Uroguanylin-decorated Nanosystems Containing Etoposide, a Potential Targeted Combination Therapy for Colorectal Cancer

Colorectal cancer is the third most frequently diagnosed cancer malignancy and the second leading cause of cancer-related deaths worldwide. Guanylyl Cyclase C (GCC), a membrane receptor that is expressed in 95% of primary and metastatic colorectal cancer tumors is a promising target for the design of new drug delivery strategies. In this work we propose the use of Uroguanylin (UroG), a natural ligand for GCC, for the preparation of targeted sphingomyelin-based nanosystems (UroG-SNs). The results from in vitro cell culture studies showed that UroG-SNs have a specic interaction with the GCC receptor, resulting in an antiproliferative response. Subsequently, the anti-cancer drug etoposide (Etp) was encapsulated into the UroG-SNs. The evaluation of the resulting system showed a synergistic effect of the two drugs, thus highlighting the therapeutic potential of this strategy for the treatment of colorectal cancer.


Background
According to World Health Organization (WHO), cancer causes more deaths than all heart diseases or strokes [1]. Colorectal cancer (CRC) is the third most frequently diagnosed cancer malignancy and the second leading cause of cancer-related deaths in the world, causing approximately 10% of deaths with an increase of over 20 million new cancer cases expected annually by 2025 [2]. Moreover, the presence of local or distant metastasis remains the leading cause of death among cancer patients, with an overall mortality above 50%. These facts emphasize an unmet clinical need for the effective targeting of colorectal cancer metastasis [3]. Guanylyl Cyclase C receptor (commonly referred as GCC or GUCY2C) is expressed at the apical membrane of enterocytes from duodenum to distal rectum and also by primary and metastatic colorectal cancer cells, however, it is not expressed in healthy extraintestinal tissue such as liver and lungs, where colorectal cancer cells usually metastasize [4][5][6]. GCC is activated upon binding to the paracrine hormones Guanylin (Gn) and Uroguanylin (UroG) as well as to the enterotoxigenic Escherichia coli heat stable enterotoxin (ST) [7]. GCC-paracrine hormones axis is considered a key regulator of several cellular processes such as differentiation, apoptosis, proliferation and migration [7,8]. Numerous works have reported that the levels of mRNA of both guanylin and uroguanylin hormones were markedly downregulated in adenocarcinomas even at rst stages, but high levels of the GCC receptor continue to be expressing [9][10][11]. With respect to its use in therapy, there are several studies in T84, HT29 and CaCo2 colorectal cancer cell lines showing that both Gn, UroG and the ST enterotoxin can inhibit cell proliferation based on GCC activation [5,12]. This clinical evidence prompted several GCC agonists already approved by the FDA (Linaclotide, Plecanatide and Dolcanatide) as potential candidates for oral cancer chemoprevention [13]. In line with these discoveries, some authors have reported the development of radiotracers, based on chemical modi cation of the endogenous agonists (UroG, Gn and ST), and exploiting their ability to target GCC for PET and SPECT molecular diagnosis [14][15][16]. To the best of our knowledge, nanosystems targeted to GCC to deliver anticancer therapies to colorectal cancer have not been reported to date. The use of nanosystems for the development of anticancer therapeutics has several advantages, such as an increase in the therapeutic effectiveness, diminishing the administered dose, and/or decreasing the secondary effects by means of targeting strategies (active/passive) that increase drug accumulation into the tumors [17]. On the other hand, nanosystems offer the possibility to include a variety of anticancer drugs in a single carrier for the development of combination therapies [18].
The objective of this work was the design, development and characterization of an actively targeted therapy consisting of drug-loaded sphingomyelin nanosystems (SNs) functionalized with Uroguanylin (UroG). For this, the rst step was the preparation of a derivative of the natural hormone UroG by conjugation to a PEG-lipid moiety (UroGm) for its facile incorporation into SNs [19]. In a second step, the anticancer drug etoposide [20] was encapsulated into SNs, and the potential of this combination therapy for interfering with colorectal cancer cells proliferation was investigated.

Synthesis and characterization of an UroG derivative
The conjugation of UroG to an amphiphilic molecule, poly(ethylene glycol) (PEG) with a hydrophobic stearic acid derivative (C 18 ), was explored in order to promote a successful insertion/anchoring of the peptide in SNs while exposing UroG linked to the PEG section in the outer part, thus making it accessible for receptor recognition. The primary amino group of UroG was linked to the terminal carboxylic group of the amphiphilic surfactant COOH-PEG 12 -C 18 . Activation of the carboxyl group through DMTMM is a simple reaction where DMTMM chloride generates the activated ester releasing 4-methylmorpholine in the rst step. An amide bond is then formed between the activated ester and the amine present (Fig. 1A).
Uroguanylin reaction progress was monitored by HPLC, evidenced by a decreasing intensity of the original peptide peak along reaction time and its shifting from t R (UroG) = 13.8 min to t R (UroGm) = 14 min ( Fig. 1B). Once the puri cation of the product was done, HPLC analysis indicated a 75.45% conjugation yield. In order to certify the conjugation and the identity of the conjugate, MALDI-TOF analyses were subsequently carried out (Fig. 1C). These data provided the molecular weight (MW) of the conjugate, approximately 2560 Da, which corresponds to the formation of a 1:1 conjugate. Next, this data was further corroborated by NMR analysis through: 1 H-NMR ( Figure S1A) and TOCSY ( Figure S1B) experiments. The diffusion coe cients (D) of the single reagents (UroG and PEG 12 -C 18 ) and the reaction product (UroG-PEG 12 -C 18 conjugate, UroGm) were also determined by diffusion NMR (DOSY-NMR) experiments (Fig. 1D). This parameter is directly related with many intrinsic properties of the molecule such as MW, size, shape and charge [21,22]. In the present study, a lower D value of the conjugate was obtained in comparison with non-modi ed peptide (D UroG = 3.7 × 10 -10 m 2 s -1 , D UroGm = 0.45 × 10 -10 m 2 s -1 , D C18-PEG12 = 0.46 × 10 -10 m 2 s -1 ) as expected since lower diffusion coe cients correspond to higher MW species. Although it was not possible to calculate the coe cient through the peptide peaks in the UroGm sample, due to a de cient signal-to-noise ratio, the PEG-lipid signals were highly robust to allow the calculation of the coe cient in a consistent manner. Finally, TOCSY analysis proved the presence of UroG in the conjugate sample ( Figure S2). Conjugation is also proved due to the observation of broader peaks in the 1 H-NMR spectra, a well-established characteristic after polymer conjugation, as well as the shifting of NH-signals of the UroG in comparison with the pure peptide. Overall, the results of the three characterization techniques led us to conclude that conjugation prompted a structurally well-de ned peptide-PEG-lipid (UroG-PEG 12 -C 18 ), certifying that UroG peptide was successfully conjugated through an amide bond.

Development Of A Targeted Nanosystem
Sphingomyelin-based nanosystems (SNs) composed by an oleic acid core and stabilized by sphingomyelin were prepared by spontaneous emulsi cation using ethanol injection method [23]. UroGm was incorporated into the aqueous phase, followed by the addition of the lipids dissolved in ethanol. The physicochemical properties of the obtained UroGm-SNs were exhaustively studied using a wide panel of analytical techniques. UroGm-SNs showed a nanometric size below 150 nm and a negative surface charge (Table 1). A decrease in the mean particle size was observed for the decorated UroGm-SNs (131 ± 12 nm) with respect to plain SNs (149 ± 10 nm). This variation could be associated to the ability of the PEG 12 -C 18 to act as a capping agent due to the O-H bond present at the end of the molecule, which rises the hydrophilic character of the UroGm-SNs. The conjugated peptide could then behave as a new surfactant molecule due to its amphiphilic character [24]. Variation in charge towards more negative values was also observed indicating an e cient association of the negative conjugate to the nanosystem surface. Monodisperse populations were obtained for both SNs and UroGm-SNs, according to the homogeneity values, polydispersity index < 0.2 and SPAN value < 1. Particle concentration measurements established an average of 5 × 10 11 particles/mL, irrespective of the presence of peptide.Morphological examination was subsequently performed by Field Emission Scanning Electron Microscopy (FESEM). The images in Fig. 2A showed a de ned spherical shape which corroborated the same size values obtained by the other technics, thus highlighting the robustness of the system. Interestingly, the morphology of the UroGm-SNs was found to be more irregular than non-decorated SNs, which may be due to the presence of a more hydrophilic part (corresponding with the PEG chain and the UroG peptide moieties) decorating the surface of the nanosystems. Stability of SNs and UroGm-SNs was next assessed at 37ºC for 4 hours (Fig. 2B). The results show a good colloidal stability in suspension at these conditions for both nanosystems (SNs and UroGm-SNs). Nevertheless, signi cant differences were found when incubated with supplemented and non-supplemented cell culture medium (DMEM). SNs suffered a major increase in size during the rst hour of incubation in these media, thus suggesting a strong interaction with serum proteins (FBS). However, the size of the decorated nanosystems (UroGm-SNs) remained constant at the same tested conditions, indicating an improved stability due to the presence of the amphiphilic UroGm at the interface. Additional evidence of the incorporation of UroGm to SNs was obtained by NMR analysis. Figure 2C shows the appearance of a signal from the PEG 12 peak (δ 3.69-3.71) in the spectra of the UroGm, which is also observed in spectra of the functionalized UroGm-SNs, but not in that of non-functionalized SNs. For an accurate integration of UroGm, NMR signals corresponding to PEG 12 peak were normalized to an internal control (TSP). Calculation of the precise amount of UroGm was done as well by NMR, revealing an actual concentration of UroGm in the formulation of 2.08 ± 0.14 µg/mL. Following Eq. 1A and B (as detailed in methods section), we estimated a density of 0.012 UroGm molecules/nm 2 .

Evaluation Of The Targeting And Therapeutic Capacities Of Urog
The next experiments were intended to determine the therapeutic potential of the UroGm amphiphilic derivative and the targeting capacity of UroGm-decorated SNs. All experiments were carried out in a metastatic colorectal cancer cell line constitutively expressing the GCC receptor, SW620 [25] (expression was con rmed by immuno uorescence and western blot assays, as presented in supplementary methods and Figure S3). Firstly, the successful functionalization and the effective targeting capacity of UroGm-SNs were studied. The results showed that, after 1 h of incubation, the green uorescence intensity (associated with TopFluor®−sphingomyelin) was higher for cells incubated with UroGm-SNs than for the ones incubated with the non-decorated nanosystems (Fig. 3). However, after 4 h of incubation, both nanosystems reached similar levels of green uorescence intensity. These results suggest that despite the chemical modi cation of UroG, the new conjugate preserves the GCC targeting capacity, resulting in a faster initial internalization of the surface-decorated UroG-SNs.
Secondly, a tumor colony forming assay (CFA) was used to quantitatively evaluate the capacity of UroGm to impede the ability of a single cell to grow into a large colony through clonal expansion [26,27]. After addition of increasing concentrations of UroGm in solution, from 50 nm to 1 µM, it was observed that the number of colonies signi cantly decreased following a concentration dependent pattern ( Figure S4). Subsequent experiments were carried out to determine the potential of UroGm-SNs for the development of anticancer therapeutics to colorectal cancer. Results from CFA manifest an augmented antiproliferative activity of UroGm when it is loaded into SNs, something that is not seen with the free molecule (Fig. 4A), and that con rms the relevance of including this macromolecule in a delivery system for improved therapeutic effects. Additionally, results from a cell viability study (MTT) show a markedly decrease in cell viability of SW620 cells treated with UroGm-SNs at a particle concentration of 0.1 mg/ml in comparison with control cells (Fig. 4B). A dose-response assay with blank SNs exhibit no unspeci c effects due to the nanosystems forming components at the same tested concentration (0.1 mg/ml) ( Figure S5).

Development Of A Combination Nanotherapy
After proving the targeting and therapeutic capacity of UroGm-SNs, we decided to explore this bifunctional nanosystem for the development of a combination therapy. For this purpose, we selected the Topoisomerase II inhibitor etoposide (Etp), indicated for the treatment of metastatic colorectal cancer [28]. Given its hydrophobic nature, Etp could easily be incorporated in our lipid SNs. The resulting formulation (UroGm-Etp-SNs) as displayed in Table 2, showed a homogeneous distribution of particles with a similar size to UroGm-SNs and a negative zeta potential. Effective incorporation of UroGm and Etp into the nanosystem was studied by NMR and HPLC respectively. Results show similar peptide concentration (2.30 ± 0.12 vs 2.08 ± 0.14 µg/mL) and ligand density (0.010 vs 0.012 molecules/nm 2 ) in UroGm-Etp-SNs and UroG-SNs. Etoposide concentration in UroGm-Etp-SNs was 40.51 ± 5 µg/mL. Therefore, it is possible to conclude that etoposide was successfully included in the formulation, without modifying substantially the properties or the ligand density of UroGm-SNs. The potential of this combination nanotherapy was assessed for the treatment of metastatic colorectal cancer using the same cell line as in previous assays. CFA was performed to evaluate the exact concentration range at which a combination effect of both drugs is obtained. We observed that by combining separated drugs at subtherapeutic concentrations (as previously determined, ≤ 500 nM of etoposide and ≤ 50 nM of UroGm, Figures S4 and S6), a potentiated effect was achieved ( Figure S7). As shown in Fig. 5, a concentration of 50 nM / 500 nM of UroGm / Etp is able to reduce colony formation down to 4 times lower than the control. These results expose the great potential of this targeted therapeutic alternative for metastatic colorectal cancer treatment.

Discussion
Nanosystems intended for cancer treatment have been mostly designed relying on the intrinsic capacity of these small particles to enhance their circulation times and eventually undergo passive accumulation into the tumor tissues due to the enhanced permeability and retention effect (EPR effect) [29]. Recent studies highlighted the need for targeted therapies that could enhance the accumulation of nanoparticles in the tumor [30,31]. By means of active targeting, nanoparticles can theoretically achieve higher levels of drug concentration in tumour tissues via receptor-mediated endocytosis [17]. In fact, the use of targeting ligands has been shown to reduce side effects and improve the therapeutic output [32][33][34]. Typically, well-known receptors involved in tumour progression, such as HER2, folate receptor, CD44, and EGFR, have been exploited for that purpose [35]. One of the problems associated to these strategies relies on the fact that most of these receptors are non-speci c for cancer cells but ubiquitously expressed in the body.
In addition, competition with endogenous ligands may frequently hamper the potential of this approach. Therefore, the identi cation of selective markers on tumor cells becomes critical to enable selectivity for tumor tissue over normal cells [36]. Considering this, we selected the Guanylyl Cyclase C (GCC) receptor for targeting, since is only expressed at the apical membrane of enterocytes, as well as by primary and metastatic colorectal cancer cells, but not by healthy extraintestinal tissue [4][5][6]9].
Based on this previous knowledge, the objective of this work was to design a targeted therapy that could be used in combination with an anti-cancer drug in order to achieve a synergistic therapeutic effect. In a rst step, we selected the natural hormone uroguanylin (UroG) since it is a ligand of the GCC receptor expressed on colorectal cancer cells [7], and speci cally inhibits cell proliferation [5,12]. We synthesized a new uroguanylin derivative (UroGm) in order to provide a rmer association of the hormone to SNs, and evaluated the activity of the resulting surface-decorated formulation (UroGm-SNs). In addition, the anticancer drug etoposide was encapsulated in the SNs to develop a combination therapy approach (UroG-Etp-SNs) that could provide improved antitumoral effects in colorectal cancer cells. SNs were produced using a very mild and simple technique (ethanol injection method), an optimized low energy manufacturing technique traditionally used for liposome preparation [19,37]. In addition, without changing this production methodology, the inclusion of the targeting molecule (UroGm) into the nanosystem was expected to happen through the C 18 lipophilic segment of the conjugate [38,39]. In that way, the hydrophilic targeting moiety would be oriented towards the external aqueous phase.
Nanosystems coated with both proteins and PEG chains are well known to avoid aggregation and to be stable in cellular media [40]. PEGylated lipids, and particularly PEG-DSPE (phosphatidylethanolamine), have been widely used in nanoformulations as surface stabilizers, mainly to improve their circulation half-life [41]. In the last decade, the bifunctionality of PEG has allowed further exploitation for the conjugation of bioactive molecules such as antibodies or peptides through a great variety of linkers, constructing cell-speci c targeting nanocarriers [42]. Furthermore, peptide-PEG-lipid conjugates can be exhaustively characterized, a fact that positively contributes to address later regulatory processes and clinical translation [43]. The selection of an amide bond as the linkage functional group was based on its simplicity, non-immunogenicity and was consistent with reported studies considering the importance of the carboxy-terminal conserved domain (cysteine domain) for the proper folding and bioactivity of the hormone [15,44]. Besides, the amide group provides a stable union between the peptide and the PEGylated lipid, avoiding a prompt release of the targeting moiety from the nanosystem under multiple biological scenarios, enhancing its half-life and subsequent accumulation via EPR effect [45]. The in vitro cell culture studies showed that the hydrophobized derivative (UroGm) preserves the native UroG ability to e ciently target the GCC receptor and to decrease cell tumorigenicity for concentrations in the same order of magnitude as published by other authors [46,47]. This fact con rms that the biological function of the hormone remains intact despite the chemical modi cation. Although many reports in the literature have shown diverse ways to conjugate ligands to the nanosystems surface, the majority of them do not specify the ligand density obtained [30,44,[48][49][50]. The values of ligand density observed in this work are in line with those reported for different nanosystems, oscillating from 0.225 to 0.005 molecules/nm 2 [30,44]. The use of combination therapies involving the incorporation of two anticancer agents into a nanosystem may provide bene ts in the sense that different drugs may attack cancer cells at varying stages of their growth cycle [51]. Considering this, combination nanomedicine should be designed in such a way that it targets multiple signaling pathways with limited toxicity [36]. In this work, the decoration of SNs with UroGm combined with the encapsulation of the anticancer drug etoposide was selected to promote a synergistic effect of both drugs. This synergy could be justi ed due to the joint activation of cGMP-AKT axis by the UroGm and the DNA damage response triggered by Etp [52,53], and to an improved uptake of Etp due to its encapsulation into a nanosystem [54,55].

Conclusion
In this work we have shown the potential of UroG to be used in new targeted therapeutics for colorectal cancer. UroGm-SNs can interact with GCC-expressing colorectal cancer cells in an e cient way, due to the exposure of the UroG ligand on the nanoparticle surface, while simultaneously inducing a therapeutic response. Moreover, the developed UroGm-Etp-SNs could mediate the delivery of a second drug for the development of combination therapies. From our knowledge there are no studies using the UroG hormone either alone or in combination with other drugs for the development of targeted nanomedicines. Therefore, this strategy might represent a useful approach for the development of a new treatment for metastatic colorectal cancer.  [15]. DMTMM (120 eq, 276.72 g/mol) was added over C 18 -PEG 12 -COOH solution (100 eq, 825 g/mol) under magnetic stirring and left for 10 minutes at room temperature (RT) to promote the activation of the carboxylic groups. Then, 200 µL of UroG stock solution (1 eq, 1667,9 Da) were added dropwise to the previous mixture. HEPES buffer was used to adjust the pH to 7.6 with a nal buffer concentration of 150 mM. The reaction was allowed to proceed for 8 h at RT. For puri cation, the reaction volume was dialyzed against deionized water 3 times for 20 h by using a MiniDyalisis Kit (MWCO 1 kDa) and then analyzed by HPLC, NMR and MALDI-TOF techniques (detailed information in supplementary materials).

Preparation Of Urogm-sns
Sphingomyelin nanosystems incorporating the modi ed Uroguanylin (UroGm) were prepared by ethanol injection technique. Brie y, UroGm was dissolved in water at a concentration of 0.5 mg/mL. On the other hand, oleic acid and sphingomyelin were dissolved in ethanol at a concentration of 200 mg/mL and 40 mg/mL respectively. Subsequently, 50 µL of the oily phase (composed by 2.5 mg of oil and 0.5 mg of surfactant) were injected into 450 µL of ultrapure water (containing the appropriate quantity of UroGm) under continuous magnetic stirring and nanosystems were spontaneously formed. Increasing amounts of UroGm were added to the formulation in order to explore the maximum loading capacity (data not shown) establishing a nal amount of 10 µg of UroGm per formulation as the best condition. Formulations were then isolated by centrifugation (20000 RFC for 45 minutes at 15ºC) using an Eppendorf 5417R centrifuge (Eppendorf, Germany) to purify the nanosystem.

Physicochemical Characterization
Particle size and polydispersity index (PdI) were determined by Dynamic Light Scattering (DLS), and Zpotential values by Laser Doppler Anemometry (LDA), using a Zetasizer NanoZS® (Malvern Instruments, UK). Measurements were performed at 25 ºC with a detection angle of 173º upon 1/10 dilution with ultrapure water (MilliQ®). Nanosystems were additionally characterized by Nanoparticle Tracking Analysis (NTA), a method to measure particle size based on imaging of individual nanosystems. Experiments were conducted with a NanoSight NS3000 System (laser operating at λ = 488 nm) (Malvern Instruments, UK). Brie y, nanosystems were injected in the sample chamber at a 1000-fold dilution in ultrapure water. Five captures, with a camera level of 14, were used to determine several parameters such as average size, homogeneity and particle concentration. Colloidal stability of the nanosystems was determined after being stored at 4 and 37ºC, as well as after incubation in biological media (DMEM high glucose, Sigma Aldrich) supplemented or not with 1% v/v fetal bovine serum (FBS, Gibco).

Morphological Examination
Morphological examination of the formulation was performed by Field Emission Scanning Electron Microscopy (FESEM) Ultra Plus (Zeiss, Germany) con gured with InLens and STEM modes and operating at 20 kV. For the preparation of FESEM samples, 20 µL of the nanosystem suspension were mixed with 20 µL of 2% (w/v) phosphotungstic acid and stained for 6 hours. The mixture was placed onto a copper grid with a formvar-carbon lm, washed with 500 µL of ultrapure water and dried overnight in a desiccator under vacuum.
Ligand Density Calculation E cient incorporation of UroGm into the nanosystems surface was determined by NMR. To achieve an accurate quanti cation, a fraction of the non-isolated UroGm-SNs were collected to quantify the precise total amount of UroGm presented in the formulation. After isolating the nanosystem, both the supernatant (where the decorated UroGm-SNs are located) and the undernatant (containing the free compounds in solution) were collected for further analysis. These three fractions (i.e. total, supernatant and undernatant) were freeze dried to remove traces of ethanol that were found to interfere with the analysis (peaks of ethanol overlap with the peak of the PEG in the 1 H-NMR spectrum), and eventually dissolved in 500 µL of deuterated DMSO (99.8% D). NMR experiments were conducted at 25 ˚C on a Bruker NEO 17.6 T spectrometer (proton resonance 750 MHz) (Bruker, US), equipped with a 1 H/ 13 C/ 15 N triple resonance probe and shielded PFG z-gradient. All the spectra were processed with MestreNova software v12.0 (Mestrelab Research Inc., Spain). The chemical shifts were referenced automatically with respect to the deuterium lock. Samples were prepared in 5 mm thin wall NMR tubes. A 1D proton spectra ( 1 H) was acquired for each sample using the pulse-acquisition sequence. The spectrum was acquired under quantitative conditions by using a low excitation tilt pulse angle of only 30 degrees, an inter-scan delay (d 1 ) of 6 s and an acquisition time (aq) of 2.75 s. The proton spectrum was processed with Fourier transformation and the phase and baseline were carefully corrected. For control, plain SNs were also prepared and characterized following the same methodologies, without the addition of UroGm in the aqueous phase. Surface density of UroGm molecules was subsequently calculated as the number of molecules per surface unit of nanosystem (nm 2 ). Firstly, the number of UroGm particles were calculated with Eq. 1A from the previously NMR determined concentration. On the other side, nanosystems surface area were calculated using Eq. 1B, considering SNs morphology as perfect spheres, and with the concentration and radium parameters obtained from NTA measurements.

Equation 1
Formulas for calculation of number of particles (A) and surface area of a sphere (B).

Preparation Of Dual-loaded Sns
UroGm-functionalized sphingomyelin nanosystems (UroGm-SNs) were additionally loaded with the chemotherapeutic drug etoposide (UroGm-Etp-SNs). In this case, up to 250 µg of etoposide (40 mg/mL in DMSO) were placed into the organic phase within the 50 µL of ethanol and injected into the 450 µL of ultrapure water containing UroGm. Nanosystems were isolated using the same conditions as previously described. Encapsulation e ciency was determined by direct quanti cation of etoposide in the nanosystem using an isocratic HPLC method optimized from the literature [57]. Analyses were performed in an HPLC system 1260 In nity II Agilent (Agilent Technologies, US) equipped with a pump G7111A, an autosampler G7129A and an UV-Vis detector G7114A set at 254 nm. Separation was achieved on an In nityLab Poroshell 120 EC-C18 (100 mm In vitro studies

Cell Viability Studies
Cell toxicity analyses were performed to determine the viability of metastatic colorectal cancer cells SW620 (ATCC® CCL-227™) upon exposure to increasing concentrations of SNs (from 0.01 to 10 mg/mL) in a nal volume of 150 µL (25 µL corresponding to the nanosystem and 125 µL to complete medium).
Etp-SNs were also tested to evaluate the effect of encapsulating of the cytostatic drug. Cells were seeded at a density of 10.000 cells/well in 96-well plates 24 h before the experiment. After 48 hours of incubation with SNs and Etp-SNs, medium was removed and 100 µL of tetrazolium dye (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, MTT) solution (5 mg/mL in PBS, MTT Alfa Aesar, Germany) were added to each well. After 3 hours of incubation this solution was also removed and formazan crystals were solubilized with 100 µL of DMSO and maintained at 37ºC for 15 minutes protected from light. Results were obtained by measuring absorbance at 570 nm in a microplate spectrophotometer (Multiskan EX, Thermo Labsystems). Cell viability in percentage (%) was calculated in comparison with control wells containing untreated cells.

Cellular Internalization Studies
Internalization studies in SW620 metastatic cancer cells were performed by confocal microscopy (Leica Colony Forming Assays SW620 colorectal cancer cells were plated in triplicate at a density of 600 cells/well in 12-well plates and cultured in a humidi ed incubator (37 ºC, atmosphere of 5% CO 2 and 95% RH). Drug treatments were maintained in contact with cells for the complete duration of the experiment (15 days). After this period cells were stained with an MTT solution (5 mg/mL) for 3 to 4 hours and subsequently dried and scanned. Obtained images were analyzed using ImageJ software. In vitro differences were statistically determined by one-way ANOVA (GraphPad PRISM, version 6.0, GraphPad Software, Inc., US) Consent for publication.

Not applicable
Availability of data and material.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare no competing interest. Funding.