This is the submitted version of the following article: Marcelo G.A., Montpeyo D., Novio F., Ruiz-Molina D., Lorenzo J., Oliveira E.. Luminescent silicon-based nanocarrier for drug delivery in colorectal cancer cells. Dyes and Pigments, (2020). 181. 108393: - . 10.1016/j.dyepig.2020.108393, which has been published in final form at https://dx.doi.org/10.1016/j.dyepig.2020.108393 © https://dx.doi.org/10.1016/j.dyepig.2020.108393. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ Luminescent silicon-based nanocarrier for drug delivery in colorectal cancer cells Gonçalo A. Marcelo a, David Montpeyo b, Fernando Novio c,d, Daniel Ruiz-Molina c, Julia Lorenzo b,**, Elisabete Oliveira a,e,* a a BIOSCOPE Group. LAQV@REQUIMTE. Chemistry Department. Faculty of Science and Technology, University NOVA of Lisbon, Caparica Campus, 2829-516, Caparica, Portugal b Institut de Biotecnologia i Biomedicina, Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain c Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and The Barcelona Institute of Science and Technology, Campus UAB, Bellaterra, 08193, Barcelona, Spain d Departament de Química, Universitat Autònoma de Barcelona, Campus UAB, Cerdanyola del Vallès, 08193, Barcelona, Spain e PROTEOMASS Scientific Society, Rua dos Inventores. Madam Parque, Caparica Campus, 2829-516, Caparica, Portugal A R T I C L E I N F O Keywords: Luminescence, Silicon quantum dots, Mesoporous silica nanoparticles, Drug delivery, Colorectal cancer cells, Doxorubicin A B S T R A C T Nanocarriers sensitive to exogenous or endogenous stimuli emerged as an attractive alternative to target drug delivery, with inorganic silica mesoporous nanoparticles (MNs) playing a core role in the development of a new generation of non-toxic and tuneable nanocarriers. A sensitive nanovector (NANO1) comprising luminescent silicon quantum dots (SiQDs) and functionalized with MNs was synthesised and loaded with doxorubicin (DOX). NANO1 nanoparticles have a size of 74 ± 10 nm and DOX loading percentages of ca. 43%. As a control sample, a similar nanocarrier (NANO2), without SiQDs, was also synthesised and loaded with DOX. Release profile studies, in PBS, revealed the strong NANO1@DOX pH-dependant behaviour, with a pH 5.0 favouring the release of DOX to percentages of ca. 70%. Cytotoxicity assessments of both free and DOX-loaded nanocarriers were evaluated in human cell lines of colon, revealing both free drug and drug-loaded nanoparticles to be concentration-dependent. 1. Introduction Colorectal cancer (CRC) is the second most common cancer and the fourth cause of cancer deaths in the world, with nearly 1.4 million new cases diagnosed in 2012 [1]. The development of new nanotech-based carriers to increase (i) specificity to target tumour cells, (ii) delivery capacity to the tumour site, therefore maximising the efficacy of the therapy and (iii) minimising undesirable side effects, is crucial [2,3]. Nanocarriers sensitive to exogenous or endogenous stimuli emerged as an attractive alternative to target drug delivery, but only a few stimuli-responsive nanosystems have reached clinical stage due to their complex design and challenging synthetic scale-up [4]. From all nanoparticles systems that have been used as drug delivery agents, luminescent inorganic silica mesoporous nanoparticles (SiQDsMNs) emerged as a promising multifunctional new generation of nanocarriers. Their use as drug delivery devices is due to their low toxicity, high specific surface area, large pore volume, tunable pore structures, and size [5–7]. Usually, to facilitate their tracking in biological systems, nanocarriers are labelled with a luminescent dye or, in this case, a luminescent inorganic quantum dot. Based on previous studies, silicon quantum dots are non-toxic biocompatible emissive materials, with numerous in vivo experiments revealing that silicon degrades to silicic acid, that is later excreted in urine [8–11]. The first reports on silicon nanocrystals were published two decades ago [12,13]. However, there are persistant challenges on the working procedures for this type of material [14], as i) the feasible preparation of high-quality colloidal silicon nanocrystal, ii) the easy control of their size and optical properties and iii) the avoidance of surface oxidation on silicon quantum dots. Regarding the available synthetic methods for SiQDs, most methods reported to date rely on relative harsh conditions (strong acids, toxic reagents, organic solvents, high temperature, inert atmosphere), specialised equipment, complex experimental procedures, not easily approachable by non-experts and with scaling up problems [14]. For a successful application in biological systems, SiQDs must be water-soluble, avoiding then any post-treatment and expensive processes. Recently, Y. Zhong and co-workers reported an aqueous synthetic method to prepare fluorescent SiQDs under microwave irradiation at 160 �C [15]. Later, the same group developed a photochemical strategy towards the synthesis of large-quantity of SiQDs under UV irradiation [16]. Fabrication of ultrabright water-dispersible SiQDs through a designed chemical surface modification [17], as well as, one-step synthesis of water- dispersible SiQDs for time-resolved imaging of living cells [18] and for selective sensing of heparin and cell imaging [19] were already reported by other authors. Based on these previous studies, in this work, SiQDs were performed in a one-step synthesis in water using (3- aminopropyl) trimethoxysilane (APTMS) and sodium ascorbate (SA) [18]. Despite all reported synthetic methods of silicon quantum dots in water [15–19], nanocarriers resulting from the combination of silicon quantum dots and silica mesoporous nanoparticles are yet scarce [20,21]. Additionally, from all nanocarriers developed to date that are under the preclinical stage for CRC [22–35], none combines luminescent quantum dots (silicon quantum dots) and mesoporous silica nanoparticles. Taking this into account, herein it is presented, for the first time, the synthesis of a sensitive nanocarrier combining silicon quantum dots and silica mesoporous nanoparticles and consequent surface functionalization, as well as its cytotoxic evaluation in colorectal cancer cells and drug delivery capacity (via pH stimuli) in saline buffer (at pH 7.4 and 5.0). To surmise, the delivery of anti-cancer drugs in fully non-toxic materials (non-use of toxic luminescent QDs) comprising SiQDs and mesoporous matrixes, has, according to literature [36,37], never been reported. Thus, the novelty of the work stands, despite a recent report by the authors on the sole toxicity of mesoporous SiQD-based systems [21], on the foremost development of similar nanosystems in the controlled delivery of DOX in specific cancer colorectal cell lines, for future applications as building blocks in targeted nanotherapies. 2. Experimental 2.1. Chemicals and starting materials (3-aminopropyl)triethoxysilane (APTES, H2N(CH2)3Si(OC2H5)3, 99%), (þ)-sodium, L-ascorbate (SA, C6H7NaO6, >98%), tetraethyl orthosilicate (TEOS, SiC8H20O4, 98%), ammonia (NH3, >99.9%), cetyltrimethylammonium bromide (CTAB, C19H42BrN, >98%), (3- mercaptopropyl)trimethoxysilane (MPTS, 95%), aldrithiol-2 (98%) (2,20 -Dipyridyl disulfide), cysteamine (>98%), were purchased from Sigma-Aldrich. Ammonium nitrate (NH4NO3, 99.9%) was purchased from Alfa Aesar. Methanol (MeOH) and ethanol (EtOH) were produced by Carlo Erba Reagents. All solvents and reagents were of analytical reagent grade and were used as received. 2.2. Synthetic procedures Silicon quantum dots (SiQDs) were prepared by adding 2 mL of 3-aminopropyltriethoxysilane (APTES) to 4 mL of miliQ water, followed by the addition of 4 mL of an aqueous solution of sodium ascorbate (0.1 M). The final mixture was stirred for 2 h at 50 ºC, leading to the formation of green emissive SiQDs, as reported in literature [18]. To the previous mixture (SiQDs) was added 10 mL of an aqueous solution of CTAB (0.1 M) and kept under stirring for 30 min at 50 ºC. Ammonia (500 μL) was added and the mixture was stirred for a further 10 min. Then, 240 μL of TEOS was added dropwise and the final mixture left under stirring for 2 h at 70 ºC. After 2 h, 3- Mercaptopropyltrimethoxysilane (1 mL) was added and after 30 min of stirring the resulting nanoparticles were washed with water and centrifuged, resulting in the nanoparticles SiQDsMNsSH. The SiQDsMNsSH nanomaterial (200 mg) was treated with a methanol solution containing the precursor PDEA (2-(pyridyldisulfanyl) ethylamine) (200 mg), which was previously synthesised accordingly to Refs. [38,39], for 24 h under stirring. The resulting nanomaterial SiQDsMNsS-S-NH2 (NANO1) containing the 2-(propyldisulfanyl) ethylamine linker was centrifuged, washed several times with methanol and dried. Template removal was achieved by solvent extraction, by resuspending the nanoparticles in a 20 mL methanolic solution containing 60 mg ammonium nitrate, that was stirred for 2 h at 60 ºC. The procedure was repeated twice and the nanoparticles centrifuged and washed in water. As a control sample the same synthesis was repeated, but without the addition of silicon quantum dots (SiQDs), resulting in MNsS-S-NH2 (NANO2) nanoparticles. 2.3. Nanoparticles characterisation UV–Vis absorption spectra were recorded on a Jasco V-650 spectrophotometer and a NANODROP ND-1000; and Fluorescence emission in a HORIBA Scientific FLUOROMAX-4 spectrofluorimeter from Proteomass- Bioscope facility (Portugal). Infrared spectra were acquired in a FTIR Spectrometer Spectrum two, UATR TWO PERKIN ELMER. Hydrodynamic size and zeta potential were obtained by dynamic light scattering, by dispersion of the nanoparticles in miliQ water, in a Malvern Nano ZS Zetasizer instrument with a 633 nm laser diode, from Proteomass – Bioscope facility (Portugal). Transmission electron microscopy (TEM) images were obtained in a JEOL JEM 2010F operating at 200 kV. TEM images were collected using a multiscan camera and Digital Micrograph software from Gatan (CCD Gatan 895 USC4000). Surface area and pore size distribution were obtained from N2 adsorption/desorption isotherms at 77 K, in a Micromeritics ASAP 2010 (Accelerated Surface Area and Porosimetry), at the Laboratory of Analysis from FCT - UNL. Specific surface areas (SBET) were estimated with the BET method [40]. XRD spectra were acquired with RIGAKU MiniFlex II X-ray diffractometer equipped with a Cu-Kα source (30 KV/15 mA) for 2θ angles ranging from 3º to 80º. The fluorescence quantum yield of SiQDs was measured using as standard fluorescein (φ Ό 0.79) [41] in ethanol. The value of fluorescence quantum yield was corrected accordingly with the different refraction indexes. 2.4. Doxorubicin loading and in vitro release studies To a solution of doxorubicin (DOX) (1 mg/mL), previously prepared in phosphate buffer (PBS) pH 7.4, were added 24 mg of NANO1 (SiQDsMNsS-S-NH2) or NANO2 (MNsS-S-NH2) nanoparticles and kept under stirring for 24 h at room temperature. After that, the sample was centrifuged and washed four times with PBS 7.4. The supernatants were quantified by absorption in the NANODROP ND-1000, from which were obtained the encapsulation efficiency (EE%) and the loading capacity (mg/g), determined by the following equations (tDOX: the total amount of DOX/nanoparticles fDOX: the amount of free DOX/nanoparticles) [42]: The in vitro doxorubicin release from the encapsulated NANO1@DOX and NANO2@DOX was performed by suspending the nanoparticles (10 mg) in a 2 mL solution of PBS 7.4 and PBS 5.0. All suspensions were stirred and kept at 37 ºC. Aliquots were collected at different times and the released doxorubicin quantified by absorption in the NANODROP ND- 1000. 2.5. Cell culture Colon cancer cell lines HT29 and HCT116 were obtained from the ATCC (American Type Culture Collection). All cell cultures were routinely maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) in standard growth conditions (37 ºC, 10% CO2). Fig. 1. (A) Emission and excitation spectra on SiQDs and solid-state emission spectrum of NANO1 nanoparticles (λexc ¼ 370 nm, λem ¼ 510 nm). (B) Naked-eye (I) and under UV lamp (II) (λexc ¼ 365 nm) images of Silicon quantum dots in solution and of the NANO1 nanoparticles in solid state. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 2.6. Cytotoxicity and intracellular accumulation of the nanoparticles The cytotoxicity of free nanoparticles NANO1 and NANO2, as well as those loaded with doxorubicin (NANO1@DOX; NANO2@DOX) was tested in human cancer colon cells using the PrestoBlue assay. Cells were seeded into a 96-well plate at a cell density of 3.0 x 103 cell/well and incubated for 24 h before nanoparticles were added at a concentration from 1 to 300 μg/mL. After 72 h, 20 μL of prestoBlue reagent were added to each well and the plates were further incubated for 4 h at 37 ºC. The effect on cell viability was measured using a multilabel plate reader (Victor 3 PerkinElmer). Its fluorescence was quantified after excitation at λ Ό 531 nm and collection at λ Ό 615 nm, while its absorbance was monitored at λ = 570 nm. Cell cytotoxicity was evaluated in terms of cell growth inhibition in treated cultures and expressed as a percentage of the control conditions. Each experiment was repeated at least three times, and each concentration was tested in at least three replicates. For cellular accumulation determination, 105 HT29 cells were plated in 35 mm-cell culture dishes and were allowed to grow overnight. Cells in exponential growth were incubated, for 24 h, with 50 μg/mL of each nanoparticle (untreated cells and cells treated with 1.75 μM of doxorubicin were used as a control). After incubation, cells were washed twice with PBS and lysed with PBS containing 1% SDS. Fluorescence intensity of the lysates was measured in a Jasco FP-8200 spectrofluorometer at λex = 370 nm/λem = 499 nm and λex = 470 nm/λem = 589 nm. Initial fluorescence intensity was measured from 50 μg/mL of each nanoparticle in PBS containing 1% SDS (Sodium Dodecyl Sulphate), and the uptake ratio was calculated for every fluorescence-emitting nanoparticle. 2.7. CLSM in vitro imaging of nanoparticles-treated cells Cellular uptake experiments were performed on HT29 cell line. Cells were seeded at a density of about 2 x 104 cells per plate on 14 mm glass-bottom microwell dishes (MatTek, corp). After 24 h, the cells were treated with 25 μg/mL of NANO1@DOX and NANO2@DOX, and incubated for additional 24 h. Cells were washed three times with cell media, and cell nuclei and membrane were stained with Hoechst or CellMask respectively for 15 min. Cell images were taken using a laser scanning confocal microscope (LSCM) SP5 (Leica). 3. Results and discussion 3.1. Synthesis and characterisation Luminescent Silicon quantum dots (SiQDs) were synthesised in water and in open-air conditions at 50 ºC for 2 h. APTES and sodium ascorbate were used as silica source and as stabiliser/reducer. Green luminescent SiQDs showed excitation and emission maximum bands centred at ca. 370 nm and ca. 510 nm, respectively (Fig. 1). Additionally, the SiQDs presented a hydrodynamic size of 3.6 nm, a zeta potential of - 3.6 mV and a fluorescence quantum yield of 13%. The resulting SiQDs were covered with a layer of mesoporous silica nanoparticles (SiQDsMNs), functionalized with a layer of MPTS (SiQDsMNsSH) and the 2-(propyldisulfanyl) ethylamine linker finally added to the nanoparticles, leading to the final nanoparticles NANO1 (SiQDsMNsS-S-NH2) as depicted in Scheme 1 and Fig. 1. As control sample for the in vitro assays in colon cancer cell lines, the same nanocarrier without silicon quantum dots was also synthetized (NANO2). The final nanocarrier NANO1 was characterized by solid-state emission, TEM, FTIR, zeta potential, N2 isotherms and XRD. Moreover, the different coatings were confirmed by zeta potential, FTIR and N2 isotherms. As can be seen in Fig. 1B, NANO1 was obtained as a white blue emissive powder with a maximum emission band centred at 475 nm. As reported by Dasog and co-workers [37,43,44], the emission shift from green to blue upon functionalization of the SiQDs with mesoporous silica nanoparticles is attributed to the presence of nitrogen and oxygen sources leading the formation of silicon oxynitride type species. Based on TEM images NANO1 present a spherical shape with a size of 74 ± 10 nm (see Fig. 2A and B). The presence of silica element was also confirmed in the EDX spectra, depicted in Fig. 2C. To investigate the porosity of the materials, nitrogen adsorption/ desorption isotherms were performed (Fig. 2E). The luminescent un- functionalized, SiQDsMNs, showed a BET surface area of 677.7 m2/g and pore size of 4.1 nm (BJH model). The isotherm can be classified as a type IV isotherm with an adsorption step behaviour at P/P0 around 0.3–0.35, characteristic of mesoporous materials [45]. With the addition of further coatings, it is notable a decrease of surface area from SiQDsMNsSH to SiQDsMNs-S-S-NH2 (NANO1), with 96.9 m2/g falling to 13.5 m2/g, respectively. Due to the decrease of surface area, and so of the mesoporosity of the system, the consequent release through the pores of the silicon quantum dots is prevented, and the luminescent characteristics of the nanocarrier maintained. The XRD spectra (Fig. 2F) showed a broad peak at ca. 22º (SiQDsMNsSH, NANO1, NANO2), which corresponds to the amorphous peak of silica oxide. Zeta potential showed that the NANO1 had a surface charge of 44.8 ± 0.8 mV, while that of SiQDsMNs and SiQDsMNsSH was of 28.6 ± 0.6 mV and 40.6 ± 0.7 mV, respectively (Fig. 3). The above described values were obtained in miliQ water and at neutral pH of 7.0. From the general point of view, the increase of coatings functionalizing the surface stabilises the nanocarrier, turning it more positive. The different coatings were also verified by infrared spectroscopy. The FTIR spectra (Fig. 3) of SiQDsMNsSH and NANO1 show the characteristic bands of the silica structure, with a peak at 1028 cm� 1 corresponding to the Si–O–Si vibrations (asymmetric stretching). The absence of a peak at ca. 965 cm-1 for the SiQDsMNsSH nanoparticles shows that –OH groups on the surface of the nanosilica have completely reacted with MPTS. Covering by MPTS can also be confirmed by the peaks at 1117 cm � 1 (–Si-O-C-stretching), 1237 cm-1 (Si–CH2-R stretch) and 2932 cm- 1 (CH stretch). Moreover, the amino linker was confirmed by the peak at 1521 cm � 1, 1627 cm� 1 corresponded to the groups (C–N stretch) and (R–NH2 in-plane bend), respectively. Scheme 1. General synthetic pathway of the NANO1 and NANO2 nanoparticles. Fig. 2. TEM images (A, B), EDX spectrum (C) and size distribution (D) of NANO1 nanoparticles. (E) Nitrogen adsorption –desorption isotherms of NANO1, SiQDsMNsSH, NANO2 and SiQDsMNs nanoparticles. (F) XDR spectra of NANO1, NANO2, and SiQDsMNsSH. Fig. 4. The release profile of doxorubicin loaded into the NANO1 and NANO2, at pH 7.4 and pH 5.0 PBS buffers. Chemical structure of doxorubicin. Fig. 3. (A) FTIR spectra of SiQDsMNsSH and NANO1 nanoparticles. (B) Zeta potential, BET surface, pore volume and size data of the nanoparticles. 3.2. Doxorubicin loading/release Doxorubicin, a common drug used in cancer treatment, was chosen as a model drug to investigate the potential application of the luminescent nanocarrier NANO1. Doxorubicin loading into NANO1 and NANO2 was performed according to section 2.4, and from equations (1) and (2) were obtained encapsulation efficiencies of 41%–43% and loading capacities of 18 mg and 20 mg of doxorubicin per gram for NANO1 and NANO2, respectively. According to the obtained results, the total coverage of NANO1’s surface and the presence of a core of SiQDs, led to a reduction in the system’s mesoporosity, implying that DOX interactions and loadings occur mostly at the nanoparticles’ surfaces via strong electrostatic and hydrogen bonding. In NANO2, despite surface coverage, the absence of a SiQD core, lead to a slightly higher surface area, with some DOX also adsorbing onto the pores’ walls, thus justifying the marginally superior loading ratio. Drug release assays were performed in PBS at two different conditions: pH 7.4 and pH 5.0 (Fig. 4). Whilst at pH 7.4 no significant drug release was observed, for NANO1 (ca. 5%), this phenomenon was favoured at pH 5.0 where release percentages of ca. 70% were obtained. As previously reported for mesoporous silica nanoparticles [46], a burst release of DOX (5 min) was observed for NANO1. In contrast, NANO2 release percentages of ca. 20% and 50%, lower than those of NANO1, were obtained at pH 7.4 and pH 5.0 only after 30 min of continuous release. The observed release behaviour is partly attributed to DOX solubility differences, and the attractive - repelling forces developed between the drug and the modified nanoparticles at the respective pH values. The nanoparticles have a positive charge, while DOX is positively charged at low pH and partly neutral at physiological pH conditions (pKa ¼ 8.25). The repulsion between DOX and the organic coating and that of DOX with the pores’ wall is different. The repulsion at acidic pH is higher between the positively charged DOX and the positively charged surface coating, which justifies the higher percentage release in NANO1 at pH 5.0. Thus, the release of DOX from the NANO1 nanocarrier is favoured by acidic conditions (Fig. 4). 3.3. Dose-dependent of cytotoxicity of nanoparticles The viability of cell cultures, after exposure to free doxorubicin and the different DOX-loaded, or non-loaded, nanoparticles, was examined using PrestoBlue (Cell viability reagent [47]). The assay measures the amount of resazurin reduction by mitochondrial dehydrogenase and assumes that cell viability (corresponding to reductive activity) is proportional to the production of resorufin which is measured spectrophotometrically (570 nm), or fluorescently, by exciting at 531 nm and collecting the emission at 615 nm. Exponentially dividing cells were treated with the different concentrations (from 1 to 300 μg/mL) of each nanoparticle for 72 h, as described in the experimental section. Control DOX-treated cells were exposed to 1.75 μM of doxorubicin to simulate the equivalent concentration of the drug contained inside the tested nanoparticles. The absence of the cytotoxicity of NANO1 and NANO2 can be observed in Fig. 5. No significant effect on cell viability was detected at concentrations up to 75 μg/mL and 150 μg/mL, in tumoral cell lines HCT116 and HT29, respectively. Fig. 6 shows the dose-response effect of the two cancer cells lines against free DOX, NANO1@DOX and NANO2@DOX nanoparticles (NPs). As expected for all cell lines, we can confirm that as DOX concentration increased, cell survival decreased. Indicating, thus, that the effect of both free drug and drug-loaded NPs is concentration-dependent. ANO2@DOX and DOX show similar cytotoxic behaviours. With, IC50 values of free DOX and NANO2@DOX NPs being very similar: 1.54 ± 0.13 and 0.75 ± 0.28 for DOX; and 0.37 ± 0.02 and 2.36 ± 0.02 μM for NANO2@DOX. Conversely, NANO1@DOX showed less cytotoxicity, with IC50 values of 8.92 ± 0.87 and 9.35 ± 0.84 μM, respectively (Table 1). 3.4. Cellular uptake of doxorubicin-loaded nanoparticles After 24h of exposure to nanoparticles, cells were stained and imaged by LSCM. Analysis of z-stack images (Fig. 7 B and C) and their orthogonal sectioning demonstrated that both NANO1@DOX and NANO2@DOX are internalized by HT29 cells (Fig. 7 D and E). The red fluorescence from doxorubicin is mostly localized in the cytoplasm, between the blue fluorescence of Hoechst, that signals nuclei position in cells and the green fluorescence of cellular membrane staining. From these images, we can see that DOX was internalized by the cells, arriving at the nuclei of some of them. The fluorescence of SiQDs contained within NANO1 and NANO1@DOX, as well as DOX fluorescence from NANO1@DOX and NANO2@DOX, inside the cells, were quantified by analysing cell lysates in a spectrofluorometer (excitation wavelengths: 370 and 490 nm; emission wavelengths: 499 and 589 nm respectively for SiQDs and DOX). Table 2 displays the relative cellular internalisation, in percentage, of added nanoparticles and DOX to the cell culture medium. It is interesting to notice that the magnitude of detected fluorescence for DOX, in the case of both nanoparticles, is higher than that of free drug. The relative cellular internalisation of NANO1 and NANO1@DOX was around 30%, with values for the calculated uptake, at 370 nm for the SiQDs and 470 nm for the DOX, being very similar. The percentage of uptake obtained for NANO2@DOX was around 24%, which is slightly lower than the uptake obtained for NANO1@DOX. In the attempt to correlate the obtained uptakes and cytotoxicity, we can affirm that, although, with the lowest uptake percentages, DOX and NANO2@DOX have the most significant cytotoxic effects against cancer cell lines, with small differences in terms of IC50. While DOX, with the lowest uptake (ca. 14%), has the best IC50 for HT29 cells, on the other hand, NANO2@DOX with a higher uptake showed outstanding and unmatched cytotoxicity against HCT116 cells. Considering NANO1@DOX high uptake but low cytotoxicity, and according to a fluorescence evaluation-based uptake, it is possible that for NANO1@DOX we are detecting fluorescence of DOX that is attached to the nanoparticles. Not being a free drug, DOX-NP complexes lack the capacity to arrive at its target and induce cellular death, within the in vitro tested timescale. Suggesting a possible NANO1@DOX controlled release of DOX, overtime (for t > 72 h). 4. Conclusions In this study, DOX-loaded nanoparticles based on silica were successfully synthesised, with sizes around 70–80 nm, with pH-dependent behaviours. The in vitro biological effects of the DOX loaded nanoparticles were evaluated on human cancer cell lines of colon. The results indicate that doxorubicin-loaded NPs induced noticeably cytotoxicity and cellular uptake on cancer cells with cytotoxic effects comparable to that of free drugs. The luminescent nanocarrier herein presented (NANO1) showed low toxicity and very promising results on the load and release of DOX, while keeping its fluorescent in cellular environment. Based on these results we envisage the addition of a targeting molecule at the surface of this nanocarrier, in order to make it more selective for a specific type of cell, increasing its range of applications and specificity, as well as promoting a decrease in prescribed DOX, and anti-cancer drugs, in cancer therapies. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Gonçalo A. Marcelo: Validation, Investigation, Data curation, Visualization, Writing - original draft, Writing - review & editing. David Montpeyo: Validation, Data curation, Investigation. Fernando Novio: Validation, Investigation, Resources, Writing - original draft, Writing - review & editing, Funding acquisition. Daniel Ruiz-Molina: Resources, Writing - original draft, Writing - review & editing, Funding acquisition. Elisabete Oliveira: Conceptualization, Methodology, Validation, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Funding acquisition, Visualization, Supervision, Project administration. Acknowledgements This work was supported by the Associate Laboratory Research Unit for Green Chemistry - Clean Processes and Technologies - LAQV which is financed by national funds from FCT/MEC (UID/QUI/50006/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER- 007265); by the Scientific Society PROTEOMASS (Portugal) (General Funding Grant) and by the Spanish Ministry of Science, Innovation and Universities project RTI2018-098027-B-C22. This work was also supported by grant RTI2018-098027-B-C21 and RTI2018-088027-B-C22 from the Spanish Government funds and by the European Regional Development Fund (ERDF). The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, funded by the Spanish Research Agency (AEI, grant no. SEV-2017-0706). F. N thanks to COST Action CA17121. F. N and J. L thanks to COST Action CA15138. G.M thanks to FCT/MEC (Portugal) for his doctoral grant PD/ BD/142865/2018. E. O thanks FCT/MEC (Portugal) for the individual contract, CEECIND/00648/2017. Appendix A. Supplementary data Supplementary data to this article can be found online References [1] Ferlay Js I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Brav F. Cancer incidence and mortality worldwide: JARC CancerBase Nº11. GLOBOCAN; 2012. [2] Chauhan VP, Jain RK. Strategies for advancing cancer nanomedicine. Nat Mater 2013;12(11):958–62. 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