoxicity and biological interactions in J774A.1 macrophages and ovarian cancer SKOV-3 cells.

2 Results

2.1 FUT4-Derived T-Cell Epitopes Have MHCI/II Affinity

We designed a multiepitope peptide that interacted with the haplotypes H2-Db and H2-Kb in the C57BL/6 mouse, which was used as an experimental immunocompetent ovarian cancer model [25]. Three predicted epitopes with the lowest ∆G for MHC I, three with the lowest ∆G for MHC II, and promiscuous epitopes were chosen for docking. Additionally, to confirm that MHC I epitopes were generated by proteasomal cleavage, they were analyzed in the PCPS server (Table 1; http://imed.med.ucm.es/Tools/pcps/).

Table 1 Immunoinformatic predictions of affinity peptides derived from FUT4
Full size table

All epitopes for MHC I were found to exhibit 100% proteasomal processing (Table 1). This is essential since the proteasome is responsible for proteolytic degradation and MHC I presentation [26]. Epitopes RVRYYHQL, LWRNAFLAGAVPVVL, and RYFRWRRSFAVHITS were discarded because their interaction sites did not coincide with the specific MHC pocket [27]. The final epitopes selected were (1) LAFENSRHV, (2) ARYKFYLAF, and (3) RRYFRWRRSFAVHIT. MHCI/II-peptide docking was conducted, and the results are depicted in Fig. 1.

Fig. 1
figure 1

Protein–protein docking. A ARYKFYLAF vs. MHC I, B LAFENSRHV vs. MHC I, C RRYFRWRRSFAVHIT vs. MHC II. The MHC hydrogen bonds are shown. In the upper quadrant, the peptide-MHC structure is observed in a simplified form.

Full size image

These peptides were conjugated in a multiepitope peptide with serine (S) at the -C and -N termini to serve as a link for bioconjugation. The peptide sequence was as follows: SARYKFYLAFENSRHVDYRRYFRWRRSFAVHITSFWS (designed as PEP37).

2.2 PEP37 Was Correctly Bioconjugated to P-CNTs

Previously, for bioconjugation, we synthesized unpurified CNTS (UP-CNTs), purified them (obtaining P-CNTs), and characterized them to determine their physicochemical properties and ensure their purification, then performed bioconjugation and characterized the resulting f-CNTs). UP-CNTs showed variations of micrometers in length (Fig. 2A), which decreased after the purification process to < 1 μm (Fig. 2B). Electrodense regions present in the UP-CNTs, attributable to amorphous carbon or catalyst impurities, disappeared in the P-CNTs [28]. The length and diameter of the f-CNTs did not show apparent differences from P-CNTs; nevertheless, the length distribution revealed that f-CNTs were smaller than P-CNTs (Supplementary Figure 3A), and the aspect of their surface was quite different, showing an amorphous structure surrounding the CNT walls, which was attributed to the bioconjugated PEP37 (Fig. 2C).

Fig. 2
figure 2

High-resolution scanning electron microscopy of CNTs. Zoom X50,000 and X200,000 (upper charts) in transmitted and secondary electron modes (right and left, respectively). A UP-CNTs, B P-CNTs, and C f-CNTs.

Full size image

The stability of the nonconjugated and bioconjugated CNTs was analyzed, showing a decrease in ζ potential in f-CNTs (-27, SD ± 0.7) compared to the nonconjugated P-CNTs (-24.15 SD ± 0.7 1.15); this difference relates to an increase in stability and a decrease in agglomeration associated with the dispersion in aqueous medium (Supplementary Figure 3).

The changes in the f-CNT surface compared to the P-CNT surface were observed by atomic force microscopy (AFM) and high-resolution transmission electron microscopy (HR-TEM). The P-CNTs showed smooth walls with a regular molecular layer < 10 nm and little or no amorphous material (Fig. 3A); in contrast, the f-CNTs showed amorphous material in their structure (Fig. 3C, outlined by a red dotted line). The AFM results revealed the height of CNTs, which are displayed as a heatmap (brown–yellow from low to high); P-CNTs (Fig. 3B) showed a regular surface structure with a maximum height of 14 nm corresponding to the plane structure of P-CNTs, whereas the f-CNT surface showed an irregular rough structure with a maximum height of 47 nm (Fig. 3D), resulting from the bioconjugated peptides on the f-CNT surfaces. Distortion of the CNT surface was evidenced in the Raman spectra (Fig. 3E). The typical Raman spectra of CNTs are shown in Fig. 3E as a black line, the D band (defects), and the G band (graphite). The obtained IG/ID ratio describes the structural defects on the CNT- surface. The IG/ID ratios of f- and P-CNTs are lower than those of UP-CNTs (0.41, 0.43, and 0.86, respectively), indicating different functional groups [29,30,31,32]. Multiple bands between 400 and 1200 cm−1 in the Raman spectrum of f-CNTs coincided with signals obtained from the Raman spectrum of the peptide alone (Fig. 3E, pink line).

Fig. 3
figure 3

Physicochemical characterization of CNTs. HR-TEM and AFM images of P-CNTs (A and B) and f-CNTs (C and D), amorphous material on the surface of f-CNTs (red dotted line, C), which is not present in the P-CNTs (A). Raman spectra displaying the D (yellow box), G (pink box), and G’ (green) bands (E). ATR-FTIR of the different CNTs (F).

Full size image

The attenuated total reflectance by Fourier transform infrared (ATR-FTIR) spectra of the UP-CNTs showed no peaks (Fig. 3F); after the purification process, P-CNTs displayed several peaks corresponding to C-O (1100 cm−1) and O-H (3000–3500 cm−1), which are products of the oxidation process[33]. In f-CNTs, the O-H peaks increased, and two signals corresponding to amine I (1650 cm−1) and II (1450 cm−1) appeared [34]. Peaks corresponding to the C=O bond (1700 cm−1) and C-O bond (1100 and 1300 cm−1) were also detected on f-CNTs, corroborating ester bond (COO-R) formation. C-N, C-H, and amine signals that appeared in the peptide spectrum (Fig. 3F, purple line) were also detected in f-CNTs.

Protein quantification revealed no protein in the P-CNTs, whereas in the f-CNTs, 7.77 mg of protein/100 mg of f-CNTs was measured, an efficiency of ~77%. XPS analysis of the different CNTs (Table 2) showed that the UP-CNTs consisted of carbon and a low amount of oxygen, whereas in the P-CNTs, oxygen increased to 11.81% in weight. In the f-CNTs, the oxygen rose even more, and nitrogen and sulfur appeared in the analysis, resulting in a 7.62% increase in weight attributable to the bioconjugated peptide, which is consistent with the protein quantification.

Table 2 X-ray photoelectron spectroscopy (XPS) analysis of different CNTs
Full size table

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and silver staining revealed at least three different protein bands for f-CNTs (> 250, 90, and 50 kDa) (Fig. 4A). This result suggests polypeptide formation because free COOH can react with other OH groups in the same or another peptide, forming a polypeptide structure. Thus, the molecular weight depends on the CNT length, as shown in Fig. 4B. Controls without enzyme (wo/Enz) or peptide (wo/Pep) did not show protein in the samples (Fig. 4A).

Fig. 4
figure 4

A SDS–PAGE of the f-CNTs) and B a representative scheme of the length of the CNT-dependent amount of peptide of the three bands. C Kinetic release of PEP37 after treatment with esterase enzyme. The upper inset in the graph shows the silver-stained SDS–PAGE of the released peptide. MWM, molecular weight marker; f-CNTs, bioconjugated CNTs; wo/Enz, control without enzyme and wo/Pep, control without peptide.

Full size image

We performed a kinetic release of PEP37 from f-CNTs by enzymatic hydrolysis. Fig. 4C shows that PEP37 was released from the f-CNTs beginning 15 min after treatment, and the amount released continued to increase over time.

2.3 Bioconjugated CNTs Did Not Induce Cytotoxicity in SKOV-3 or J774A.1 Macrophages

Cytotoxicity analysis in SKOV-3 cells, used as a human cancer model, showed that UP-CNTs significantly decreased cell viability at all concentrations when evaluated after 1 h of interaction (Fig. 5B). At 24 h, 60 μg/mL UP-CNTs caused a 50% reduction in cell viability, which was dependent on reactive oxygen species (ROS) production (Fig. 5C). P-CNTs also affected cell viability and ROS production but to a lesser degree.

Fig. 5
figure 5

Cytotoxicity assays in the SKOV-3 cell line. A Annexin V-PI apoptosis assays. B Cell viability at 1 h of interaction, C cell viability at 24 h of interaction, and D ROS production assay. Each bar represents the mean of three independent experiments performed in triplicate ± SD. *, difference vs. negative control without stimulus: # difference vs. death control (5% DMSO), p < 0.05 ANOVA and Dunnett. E Representative images of actin cytoskeleton disruption. In DIC images, yellow arrows indicate CNTs inside cells stained with DAPI (nuclei-blue) and AF488-phalloidin (F-actin-Green). DIC, differential interference contrast imaging.

Full size image

The f-CNTs showed a nonsignificant cell viability reduction at concentrations < 6 μg/mL. At 60 μg/mL, f-CNTs decreased cell viability by 15%, which was not dependent on ROS production (Fig. 5D). Additionally, we analyzed live cells, apoptosis, and necrosis at 6 μg/mL and found significant differences in the presence of any of the CNTs, suggesting that concentrations < 6 μg/mL did not induce apoptosis in SKOV-3 cells (Fig. 5A and Supplementary Figure 1A).

The actin cytoskeleton was analyzed to evaluate whether the cytotoxic effect of CNTs was due to disruption of actin polymerization, as reported previously for the SKOV-3 cell line [35]. The UP-CNTs induced cytoskeleton disruption with peripheral circle reorganization even at 6 μg/mL (Fig. 5E); the differential interference contrast (DIC) images confirmed that UP-CNTs localized in needle-like forms in the cytoplasm and nucleus. The P-CNTs caused similar cytoskeleton alterations, but only at the highest concentration; DIC images revealed intracellular and perinuclear bundles of P-CNTs (Fig. 5E, arrows). Interestingly, f-CNTs did not cause actin cytoskeleton reorganization (Fig. 5E); DIC images suggest that the f-CNTs did not enter the cells as the other CNTs did.

The specific uptake of f-CNTs by antigen-presenting cells (APCs) was analyzed in murine J774A.1 macrophages. The same experimental protocol for SKOV-3 was performed to evaluate cytotoxicity.

At 1 h of interaction, UP-CNTs and P-CNTs significantly decreased cell viability by 0.6 and 60 μg/mL, respectively (Fig. 6B). In contrast, f-CNTs did not show a significant difference in cell viability even at 60 μg/mL (Fig. 6C). At 24 h, the UP-CNTs decreased cell viability at 6 and 60 μg/mL (Fig. 6C). Neither P-CNTs nor f-CNTs significantly reduced cell viability at any of the concentrations evaluated when interacting with macrophages; however, a small increase in cell viability was observed in macrophages exposed to 0.6 μg/mL f-CNTs (Fig. 6C).

Fig. 6
figure 6

Cytotoxicity assays on the J774A.1 macrophage line. A Annexin V-PI apoptosis assays, B cell viability at 1 h of interaction, C cell viability at 24 h of interaction, D ROS production assay. Each bar represents the median ± SD of three independent experiments performed in triplicate. *, difference vs. negative control without stimulus; # difference vs. death control (5% DMSO), p < 0.05 ANOVA and Dunnett’s test. E Representative images of actin cytoskeleton disruption. In DIC images, yellow arrows indicate CNTs inside cells stained with DAPI (nuclei-blue) and AF488-phalloidin (F-actin-Green). DIC, differential interference contrast imaging.

Full size image

ROS production (Fig. 6D) by macrophages interacting with CNTs was lower than that of the SKOV-3 cell line but showed no significant differences; an increase was observed only in macrophages exposed to 60 μg/mL UP-CNTs or P-CNTs. These results suggest that the cytotoxicity of CNTs to macrophages is not induced by ROS (Fig. 6D). To confirm the type of cell death, we analyzed apoptosis and necrosis (Fig. 6A and Supplementary Figure 1A and B). Macrophages exposed to UP-CNTs and P-CNTs displayed a decrease in cell viability and an increase in late apoptosis at 6 μg/mL. On the other hand, we did not observe any cell death in macrophages exposed to f-CNTs (Fig. 6A and Supplementary Figure 1B).

Compared to UP-CNTs or P-CNTs, which induced pathological rearrangement in the actin cytoskeleton of macrophages, f-CNTs induced the conventional morphology of activated macrophages (Fig. 6E).

2.4 Uptake of f-CNTs by Macrophages Is a Time-Dependent Process

To elucidate the macrophage f-CNT uptake mechanism, we analyzed lysosome formation after f-CNT interaction with cells. Lysosome formation increased after interaction with f-CNTs at 15, 30, and 60 min (Fig. 7A and B). Fluorescence analysis of confocal images showed a significant increase in lysosome formation after 30 min of interaction with f-CNTs (Fig. 7B). These data suggest that the uptake of f-CNTs by macrophages is regulated by endosome formation, and consequently, lysosome development is a time-dependent event.

Fig. 7
figure 7

Macrophage uptake of f-CNTs. A Lysosome formation in macrophages after f-CNT interaction for 0, 30, and 60 min. B Arbitrary units of fluorescence. Each bar represents the median of arbitrary fluorescence units of twenty images independently analyzed. *, a significant difference vs. medium; **p < 0.005; ns, no significant difference. C Cellular localization of f-CNTs in macrophages. D Cellular localization of f-CNTs in SKOV-3 cells.

Full size image

The endocytosis of f-CNTs was monitored using an anti-f-CNT antiserum (Supplementary Figure 2) to localize f-CNTs inside cells. In permeabilized macrophages, f-CNTs were observed in vesicle-like structures, suggesting phagocytosis-mediated uptake (Fig. 7C). The FITC signal on the surface of nonpermeabilized macrophages (Fig. 7C) suggests MHC antigen presentation; nevertheless, additional experiments are necessary for confirmation. SKOV-3 cells did not show a signal inside the cells, confirming that f-CNTs did not enter the cells by passive diffusion (Fig. 7D).

2.5 f-CNTs Activate and Polarize Murine Macrophages to a Proinflammatory M1 Phenotype

Macrophage polarization was evaluated by analyzing M1 and M2 membrane markers and cytokine secretion patterns by flow cytometry. Assays on J774A.1 macrophages revealed that f-CNTS significantly increased CD80 expression at 0.1 μg/mL and CD80, CD86, and MHC II at 6 μg/mL, which is consistent with M1 subset markers (Fig. 8A, B, and C). Controls with LPS increased the M1 subset markers to a similar level. In cells exposed to P-CNTs or the peptide alone, significant changes in M1 subset markers were not detected. Regarding M2 polarization markers, no significant differences were observed in CD163 or CD209 after 24 h of interaction with P-CNTs or peptide alone (Fig. 8D and E). Additionally, P-CNTs and the peptide were used as controls to determine if the peptide or the P-CNTs per se induced the response. Differences in the markers were not detected with either P-CNTs or the peptide alone.

Fig. 8
figure 8

M1 and M2 polarization assays on J774A.1 macrophages. A CD80, B CD86, C MHC II, D CD209, and E CD163 expression by flow cytometry. F Nitrite production, G and H ARG-1 expression by Western blot. I Cytokine quantification by flow cytometry. J IFNγ/IL10 ratio. The normalized expression was calculated as the mean fluorescence intensity normalized to 100% in the nonstimulated cells. Each bar represents the mean ± SD of three independent experiments with duplicates. LPS, lipopolysaccharide; PEP, peptide alone. *, a significant difference vs. medium; **p < 0.005; ns, no significant difference.

Full size image

Nitrite production is related to nitric oxide production in stimulated macrophages; in macrophages exposed to 6 μg/mL f-CNTs for 24 h, a significant increase in nitrite was observed (Fig. 8F), suggesting the activation of inducible nitric oxide synthase (iNOS). In contrast, the expression of ARG-1, which is associated with M2 macrophages, was decreased in macrophages exposed to f-CNTs, which is consistent with M1 subset markers (Fig. 8 G and H).

To confirm that f-CNTs were recognized and internalized by macrophages and consequently able to elicit an immune response, we analyzed the cytokines secreted by cells that interacted with 6 μg/mL f-CNTs for 24 h. A proinflammatory cytokine pattern consistent with an M1-like macrophage marker subset was detected in f-CNT-treated macrophages (Fig. 8I), with a significant decrease in IL-4 and IL-10 and an increase in IL-6. Additionally, a tendency for IFN-γ, IL-2, and the IL-10/IFN-γ ratio to increase was observed (Fig. 8J), suggesting polarization toward M1-like macrophages.

Our results suggest that f-CNTs induced macrophage activation, represented by an M1-like subset of markers and that this induction was dependent on the bioconjugate, since the P-CNTs or peptide alone were unable to modify these markers. Additionally, f-CNTs potentiated the response since, even at low concentrations, such as 0.1 μg/mL, they increased CD80 expression.

3 Discussion

3.1 Peptide Design and f-CNT Physicochemical Characterization

The potential immunogenicity of FUT4 has not been studied before. However, using bioinformatic approaches, we designed a multiepitope peptide based on the FUT4 mouse sequence, which induced a good antibody production response. For this purpose, we considered the mouse C57BL/6 haplotypes (H2-Db and H2-Kb), a widely used immunocompetent mouse model of ovarian cancer [25, 36, 37].

A critical issue with using a nanomaterial in nanomedicine is correct characterization; here, we fully characterized the f-CNTs and confirmed the appropriate peptide bioconjugation of 8% w/w by an enzymatic method. The physicochemical characterization was performed in the same way as other research groups that have bioconjugated peptides and proteins. Ng et al. (2016) bioconjugated CNTs with insulin via diimide-activated amidation and analyzed the structure of the bioconjugated CNTs by TEM and STEM, showing that the insulin was displayed on the surface of the CNTs as spherical and amorphous materials, similar to our results [38]. Similar AFM results have been reported by Muzi et al. (2016) and Fu et al. (2002) for bovine serum albumin-functionalized CNTs [39, 40], showing amorphous material attributable to the protein on the surface of the CNTs.

Furthermore, according to the SDS–PAGE results, the polypeptide structure of f-CNTs, being a large biomolecule, could be a suitable biostructure to increase interaction with the immune system, facilitating its recognition and phagocytosis by antigen-presenting cells [41].

3.2 Cytotoxicity Assays on SKOV-3 and J774A.1 Macrophages

Montes-Fonseca et al. (2012) reported a decrease in cytotoxicity in J774 macrophages exposed to P-CNTs, which was a purification-dependent process. Hydroxyl and COOH radicals introduced into P-CNTs by the purification process increased their solubility and reduced the amount of residual catalyst, directly impacting their cytotoxicity [28]. Our results corroborate these previous findings because f-CNTs did not decrease cell viability at the tested doses at 1 or 24 h of interaction in the SKOV-3 or J774A.1 cell lines. The ability of bioconjugation to as protein-decrease CNT cytotoxicity in in vivo models is critical for their potential biomedical application [42, 43].

Oxidative stress, mediated by ROS production, has been reported as a key mechanism in nanoparticle cytotoxicity [44,45,46,47,48]. Glutathione is the most important antioxidant for the self-protection of macrophages against ROS damage. In this work, macrophages produced less ROS than SKOV-3 cells exposed to CNTs. Nevertheless, Zhang et al. (2018) tested CNT size-dependent cytotoxicity and showed that CNTs induce high ROS production in macrophages followed by glutathione depletion, which confirmed the increase in ROS [41]. However, our results showed that high doses of UP- and P-CNTs increased ROS production, which was linked to reduced cell viability and cytoskeleton damage but did not occur in cells exposed to f-CNTs. These results agree with the findings of other authors. Gaddam et al. (2017) demonstrated that ROS production induced by carbon nanomaterials is directly related to their cytotoxicity, which is mediated by apoptosis induction. Additionally, they reported that these mechanisms are dependent on the cell type [48].

There are few studies regarding alterations induced by CNTs on the actin cytoskeleton. Zhang et al. (2020) studied the association between the actin cytoskeleton and CNTs in an in vitro model using SKOV-3 cells; they observed an association between exposure to raw CNTs and cytoskeleton and migration disruption [35]. In our work, comparable effects were found in SKOV-3 and J774A.1 macrophages exposed to UP-CNTs, but exposure to P-CNTs and f-CNTs showed smaller effects. Thus, f-CNTs did not induce disruption of the actin cytoskeleton in the cell, which was consistent with low ROS production and high cell viability. The last could be explained by the hydrophilic surface surrounding the peptide bioconjugated to CNTs and obstructing passive transport through the cell membrane. This could suggest that these f-CNTs enter the cell by a controlled pathway such as phagocytosis, decreasing the number of CNTs inside the cell and impacting their cytotoxicity.

The morphological changes induced in macrophages by UP-CNTs were clear: disruption of the cytoskeleton at 6 and 60 μg/mL and DAPI signal outside the cells, suggesting the presence of apoptotic bodies. The induction of apoptosis was confirmed by Annexin V assays; similar effects were produced by P-CNTs, but only at the highest dose of 60 μg/mL. Notably, the macrophages that interacted with f-CNTs showed morphological changes characteristic of activated macrophages, such as pseudopods, increased size, and increased vesicle formation [49]. These findings are relevant to the possibility of using f-CNTs as an adjuvant because recognizing and activating APCs is a critical step for activating the immune system.

3.3 Macrophage Uptake of f-CNTs, Activation, and M1 Polarization

An effective vaccine must induce a robust immune response, either at the cellular (by stimulating cytotoxic T cells) or humoral level (through antibody production). Regardless of the response, antigen recognition, internalization, processing, and presentation by APCs to T lymphocytes are needed. An essential factor in designing a cancer vaccine is that most antigens need additional molecules acting as adjuvants for complete processing, thus improving their stability and antigenicity [50]. Here, we propose using CNTs as stimulating particles to ensure an effective immune response against cancer antigens. The internalization of CNTs in cells could rely on direct translocation by the insertion of nanoparticles or on diffusion by endocytic–phagocytic mechanisms [51, 52]. Additionally, CNT uptake depends on the CNT synthesis method, size, and functionalization [53]. Several reports of CNT uptake agree that CNT uptake depends on the nature of the CNTs; therefore, it is crucial to characterize the uptake of f-CNTs in phagocytic cells.

Regarding cancer vaccines, the endocytic pathway is an effective and controlled way to release a nanovehicle. In this work, we bioconjugated the peptide through an ester bond [29], which can be processed later by a lysosome esterase for release inside the cell cytoplasm, allowing antigen presentation. Our results showed that f-CNTs induce morphological changes related to activation in macrophages; additionally, we proved that f-CNT uptake is related to lysosome formation in a time- and cell type-dependent manner because f-CNTs did not enter SKOV-3 cells. The activation and polarization of macrophages in the tumor microenvironment are key to evoking an antitumoral or protumoral response. M1 macrophages have been considered antitumoral, while M2 macrophages are protumoral [54]. In this way, we analyzed the role of f-CNTs in macrophage polarization. The results highlighted an increase in proinflammatory cytokines and a decrease in anti-inflammatory cytokines. Contrasting results have been reported regarding M1/M2 polarization by CNTs. Kinaret et al. (2020) reported a mixture of M1/M2 macrophage activation with nonbioconjugated CNTs [55], whereas Meng et al. (2015) reported similar results, an M1/M2 mixture that is capable of initiating phagocytosis and upregulation of CD14, CD11b, TLR-4/MD2, and CD206, and in contrast to our results, they reported that MHC II expression was not altered [56]. Thus, we presume that the bioconjugated peptide in the f-CNTs used in our study is responsible for macrophage M1 polarization. CD80, CD86, and MHC II were upregulated without changes in M2 markers, suggesting that these macrophages are M1 polarized.

On the other hand, Firouzi-Amandi et al. (2018), using a different approach related to tissue regeneration, analyzed the opposite effect of polarizing macrophages to the M2 subtype using chrysin encapsulated with PLGA-PEG nanoparticles; they showed a decrease in the inflammatory function of macrophages previously activated by LPS/IFN-γ, which was demonstrated by the reduction in proinflammatory cytokines (TNF-α, IL-1β, and IL6), in addition to an increase in the expression of ARG-1 and a decrease in iNOS. Our work shows a completely opposite pattern, which demonstrates polarization toward a proinflammatory M1-type profile[57].

Wu et al. (2019) reported that oxidized CNTs prevent tumor metastasis in Lewis lung carcinoma (LLC) or B16F10 melanoma-bearing mice by switching M2-polarized tumor-associated macrophages (TAMs) to M1 via TLR4 activation. In that paper, the authors reported an increase in iNOS and CXCR10 (M1 markers), a decrease in CD206 and arginase (M2 markers), and an increase in Th1 cytokine expression with a reduction in Th2 cytokines. Finally, they attributed this M1 polarization to the TLR4/NF-kB p65 signaling pathway and suggested that using multiwalled CNTs to target TAMs is a potential therapeutic approach against tumor metastasis[58]. In our work, we did not explore the role of CD206 or CXCR10; nevertheless, our data suggest an evident M1-like activation pattern in macrophages. Moreover, we provide explanations for the role of CNTs in the M1 polarization process induced by tumor antigens and, thus, their potential role in cancer immunotherapy.

4 Conclusion

The results confirm that the designed FUT4-derived peptide was bioconjugated to CNTs with high efficiency (>70%), and the resulting f-CNTs had low cytotoxicity to SKOV-3 and J774 cells and induced activation and M1-like polarization in macrophages. These findings are relevant in cancer immunotherapy because M1 macrophages promote an anticancer response. These results suggest that f-CNTs can be considered a safe adjuvant and nanocarrier system for cancer immunotherapy. Nevertheless, additional experiments are necessary to confirm their immunological and anticancer profile in an in vivo model.

5 Experimental Procedures

5.1 Immunoinformatic Analysis of Fucosyltransferase-4

The mouse FUT4 protein sequence was obtained from the NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/, accession: AAI37589.1, GI: 187953611).

Peptides with an affinity for MHC I and MHC II were predicted using the NetMHC 4.0 server (https://services.healthtech.dtu.dk/service.php?NetMHC-4.0)[59] and NetMHCII 2.3 server (https://services.healthtech.dtu.dk/service.php?NetMHCII-2.3)[60], respectively. The immunogenicity of these peptides was corroborated in the IEDB Analysis Resource server (http://tools.iedb.org/main/).

Sequences with the highest affinity were modeled in the PEP-FOLD server (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/)[61] and protein–protein docked with MHC-I and MHC-II using ClusPro 2.0 (https://cluspro.org/help.php)[62]. The crystallographic structures used were PDB ID 1JUF for MHC I and PDB ID 1LNU for MCH II. Peptides with the best immunogenic characteristics were used to design a multiepitope peptide (PEP37) and synthesized in GL Biochem (Shanghai, CN).

5.2 CNT Synthesis and Purification

Raw unpurified CNTs (UP-CNTs) were synthesized by spray pyrolysis, as described by Aguilar-Elguézabal et al. (2006) [63] and subsequently purified (P-CNTs) using the method of Saito et al. (2002) [64] with modifications described by Montes-Fonseca et al. (2012) [28].

5.3 Bioconjugation of PEP37 on P-CNTs

Enzymatic bioconjugation of PEP37 on P-CNTs was carried out as described (Guzmán-Mendoza et al., 2022) [29] with the corresponding enzyme reaction controls: (i) CNTs and peptides in the absence of enzyme or (ii) CNTs with the enzyme in the absence of peptide.

To demonstrate the enzymatic release of the peptide, release kinetics were performed using the same enzyme but under physiological conditions (PBS, 37 °C, pH 7.4) to promote the lipase activity of the enzyme, and measurements were performed at 0, 15, 30, 60, 120, and 240 min. After the described reaction times, the immobilized enzyme was removed with a 40 μm filter, and the solution was centrifuged at 14,000 r.p.m to separate the bioconjugated CNTs (precipitate) from the released peptide (supernatant). The peptide in the supernatant was assessed by SDS–PAGE and silver staining, and the optical density of the bands was measured in ImageJ software.

5.4 Characterization of CNTs

The topographical characteristics of the different CNTs were determined by scanning electron microscopy (SEM) in a JEOL model 7800 F scanning electron microscope; the images were acquired with transmitted and secondary electrons. Morphological and structural characterizations were performed using a high-resolution transmission electron microscope (HR-TEM) JEOL ARM200F (JEOL, JP). The acceleration voltage was set to 200 keV, and the samples were placed on copper lacy carbon TEM grids.

For AFM, a BIOSCOPE catalyst was used in ScanAsyst mode (Bruker AXS, CA, USA) coupled with a Zeiss AXIO Observer. Z1 with a Cantilever T: 3.4 μm model RTESP-300 (Bruker AXS, CA, USA).

The structural quality of the CNTs was determined by Raman microspectroscopy using LabRAM HR micro-Raman equipment (Horiba Jobn Yvon) coupled to an Olympus BX-4 microscope. Small samples of the different CNTs were placed on slides and excited with a 785 nm laser.

The functional groups present on CNTs were analyzed by Fourier transform infrared spectroscopy (FTIR) using an FTIR spectrophotometer (Carry 600, Agilent). The sample was placed in the attenuated total reflectance accessory and read at 600 to 4000 cm−1.

The chemical composition of the samples was analyzed by X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Scientific, K-alpha model). A monochromatic beam (Alkα) was used as an excitation X-ray source.

The bioconjugation efficiency of f-CNTs was calculated by determining total protein by a bicinchoninic acid (BCA) assay and indirectly by the elemental analysis of carbon, nitrogen, and oxygen using XPS analysis; P-CNTs at the same concentration or weight were used as blanks. Finally, the protein band pattern was analyzed by SDS–PAGE 7.5% and silver staining [65]. The zeta potential was determined at 6 μg/mL in PBS (pH 7.4) in a Zetasizer Nano ZS (Malvern Panalytical). All measurements were carried out at 25 °C.

5.5 Cell Lines

The cell lines used were ATCC: J774A.1 (TIB-67™) Mus musculus macrophages as a mouse model and ATCC: SK-OV-3 [SKOV-3; SKOV3] (HTB-77™) as a human model of ovarian adenocarcinoma.

5.6 Cell Viability Assay

Cells (10,000/well) were cultured in 96-well plates in DMEM with 10% heat-inactivated bovine fetal serum (FBS). Cells were exposed to UP-CNTs, P-CNTs, and f-CNTs at 0.06, 0.6, 6.0, and 60 μg/mL and incubated for 1 or 24 h. Cells without stimulus were used as growth or negative controls, or DMSO 5% was added as a death control. At 20 h, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl formazan (MTT) (5 mg/mL) was added to each well and incubated for 4 h, and for 1 h assays, cells were incubated with MTT for 3 h and then allowed to interact with CNTs for 1 h more. Cells were lysed, and absorbance was read at λ=590 nm using a VarioSkan microreader (Thermo, CA, USA).

5.7 Reactive Oxygen Species (ROS) Production Assay

Cells (20,000/well) were cultured in ninety-six-well dark plates (Costar, USA) in DMEM with 10% FBS, exposed to 0.06, 0.6, 6.0, and 60 μg/mL concentrations of different CNTs, and incubated for 1 h. Then, 2′,7′-dichlorofluorescein diacetate (DCFH-DA, 20 mM) was added (Sigma–Aldrich, USA) and incubated for 30 min. Cells were washed twice, and fluorescence was detected immediately using a FluoroSkan (Thermo, CA, USA) λEX=530 nm and λEM =485 nm [66]. The fluorescence was normalized to viable cells by determining cell viability by MTT. Cultures without stimuli or with hydrogen peroxide (0.1%) were used as negative or positive controls.

5.8 Apoptosis Assay

Apoptosis was assessed using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™. CAT: 556547). Briefly, SKOV-3 cells and J774A.1 macrophages were cultured independently on twenty-four-well plates (500,000/well) and incubated for 24 h; at the end of this time, the cells were exposed to UP-CNTs, P-CNTs, or f-CNTs (6 μg/mL). The positive control was exposed to UV light for 20 min to induce apoptosis. After 24 h of interaction, the cells were detached, washed twice with cold PBS, and resuspended in 1X binding buffer at a concentration of 1 × 106 cells/ml. A 100 μl volume of cell suspension (1 × 105 cells) was transferred to a 5-mL culture tube, 5 μl of FITC Annexin V and 5 μl PI were added, and the cells were incubated for 15 min at RT in the dark. Finally, 400 μl of 1X binding buffer was added to each tube to analyze the samples by flow cytometry. The following controls were used to set up compensation and quadrants: (1) unstained cells, (2) cells stained with FITC Annexin V (no PI), and (3) cells stained with PI (no FITC Annexin V). Samples were analyzed in a BD LSRFortessaTM cytometer. Data were analyzed using Kaluza Analysis Software (Beckman Coulter, CA, USA).

5.9 Actin Cytoskeleton Analysis

The cytoskeleton changes induced by CNTs in SKOV-3 and J774A.1 cells were assessed by confocal immunofluorescence analysis using Alexa Fluor® 488 Phalloidin (Molecular Probes, Eugene, OR, USA) and an LSM 700 Zeiss® microscope. Cells (10,000/well) were seeded in chamber slides (Thermo, CA, USA) and exposed to 6 and 60 μg/mL concentrations of the different CNTs for 24 h. After exposure, cells were washed, fixed (4% paraformaldehyde for 15 min), and permeabilized (0.1% Triton X100 in PBS). Unspecific sites were blocked with 10% FBS, washed, and incubated for 30 min with phalloidin-AF488. The slide was mounted with VectaShield-DAPI mounting medium.

5.10 Lysosome Formation and f-CNT Uptake and Cellular Localization

Cultures in chamber slides, as described above, were incubated with 50 nM LysoTracker™ Red DND-99 (Invitrogen™) for 2 h. Before reaching 2 h, f-CNTs (6 μg/mL) were added to cultures for 0, 15, 30, and 60 min of final exposure. The cells were washed twice with 1X PBS and processed for confocal microscopy.

For f-CNT uptake and cellular localization, polyclonal anti-f-CNT antiserum was prepared by C57BL6 mice immunized with 100 μg of f-CNTs (three boosts in incomplete Freund adjuvant) every 15 days. The antiserum was characterized by Western blot and immunofluorescence (Supplementary Figure 2). The CICUAL-CINVESTAV committee approved this study (ID: 0288-19), and all procedures followed the institutional guidelines.

To determine the uptake of CNTs, the SKOV-3 cell line, as a nonphagocyte control cell line, and J774A.1 macrophages, as a phagocytic cell line, were used; cells were incubated with the f-CNTs for 24 h, and f-CNTs localization was analyzed by confocal microscopy. Cultures incubated without CNTs or with preimmune sera were used as negative or nonspecific controls. FITC-affinity pure donkey anti-mouse IgG (Jackson ImmunoResearch) was used as the secondary antibody. To prevent unspecific interactions with FC receptors, Ultra-LEAF™ purified anti-mouse CD16/32 antibody (BioLegend, CA, USA) was used.

5.11 Cytokine Release in f-CNT-Stimulated J774A.1 Macrophages

For cytokine release analysis, J774A.1 macrophages were cultured in twenty-four-well plates (500,000 cells/well) and interacted with f-CNTs (6 μg/mL) for 24 h. The supernatant was collected and processed to determine IL-10, IL-4, IFN-γ, IL-2, IL-6, and IL-17 by flow cytometry using the BD Cytometric Bead Array (CBA) mouse Th1/Th2/Th17 Cytokine Kit (Becton Dickinson [BD], CA, USA). Cultures without CNTs were used as controls. All determinations were conducted in a BD LSRFortessa™ flow cytometer.

5.12 M1/M2 Polarization Marker Evaluation in J774A.1 Macrophages

J774A.1 macrophages were cultured on twenty-four-well plates (500,000/well) and incubated for 24 h; at term, cells were treated with P-CNTs, f-CNTs (0.1 and 6 μg/mL), peptide alone (6 μg/mL) or lipopolysaccharide from E. coli (LPS) (1 μg/mL; Sigma-Aldrich, USA). After 6 h, the supernatant was replaced with fresh medium and incubated for an additional 18 h.

For flow cytometry analyses, the cell pellet was incubated with Ultra-LEAF™ purified anti-mouse CD16/32 antibody. Dead cells were gated out by the zombie violet fixable viability kit (BioLegend, CA, USA). Then, CD86-FITC, MHC II-FITC, CD80-PE-Cy5 (eBioscience, CA, USA), CD163-FITC (Becton Dickinson [BD], CA, USA), and DC-SING/CD 209-PE (Abcam, MA, USA) were used to analyze the polarization pattern, and the cells were incubated for 30 min at 4 °C. Samples were analyzed in a BD LSRFortessaTM cytometer. Data were analyzed using Kaluza Analysis Software (Beckman Coulter, CA, USA).

Nitrite production was analyzed in the supernatant using the Griess Reagent Kit (Biotum, USA) following the manufacturer’s protocol.

Additionally, cells were cultured, exposed to CNTs (described above), and lysed to analyze arginase-1 (ARG-1) expression by Western blotting. α-ARG-1 (A4923) (ABclonal, USA) was used 1:2000. Images were analyzed in a Newton 7.0. (Vilber, USA).