International Journal of Molecular Sciences Article Development of New Targeted Nanotherapy Combined with Magneto-Fluorescent Nanoparticles against Colorectal Cancer Gonçalo A. Marcelo 1 , David Montpeyó 2 , Joana Galhano 1 , Ramón Martínez-Máñez 3 , José Luis Capelo-Martínez 1,4 , Julia Lorenzo 2 , Carlos Lodeiro 1,4, * and Elisabete Oliveira 1,4, * 1 BIOSCOPE Group, LAQV@REQUIMTE, Chemistry Department, NOVA School of Science and Technology, 2829-516 Caparica, Portugal 2 Institut de Biotecnologia i Biomedicina, Departament de Bioquímica i de Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 3 Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico, Universitat Politècnica de València, Universitat de València, 46022 Valencia, Spain 4 PROTEOMASS Scientific Society, Rua dos Inventores, Madam Parque, Caparica Campus, 2825-182 Caparica, Portugal * Correspondence: cle@fct.unl.pt (C.L.); ej.oliveira@fct.unl.pt (E.O.) Abstract: The need for non-invasive therapies capable of conserving drug efficiency and stability while having specific targetability against colorectal cancer (CRC), has made nanoparticles preferable vehicles and principal building blocks for the development of complex and multi-action anti-tumoral approaches. For that purpose, we herein report the production of a combinatory anti-tumoral nanoth- erapy using the production of a new targeting towards CRC lines. To do so, Magneto-fluorescent NANO3 nanoparticles were used as nanocarriers for a combination of the drugs doxorubicin (DOX) and ofloxacin (OFLO). NANO3 nanoparticles’ surface was modified with two different targeting agents, a newly synthesized (anti-CA IX acetazolamide derivative (AZM-SH)) and a commercially available (anti-epidermal growth factor receptor (EGFR), Cetuximab). The cytotoxicity revealed that only DOX-containing nanosystems showed significant and even competitive cytotoxicity when com- pared to that of free DOX. Interestingly, surface modification with AZM-SH promoted an increased Citation: Marcelo, G.A.; Montpeyó, cellular uptake in the HCT116 cell line, surpassing even those functionalized with Cetuximab. The D.; Galhano, J.; Martínez-Máñez, R.; results show that the new target has high potential to be used as a nanotherapy agent for CRC cells, Capelo-Martínez, J.L.; Lorenzo, J.; surpassing commercial targets. As a proof-of-concept, an oral administration form of NANO3 systems Lodeiro, C.; Oliveira, E. Development was successfully combined with Eudragit® enteric coating and studied under extreme conditions. of New Targeted Nanotherapy Combined with Magneto-Fluorescent Nanoparticles against Colorectal Keywords: target nanocarriers; oral formulations; colorectal cancer; target drug delivery; targeted Cancer. Int. J. Mol. Sci. 2023, 24, 6612. anticancer therapy https://doi.org/10.3390/ijms24076612 Academic Editor: Loredana Ricciardi Received: 20 February 2023 1. Introduction Revised: 28 March 2023 Colorectal cancer (CRC) is the second deadliest cancer and the third most common Accepted: 29 March 2023 malignancy worldwide [1]. Due to its characteristics, it can become highly aggressive and Published: 1 April 2023 develop resistance to several therapeutic strategies [2,3]. All these aspects contribute to the alarming rising prevalence of CRC worldwide [4], making the development of more efficient anticancer therapies of utmost importance. Nanoparticle-based materials have been widely developed and successfully employed Copyright: © 2023 by the authors. as anticancer tools or as platforms to improve the efficacy of already known therapies, Licensee MDPI, Basel, Switzerland. This article is an open access article with metallic/metal-oxide nanoparticles oftentimes being used to this end [5,6]. Other distributed under the terms and nanosystems have also gained particular interest, specifically those containing mesoporous conditions of the Creative Commons silica nanoparticles/structures, whose highly porous matrix renders them with unique Attribution (CC BY) license (https:// cargo delivery capacities, surface manoeuvrability, targetability or even spectroscopical creativecommons.org/licenses/by/ activity [7,8]. Amongst these, precision diagnosis and therapeutic systems are of particular 4.0/). interest, due to their increased performance and variability [9–11]. Int. J. Mol. Sci. 2023, 24, 6612. https://doi.org/10.3390/ijms24076612 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 6612 2 of 23 Specific characteristics of tumour-like environments, such as hypoxia and acidic extracellular pH, can thus be utilized as targets for specific delivery of nanoparticle-based systems. Cancer cell environments oftentimes present uncontrolled growth that results in improperly structured and functioning vasculature, low oxygenation, and upregulation of homeostasis-associated enzymes, such as carbonic anhydrases (CAs, EC 4.2.1.1) [12]. CAs are ubiquitous metalloenzymes that catalyse the simple physiological hydration of carbon dioxide to bicarbonate ions and protons (CO2 + H2 O <-> HCO3− + H+ ), with most containing in their active centre a zinc ion (Zn2+ ), which is essential for catalysis. Besides regulation of pH and CO2 homeostasis, CAs are also responsible for CO2 and HCO3− transport between metabolizing tissues and lungs, electrolytes secretion, some biosynthetic processes (i.e., glucogenesis, lipogenesis and urea genesis), bone resorption, and tumorigenicity [12]. Their broad activity spectrum arises from the 15 presently known isozymes, with CAs I, II, III, VII and XIII residing in the cytosol; CAs IV, IX, XII, XIV, and XV being associated with membranes; CAs VA and VB occurring in mitochondria; and CA VI existing only as a secreted isoform [13]. The occurrence of an elevated number of CA isoforms in cellular membranes of nu- merous organs and the association of their overexpressed activity to some types of tumours have thus sparked a special interest in CAs as potential targets for tumour diagnosis and therapy [14,15]. The human (h)CA IX is an example of a tumour-associated CA isoform, with several groups associating it with tumour activity, cell proliferation, cell adhesion and malignant cell invasion [16,17], being thus also commonly considered as a potential biomarker for tumour diagnosis [18]. More specifically, hCA IX’s overexpression levels and increased activity have been recently associated with higher rates of hypoxia survival and cell invasion in tumours [19,20]. The inhibition of CAs has been increasingly reported as a potential pathway for can- cer therapy by using CA-directed pharmacological agents as CAs inhibitors, capable of imparting CAs activity and normal cell mechanisms [21,22]. Among others, 5-acetamido- 1,3,4,-thiadiazole-2-sulfonamide, commonly known as Acetazolamide (AZM) and its ana- logues, have exhibited outstanding and selective inhibitory activities towards CAs, being extensively used clinically as diuretics, anti-glaucoma, or anti-convulsant agents [23–25]. Regarding anti-tumoral therapies, specific CA and hCA IX inhibitors have also been re- ported as potential hypoxia-activable inhibitors and targeting agents [14,26–28]. The development of novel inhibitors capable of targeting CAs, specifically hCA IX, is thus of high interest, especially if they can be conjugated with pro-drugs or with nanometric vectors, rendering them targetable to the overexpressed hCA IX in cancerous environments. Associated with CA IX action and overexpression in hypoxic and invasive tumours, such as CRC [18,19], is also the commonly overexpressed epithelial growth factor receptor (EGFR), which mediates cancer initiation, progression and proliferation [29,30]. This associ- ation makes anti-EGFR agents also good targeting probes, with several works reporting the conjugated use of several anti-EGFR antibodies, such as the commercial Cetuximab (CTX), with therapeutic molecules (drugs, pro-drugs, etc.) and nanometric vectors for either diagnosis or therapeutics in CRC [31–33]. Considering this, herein we report the synthesis of a novel AZM-derivative containing a sulfhydryl anchoring moiety, and its subsequent biofunctionalization on superparam- agnetic, fluorescent iron oxide-cored mesoporous silica nanoparticles (NANO3-SH). This system was successfully evaluated as a potential anticancer tool against CRC cell lines [34]. The development of a dual anti-EGFR/anti-hCA IX (CTX+AZM) targeting nanoprobe is also reported, and its performance as a precision therapeutic tool against CRC is compared with that of each individual NANO3-anchored antigen. Nonetheless, the method of administration of drugs for therapeutic purposes is one critical step in the development of new ground-breaking therapies. The unsuccessful accounting of possible complex interactions with the body’s solid and liquid interfaces and all related arising hindrances dictate the successful rate of said therapies, even for those showing outstanding performances in silico/in vitro. Not only that, but with the Int. J. Mol. Sci. 2023, 24, 6612 3 of 23 ever-growing pursuit of non-invasive techniques, orally administered and inhalation-based products have gained particular interest in pharmaceutical industries/markets [35,36]. These points are particularly familiar to CRC, which, due to its body location, sees many oral delivery therapies being developed [37–39], of which some might be withdrawn from clinical usage. Most withdrawals are not only to problems of stability when in contact with body fluids constituents, pH and temperature, but also to problems of long circu- lation instability and peripheral body accumulation in organs/members other that the colon [35,40]. Some of the major barriers to the development of an orally administered therapy against CRC are the passage time, intrinsic high absorption, and extreme pH condi- tions of the whole gastrointestinal tract [40]. Its success goes, thus, through the protection of the active compound of interest with an external resistant, but also manoeuvrable, agent. Such agents must be capable of both resisting extreme denaturing conditions and having a stimuli-responsive behaviour (i.e., pH, time, etc.) to correctly dispense the therapeutic compound at the targeted site/time [41]. For anti-CRC applications, the selected formula- tion must be capable of bypassing stomach extreme conditions and then small intestine absorption, before reaching the colon. For that purpose, in a proof-of-concept, NANO3- SH systems were protected with an enteric coating formulation of Eudragit® polymers (PMMA-MMA copolymers), which showed a prominent capacity to effectively deliver the nanomaterials herein developed. 2. Results and Discussion 2.1. Synthesis of AZM-SH and Anti-CAIX Action The potential of acetazolamide (AZM) and its derivatives to serve as selective vehicles for the delivery of cancer-specific drugs and their inhibition activity over cancer-related CAs has been hinted at in several works, with special attention being given to the hCA IX isoform [15,42,43]. With this in consideration, herein we propose the synthesis of a thiol moiety containing an AZM-derivative, as a possible hCA (CA IX included) inhibitor and targeting agent in the delivery of anticancer drugs. The interest in the inclusion of a terminal thiol to the original structure of AZM arises from the potential formation of disulphide bonds with anticancer drugs of interest, or with nanovectors with intrinsic anti-tumoral activity or with drug-delivery capacity, for targeted anticancer therapy. To synthesise the new compound, the amide in AZM was firstly hydrolysed to produce an available terminal NH2 group other than that of the sulfonamide moiety. By activation of the carboxylic acid moiety of the 3-mercaptopropionic acid, via Vilsmeier mechanism, and reaction with compound AZM-NH2 , the new amide derivative (AZM-SH) was obtained. Intermediate AZM-NH2 and final product AZM-SH were characterised accordingly and compared to the initial AZM (Scheme 1). The 1 H and 13 C NMR spectra of AZM-NH2 (Figure S1) confirm the total hydrolysis of the amide bond in AZM. This step is characterised by the disappearance of the 2.25 ppm -CH3 signal in the AZM 1 H spectrum (Figure S2) and a right shift of the 13 ppm signal to 8.05 ppm, corresponding to the transformation of the secondary amide moiety into the terminal primary amine in AZM-NH2 . Moreover, the loss of the acetamide partition of AZM was verified with the vanishing of its correspondent 22.83 ppm and 161.57 ppm signals in the 13 C NMR spectrum of AZM-NH2 . The successful binding of 3-sulfanylpropanoyl chloride to the amine (1) of AZM-NH2 was confirmed by 1 H and 13 C NMR spectra of AZM- SH, in DMSO-d6 and CD3 CN (Figure S3). The reconstitution of the >NH signal at 13.13 ppm (DMSO-d6 ) and the surgency of a doublet at 8.38 ppm (DMSO-d6 ), corresponding to the sulfonamide -NH2 , in the 1 H NMR spectrum of the product (Figure S3), are the first hints at the successful synthesis of AZM-SH (Figure 1a). These signals are in accordance with the chemical shifts of the same groups in AZM’s NMR spectrum (Figure S1) and with those described for other thiadiazole-derived ligands [44,45]. Int. J. Mol. Sci. 2023, 24, 6612 4 of 23 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of HS O HS O 0 ᵒC, inert atm. OH DCM, (COCl)2 Cl Catalytic DMF Mercaptopropionic acid 3-sulfanylpropanoyl chloride O 13 10 THF, 12 h, r.t., inert atm. O 10 9 O 12 9 O 7 6 S S 4 a) EtOH, HCl 37% S 5 S 5 1 H2N 8 NH reflux 4.5 h 8 2 1 H2N NH2 11 2 N 7 N b) NaOH 5 M to pH 7.0 N 6 4 N 3 O 3 1,3,4-thiadiazole-2-sulfonamide (Acetazolamide) AZM 5-amino-1,3,4-thiadiazole-2-sulfonamide AZM-NH2 15 O 14 O 12 11 S S 6 7 H2N 10 NH 4 3 13 N 9 N 1 8 2 SH 5 O N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-3-sulfanylpropanamide AZM-SH Scheme 1. Synthetic Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW Scheme workflow on workflow 1. Synthetic the preparation on the of N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-3- preparation of N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)- 5 of 25 sulfanylpropanamide sulfanylpropanamide (AZM-SH) salt.(AZM-SH) Numbering salt. Numbering system system of atom (blue)ofused atomfor (blue) the used for the assignment assignment of 1H and 13C NMR chemical shifts. 1 H and 13 C NMR chemical shifts. The 1H and 13C NMR spectra of AZM-NH2 (Figure S1) confirm the total hydrolysis o the amide bond in AZM. This step is characterised by the disappearance of the 2.25 ppm -CH3 signal in the AZM 1H spectrum (Figure S2) and a right shift of the 13 ppm signal t 8.05 ppm, corresponding to the transformation of the secondary amide moiety into th terminal primary amine in AZM-NH2. Moreover, the loss of the acetamide partition o AZM was verified with the vanishing of its correspondent 22.83 ppm and 161.57 ppm signals in the 13C NMR spectrum of AZM-NH2. The successful binding of sulfanylpropanoyl chloride to the amine (1) of AZM-NH2 was confirmed by 1H and 13 NMR spectra of AZM-SH, in DMSO-d6 and CD3CN (Figure S3). The reconstitution of th >NH signal at 13.13 ppm (DMSO-d6) and the surgency of a doublet at 8.38 ppm (DMSO d6), corresponding to the sulfonamide -NH2, in the 1H NMR spectrum of the produ (Figure S3), are the first hints at the successful synthesis of AZM-SH (Figure 1a). Thes signals are in accordance with the chemical shifts of the same groups in AZM’s NM spectrum (Figure S1) and with those described for other thiadiazole-derived ligand [44,45]. Figure 1. Figure 1. (a) (a) Stacked Stacked 1H 1 H NMR NMR spectra spectra of of AZM, AZM, AZM-NH AZM-NH2 2AZM-SH AZM-SH(DMSO-d (DMSO-d 6, 400 MHz), for δ 6 , 400 MHz), for δbetween between 6 and 6 and14 14 ppm. ppm.(b) AZM-SH structure (b) AZM-SH and and structure atomatom number assignment. number (c) Close-up assignment. of the 1of (c) Close-up H NMR 1 spectrum of AZM-SH (CD 3 CN, 400 MHz) and chemical shift assignment of the H NMR spectrum of AZM-SH (CD3 CN, 400 MHz) and chemical shift assignment of sulfanyl- sulfanylpropanamide C(2) and C(3) protons, seen as two crossed doublet-triplet (dt) signals. propanamide C(2) and C(3) protons, seen as two crossed doublet-triplet (dt) signals. Although not Although not perceptible perceptiblein inthe theDMSO-d DMSO-d6 11H H NMR NMR of of AZM-SH, AZM-SH, in in CD CD3Cl, the signals 6 3 Cl, the signals relating the sulfanylpropanamide (NHCO-CH 2-CH2-SH) C(2) and C(3) protons are visible relating the sulfanylpropanamide (NHCO-CH2 -CH2 -SH) C(2) and C(3) protons are visible between 3.45 between 3.45 and and 3.15 3.15 ppm ppm and and between between 3.053.05 and and 2.65 2.65 ppm ppm asas aa set set of of two two doublet–triplet- doublet–triplet- like signals [46], thus supporting the successful synthesis like signals [46], thus supporting the successful synthesis of AZM-SH, of AZM-SH, byby thethe inclusion inclusion of 3-sulfanylpropanoyl via amide bond formation. The obtained of 3-sulfanylpropanoyl via amide bond formation. The obtained chemical shifts and chemical shifts and crossings of crossings of the the signals signals ofof C(2) C(2) and and C(3) C(3) protons have been protons have been pointed pointed outout before, by Schuetze before, by Schuetze et al. et al.[47], [47],toto arise arise fromfrom a certain a certain degree degree of dimerization of dimerization of the 3-sulfanylpropanoyl of the 3-sulfanylpropanoyl segment segment of of thevia the molecule molecule via abond a disulphide disulphide (Figure bond 1c). The(Figure 1c). Theofdimerization dimerization the molecule is of also the molecule is also suggested by the splitting of the -NH signal at 8.38 suggested by the splitting of the -NH2 signal at 8.38 ppm, J = 24.4 Hz (Figure 1a), comprising 2 ppm, J = 24.4 Hz (Figure 1a), comprising the asymmetry created by intermolecular the asymmetry created by intermolecular hydrogen bonding between the >NH/-NH2 and hydrogen bonding between the >NH/-NH2 and -C=O moieties of adjacent molecules (Figure 2), similarly to those formed in some reported AZM dimers [48,49]. Complete conversion of AZM-NH2 into AZM-SH was also confirmed by the latter’s 13C NMR spectrum, in DMSO-d6 (Figure S3c). ATR-FTIR spectra of both intermediate AZM-NH2 and final product AZM-SH also translate into the above-mentioned Int. J. Mol. Sci. 2023, 24, 6612 5 of 23 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 -C=O moieties of adjacent molecules (Figure 2), similarly to those formed in some reported AZM dimers [48,49]. Figure 2. Suggested (I) head-head Figure and 2. Suggested (I)(II) head-tailand head-head dimeric conformations (II) head-tail dimericofconformations AZM-SH attained via of AZM-SH attain hydrogen bonding. via hydrogen bonding. Complete conversion of AZM-NH The successful 2 intoof synthesis AZM-SH was also AZM-NH the intermediate confirmed2 and by the thelatter’s final product A 13 C NMR spectrum, in DMSO-d6 (Figure S3c). ATR-FTIR spectra of both intermediate AZM- SH were also confirmed with electron spray ionization (ESI) mass spectrometry NH2 and final product analysis AZM-SH (Figure S5). alsoWith translate into the a nominal above-mentioned mass modifications and a monoisotopic mass of 268 Da of the original AZM (Figure 267.97585 S4). Assigned Da (calculated), vibration nodes respectively, (Table S1) aredetected AZM-SH-[M]-was in accordance in two ionic fo with those described [M+H]+in(268.9831 the literature m/z) andfor [M] acetazolamide and its derivatives, specifically, + (266.9853 m/z). those concerning analogous groups, such as -SO2 NH2 , amide (-C=O, >N-H and C-N-C), and thiadiazolic ring 2.2. [48,50,51]. Docking The and Modelling appearance of preferable In Vitro Inhibition Studiesasymmetrical vibrational modes, in both AZM and AZM-SH, and the presence of particularly The effect of AZM-SH as potential inhibitor of human broader CAs, signals at the hC specifically low frequenciesisoform, demonstrate their similar appetency to form intermolecular was initially predicted by modelling the docking mechanism of AZM-SHhydrogen bonds and dimeric conformations hCA IX active site. The [48,52]. Special AZM-less attention chain A of theis brought 3IAI PDBto model the vibrations [53] was used fo concerning the determination >NH (amide) of andthe-SO 2 NH best 2 groups docking in the result. Thespectrum of AZM-SH, ligand–receptor whose interaction depicted i significant broadening supports the already hinted formation of dimers via hydrogen experimentally obtained 3IAI PDB model was taken as a comparative standard intera bonding (Figureconformation 2). between AZM and hCA IX. The successful The synthesis of theZnintermediate AutoDock4 forcefield was AZM-NH preferred 2 and overthe thefinal product traditional AZM- force AutoDock4 SH were also confirmed with electron spray ionization (ESI) mass spectrometry as its performance in properly reproducing zinc-coordination geometries (MS) has been pr analysis (Figureto surpass the other’s predictions [54]. The chosen forcefield parameters and S5). With a nominal mass and a monoisotopic mass of 268 Da consider the 267.97585 Da (calculated), respectively, common biological zinc AZM-SH-[M]-was coordination geometry, detected in two ionicone the tetrahedral forms: (Zn3.1 and Z + [M+H] (268.9831 m/z)AutoDock + and [M] atom (266.9853 m/z). where types NA (nitrogen HB acceptor), N (nitrogen non-HB accep OA (oxygen HB acceptor), and SA (sulphur HB acceptor) and their interaction 2.2. Docking Modelling and In Vitro Inhibition Studies positioning regarding the ZN-type atom play an important role in the proper docki The effect ligand of AZM-SH as potential molecules inhibitor (Figure 3a). of human Moreover, CAs, specifically the inclusion the hCA IXmediatin of a TZ pseudo-atom isoform, was initially predicted by modelling the docking mechanism of attractive component of the zinc ion allows a new directional tetrahedral AZM-SH into potential fo hCA IX active site. The AZM-less chain A of the 3IAI PDB model [53] was NA atom type and more accurate docking results, with lower binding energies used for the and R determination of the best values anddocking result. The higher binding ligand–receptor efficiencies interaction depicted in the (Figure 3c). experimentally obtained As expected, the highest binding efficiency (93%)standard 3IAI PDB model was taken as a comparative interactive and the lowest binding ener conformation between AZM and hCA IX. 13.89 kcal/mol), RMSD (0.0) and inhibition constant (Ki: 65.36 pM) were obtained The AutoDock4 Zn forcefield conformation wherewas thepreferred sulfonamideover moiety the traditional formed aAutoDock4 forcefield, direct interaction (1.97 Å) wit as its performance in properly reproducing zinc-coordination geometries Zn ion in a tetrahedral orientation, via coordination by the N(13)-atom 2+ has been provenof the pri to surpass the amine. other’s predictions [54]. The Like the reported chosen forcefield AZM-hCA parameters IX interactive consider complex, the Znthe2+ ion is most common biological zinc coordination geometry, the tetrahedral one coordinated (3.06 Å) by one of the adjacent O-atoms of the sulfonamide (Zn 3.1 and Zn ), 4.0moiety (F where AutoDock atom types NA (nitrogen HB acceptor), N (nitrogen non-HB 3b). Likewise, the non-coordinating O-atom and the H(13)-atom of the amine, preseacceptor), OA (oxygen HB theacceptor), and moiety, sulfonamide SA (sulphur HB acceptor) were predicted andhydrogen to form their interaction and the bonds with po-GLU106 sitioning regarding the ZN-type atom play an important role in the proper Å) and THR199 (1.98 Å) residues of hCA IX, respectively (Figure S6a) similar docking of to ligand molecules (Figure 3a). Moreover, reported results [26,55,56]. the inclusion of a TZ pseudo-atom mediating the attractive component of the zincbonding Hydrogen ion allows wasa also new calculated directionalto tetrahedral potential occur between the for the hydrophilic =N NA atom type and more accurate docking results, with lower binding energies >N(6)H, =O(4), -S(1)H groups of the ligand, adjacent water molecules and the THRand RMSD values and higher binding PRO201, efficiencies and GLN67 GLN92 (Figurereceptor 3c). residues, forming a continuous H-bond net facing the outer surface of hCA IX active site (Figure S6b,c). Accordingly, the align of the aliphatic C(3) and C(4) of the sulfanylpropanamide segment of AZM-SH regression (Figure 3c). The obtained AZM-SH IC50 of 52.4 ± 0.5 nM (n = 3) is well within the same order of magnitude as that reported by the kit’s manufacturer for the standard AZM (IC50 = 16.3 ± 2.0 nM), and as other IC50 reported in the literature for other AZM derivatives [55,57]. The assay confirmed the final product’s efficient inhibition activity Int. J. Mol. Sci. 2023, 24, 6612 6 of 23 towards CAs, namely hCA IX, thus supporting its use as potential targeting agent in future anticancer therapies. Figure Figure (a) Tetrahedral 3. (a)3. Tetrahedral zinczinc geometry, with geometry, with TZ TZ pseudo-atom pseudo-atom located at the located unoccupied at the cornercorner of unoccupied of the ideal coordination tetrahedral geometry. (b) AZM-SH zinc coordination the ideal coordination tetrahedral geometry. (b) AZM-SH zinc coordination via NA (nitrogen via NA (nitrogen acceptor) acceptor) atom-type atom-type interactionwith interaction withthe theattractive attractive component component(TZ (TZpseudo-atom) pseudo-atom) of the of zinc ion. ion. (c) the zinc (c) CAs relative activity in the presence of AZM-SH and respective CAs relative activity in the presence of AZM-SH and respective Boltzmann regression Boltzmann regression for the for the determination determination of of itsitsICIC . Docking performances . Docking 5050 performances forfor similar conformational similar conformationalmodulations, using using modulations, AutoDock4 and AutoDock4 AutoDock4 and AutoDock4Zn forcefields. Zn forcefields. As expected, the highest binding efficiency (93%) and the lowest binding energy 2.3. NANO3-SH Characterization (−13.89 kcal/mol), RMSD (0.0) and inhibition constant (Ki: 65.36 pM) were obtained for Asademonstrated conformation where the sulfonamide in a previous work ofmoiety formed[34], our group a direct interactionof the synthesis (1.97 Å) NANO3-SH with the Zn2+ ion in a tetrahedral orientation, via coordination by the N(13)-atom of the resulted in well-defined spherical, single core–shell particles with an average diameter of primary amine. Like the reported AZM-hCA IX interactive complex, the Zn2+ ion is also 60 nm and a shell porous matrix with pores of ca. 2.5–3.0 nm. coordinated (3.06 Å) by one of the adjacent O-atoms of the sulfonamide moiety (Figure 3b). The assessment Likewise, of NANO surface the non-coordinating O-atomcharges by DLS and and the H(13)-atom of zeta potential the amine, throughout present in the the synthetic steps moiety, sulfonamide was anwereimportant predictedtool to not to form only quickly hydrogen confirm bonds with surface the GLU106 (2.72modifications Å) and THR199 (1.98 Å) residues of hCA IX, respectively (Figure S6a) similar to other reported results [26,55,56]. Hydrogen bonding was also calculated to occur between the hydrophilic = N(8)-, >N(6)H, =O(4), -S(1)H groups of the ligand, adjacent water molecules and the THR200, PRO201, and GLN67 GLN92 receptor residues, forming a continuous H-bond network facing the outer surface of hCA IX active site (Figure S6b,c). Accordingly, the alignment of the aliphatic C(3) and C(4) of the sulfanylpropanamide segment of AZM-SH with internal hydrophobic moieties of the LEU198, LEU141, VAL121, and VAL143 residues (Figure S6d) contributes to the overall stability of the obtained conformation. A similar conformation was obtained using the standard AutoDock4 forcefield, and without the inclusion of the TZ pseudo-atom, but only for a lower binding efficiency and higher binding energies, RMSD value, and Ki (Figure 3c). Int. J. Mol. Sci. 2023, 24, 6612 7 of 23 The preferable AutoDock4Zn predicted ligand–receptor interactions and spatial ar- rangement (Figure S6) indicate that the chemical modification of the original AZM to include the sulfanylpropanamide segment, has little to no hampering impact on the in- hibition role of the final AZM-SH ligand, making it a good potential inhibitor for the CRC-associated hCA IX isoform. Experimental inhibition activity of the synthesised AZM-SH against CAs was assessed using an esterase-mediated assay and calculated after appropriate Boltzmann regression (Figure 3c). The obtained AZM-SH IC50 of 52.4 ± 0.5 nM (n = 3) is well within the same order of magnitude as that reported by the kit’s manufacturer for the standard AZM (IC50 = 16.3 ± 2.0 nM), and as other IC50 reported in the literature for other AZM derivatives [55,57]. The assay confirmed the final product’s efficient inhibition activity towards CAs, namely hCA IX, thus supporting its use as potential targeting agent in future anticancer therapies. 2.3. NANO3-SH Characterization As demonstrated in a previous work of our group [34], the synthesis of NANO3-SH resulted in well-defined spherical, single core–shell particles with an average diameter of 60 nm and a shell porous matrix with pores of ca. 2.5–3.0 nm. The assessment of NANO surface charges by DLS and zeta potential throughout the synthetic steps was an important tool to not only quickly confirm surface modifications but also evaluate NANO3’s polydispersity and stability in the selected medium. This factor has a high impact on the performance of the nanovectors by having an active role in drug- delivery and cell-uptake processes [58]. As depicted in Figure 4, the thiolation of NANO3 surface via EDC/NHS cross-linkage of SiQD available amines and TGAc carboxylic groups rendered the final NANO3-SH with increased stability in the physiological medium, trans- lated by an increase in zeta potential (ζ) from +12.4 mV to +21.5 mV and a clear reduction in its hydrodynamic diameter and polydispersity. The more robust net positive charge after thiolation makes NANO3-SH particularly interesting for cellular therapeutic applications, namely CRC therapy, as positively charged particles have been recurrently reported to have preferential binding activity to cell membranes and increased uptake [58]. Moreover, en- hanced cellular uptake has been reported for several thiol-containing nanoparticles, which covalently bind/interact with cell surface thiol groups, increasing 100-fold the membrane transfection efficiency and endocytosis [59]. The introduction of thiol terminal moieties to NANO3-SH systems sought also to cre- ate binding points for further biofunctionalization with anti-CA IX and anti-EGFR species. Consequently, it was not only important to assess the thiolation ratio but also necessary to ac- tivate these thiol moieties and catalyse further linkages. Ellman’s reagent (DTNB) has been repeatedly reported as a good visual and spectroscopical agent for the estimation of disul- phide bonds and existing thiol moieties, owing to the surgency of a quantifiable absorbance band at 412 nm (translated by a yellow solution) [60–62] (Figure 4). Thiolation was applied to bare NANO3-SH (0.33 ugSH /mgNANO ) and to its loaded counterparts, NANO3@D- SH (0.28 ugSH /mgNANO ), NANO3@DO-SH (0.25 ugSH /mgNANO ), and NANO3@O-SH (0.38 ugSH /mgNANO ), rendering them with 0.25, 0.25, 0.19 and 0.29 nmol possible S-binding, active sites for further modification with thiol-containing biomolecules/small molecules such as the CTX-SH and AZM-SH synthesized herein. A last assessment of the NANO3-SH physical stability was performed by expos- ing the nanosystem to simulated in-body conditions, this is, at 37 ◦ C under continuous stirring in 0.01 M PBS at physiological pH, for one month. From the TEM micrographs (Figure S7), a total decomposition of the structured material is seen with all magnetic cores segregated from the present silica, and the latter spread through the whole field with no structured order. Int. Int.J.J.Mol. Mol.Sci. 2023,24, Sci.2023, 24,6612 x FOR PEER REVIEW 98 of of 23 25 Figure4.4.(a) Figure (a)DLS DLShydrodynamic hydrodynamicdiameter diameterof ofNANO2 NANO2to toNANO3-SH NANO3-SHsystems; systems;(b) (b)respective respectivesurface surface ζ;ζ;(c) (c)molecular molecularinteraction interactionof ofEllman’s Ellman’sreagent reagentwith withthiol thiolmoieties moietiesandandcorresponding correspondingspectroscopic spectroscopic response in response in obtained supernatants supernatants(i.e., (i.e.,absorbance absorbanceband bandredred shift to 412 shift nm)nm) to 412 uponupon the formation of the forma- TNB-free tion species; of TNB-free NANO3-SH species; NANO3-SHsystems spectroscopic systems signature spectroscopic uponupon signature thiolthiol determination and determination activation. and activation. 2.4.Loading 2.4. Loadingand andRelease ReleaseAssays. Assays.Proof Proofof of Concept: Concept: Enteric Enteric Coatings Coatings andand Gastric Gastric Simulated Simulated Release Release The empty porous matrix of NANO3 was used to load both a model anticancer and an antibiotic, The empty in porous single and dual matrix of formulations, NANO3 was used to exploit to load existent both a synergetic effects and model anticancer be- tween them such an antibiotic, in assingle improvedand therapeutic activity and dual formulations, release existent to exploit parameters, as previously synergetic effects demonstrated between thembysuch our group [34]. The therapeutic as improved loading of both drugsand activity wasrelease performed in an inter- parameters, as mediate stepdemonstrated previously of NANO3-SH by synthesis, our groupvia electrostatic [34]. The loadinginteractions of both drugswith non-templated was performed in NANO2-COOH, an intermediate before step ofpores cappingsynthesis, NANO3-SH with SiQDs viaand thiolated interactions electrostatic species. Accessibility with non- to pores is high at this stage, where surface moieties templated NANO2-COOH, before pores capping with SiQDs and thiolatedare yet malleable and do not pro- species. duce a high steric Accessibility to poresimpediment is high at onthisdrug stage,migration. Moreover, where surface theare moieties correct selectionand yet malleable of loading do not media’s producepH (pH 7.4) a high wasimpediment steric important toonmaximize attractiveMoreover, drug migration. interactions between the correct the negatively selection charged -COOH of loading media’s surface pH (pH moieties, pores, 7.4) was silanol groups, important and bothattractive to maximize doxoru- bicin (DOX) (pKa-NH = 8.2, pKa(phenol) = 9.5) and ofloxacin (OFLO) interactions between the negatively charged -COOH surface moieties, pores, silanol 2 (pKa-COOH = 6.1, groups, and both doxorubicin (DOX) (pKa-NH2 = 8.2, pKa(phenol) = 9.5) and ofloxacin Int. J. Mol. Sci. 2023, 24, 6612 9 of 23 pKa(piperizinyl ring) = 8.2) [63,64], which are partly positively charged at physiological pH (Figure S8a). DOX and OFLO were successfully loaded inside NANO2-COOH, and its drug content was evaluated by UV-Vis (λDOX = 480 nm; λOFLO = 330 nm) at each surface modification step, till the final product NANO3-SH, before its biofunctionalization (Figure S8b). The obtained loaded nanosystems and its corresponding drug content are NANO3@D-SH with 287.5 µgDOX /mgNANO , NANO3@DO-SH with 274.0 µgDOX /mgNANO and 140 µgOFLO / omgNANO , and NANO3@O-SH with 182.8 µgOFLO /mgNANO . The high encapsulation ca- pacities are in accordance with those reported in previous studies for similar mesoporous systems and selected drugs, with OFLO successfully replicating the results obtained in other works by our group [65–68]. Regarding NANO3@DO-SH formulation, the rela- tively reduced DOX solubility at the selected medium pH contributes to the preferential loading of DOX, in detriment to OFLO encapsulation. The pH-dependent release capac- ity of each loaded system was performed with their non-biofunctionalized versions (i.e., NANO@drugs-SH, drugs = D, O, DO), to simulate the prominent stressful conditions in col- orectal cancer microenvironments where there is redox imbalance and high levels of GSH promote disulphide bonds breakage [69,70]. CTX-SH and AZM-SH disulphide bridges with NANO3-SH systems are mostly broken in such conditions, with NANO3@drug–SH systems being representative of the release interface during its therapeutic action, either inside or outside CRC cells. The simulation of cancer microenvironment and its role in drug delivery from NANO3@drug–SH nanosystems was achieved by studying drug release in both physiological (pH 7.4) and acidic (pH 5.0) conditions (Figure 5a,b) for 72 h. When compared with the results obtained in previous studies [34] for non-thiolated NANO3 systems, under the same conditions, one concludes that although the final functionalization with thiol moieties does not change the release tendencies for different pHs, it changes the total released quantities of drugs. This effect is particularly significant for total released OFLO from both NANO3@O-SH and NANO3@DO-SH, which see, respectively, a 10-fold and 3-fold reduction when compared to their non-thiolated counterparts. Notwithstanding, the presence of DOX in a combinatory formulation promotes, again, a higher release of OFLO (although lower than that of the aforementioned NANO3 systems). The slow release of OFLO from NANO3 systems is, however, an interesting result, as a more time-paced delivery of the antibiotic could promote an initial therapeutic burst of anticancer activity by DOX, followed by a later, long-term, and continuous antimicrobial action of OFLO. Conversely, DOX release either remains almost invariable or is enhanced. While for NANO3@D-SH released DOX at pH = 7.4 is the same as that of NANO3@D, at acidic conditions, it sees a 2-fold increase. Regarding NANO3@DO-SH, no changes were seen when compared to its non-thiolated counterpart. Overall, the release of drugs, DOX in particular, is favoured in acidic microenvironments and in DOX:OFLO dual formulations. As a proof-of-concept, NANO3@drugs-SH systems underwent an extra modification: a layered protective enteric coating with Eudragit® polymers (PMMA-MMA copolymers). These enteric polymers are well-known protective excipients for active pharmaceutical compounds, capable of regulating their release at different conditions of interest, be they time-based, pH-based or temperature-based [41,71,72]. Of all available Eudragit® poly- mers, E-S100 and E-L100 polymers were selected based on their renowned reputation for colon-targeted drug delivery [73,74] and their dissolution pH thresholds, with the former preferably dissolving at pH > 6.0 and the later at pH > 7.0. Their dissolution pH coincides with those of small intestine and colon sections (i.e., ascending, transverse, and descending), where a proper dissolution of their polymeric matrix promotes the release of encapsulated NANO3 systems and the effective/local delivery of their targeting and therapeutic agents to CRC cells. Int.Int. J. Mol. J. Mol. Sci.Sci. 24,24, 2023, 2023, 6612 PEER REVIEW x FOR 10 12 of 23 of 25 Figure Figure 5. 5. (a) (a) Ofloxacin Ofloxacin (OFLO) and (b) (OFLO) and (b)doxorubicin doxorubicin(DOX) (DOX)pH-stimulated pH-stimulated release release profiles profiles fromfrom NANO@O-SH, NANO@O-SH,NANO@DO-SH NANO@DO-SH and and NANO@D-SH, NANO@D-SH,atatpH pH7.47.4and and 5.0, 5.0, with with thethe latter latter significantly significantly boosting boosting DOX DOX release; (c) simulated release; (c) simulateddigestive digestivetrack trackrelease release from from enteric-coated enteric-coated NANO3@drugs NANO3@drugs systems (n = 3), with medium pH and exposure time fitting the biological conditions systems (n = 3), with medium pH and exposure time fitting the biological conditions of the of the selected selected sections of the digestive tract (yellow—stomach; blue—small intestine; green—colon). sections of the digestive tract (yellow—stomach; blue—small intestine; green—colon). 2.5. Biofunctionalization The biofunctionalization of the already activated NANO3-SH systems with the previously modified CTX-SH and the synthesized AZM-SH was achieved by promoting Int. J. Mol. Sci. 2023, 24, 6612 11 of 23 Two distinct encapsulation approaches were performed to assess the effective delivery to the colon and understand the protective role of the enteric matrices in NANO3 systems delivery: (i) a single encapsulation with E-S100 (i.e., NANO3@drugs-S100) and (ii) a dual 1:1 encapsulation with E-S100 and E-L100 (i.e., NANO3@drugs-SL100). The successful coating of NANO3-SH and NANO3@drug–SH particles was followed by zeta potential after the addition of each enteric coating as a total inversion to negative surface charges was expected [72]. The total inversion of their zeta potential (in deionized water, pH ~6.5), from a net-positive thiolated system to a net-negative system with average ζ = −24.0 ± 1.7 mV, confirmed indeed the successful coating by E-S100. A secondary coating with E-L100 was again confirmed by zeta potential, with obtained NANO3-SH materials showing consistent negative zeta potentials. A simulated digestive track delivery and colonic-targeted release were achieved by exposing coated NANO3 systems to different pHs and stirring times, in accordance with the conditions and passage times typical of each section. As perceived in Figure 5c, and in line with previously reported works on E-S100 and E-L100 [74,75], the overall release of drug-loaded nanoparticles in the simulated digestive fluids is higher at colonic pH than at small intestine and stomach pH. This demonstrates the capacity of the enteric coatings to protect the encapsulated NANO3@drugs-SH systems from external conditions, conditionate its release for the appropriate section of the digestive track, and increase its bioavailability. Regarding single E-S100 and dual E-SL100 formulations, the results show that at a pH of 1.75 and 6.5, the dual enteric formulation shows an overall better capacity to control/retard the release by dispensing less NANO3@drugs-SH to the medium than E-S100 formulations at the same pHs. The early relaxation and dissolution of the E-S100 expedite NANO3@drugs-SH exposure to surrounding media and the release of the loaded drugs at an early stage. This tendency is delayed in the dual-enteric formulations, for which, at only a pH of 7.4 (i.e., in the colon), the external E-L100 coating is dissolved and a higher dispensing of nanoparticles to the surrounding media is permitted. The obtained results are in accordance with other reports that use the same model drugs [76,77] supporting the application of these enteric coatings in the oral administration of a sustained delivery to the colon. 2.5. Biofunctionalization The biofunctionalization of the already activated NANO3-SH systems with the pre- viously modified CTX-SH and the synthesized AZM-SH was achieved by promoting the formation of disulphide bonds between nanoparticles and biological species. Biofunction- alized nanoparticles were obtained in three distinct final formulations: (1) single func- tionalization with thiolated CTX-SH (NANO3-CTX and NANO3@drugs-CTX), (2) single functionalization with AZM-SH (NANO3-AZM and NANO3@drugs-AZM); and (3) dual functionalization with CTX-SH and AZM-SH (NANO3-CTX and NANO3@drugs-CTX). A summary of all final formulations is depicted in Table 1. Surface-bound CTX content in NANO3 systems was assessed by forced reduction in cross-linking disulphide bonds and gel electrophoresis quantification (free CTX was used as standard) (Figure S9). Surface-bound AZM content was confirmed and determined by UV- vis spectroscopic analysis, at 412 nm, of the differential content of generated free TNB upon biofunctionalization of the activated (TNB containing) NANO3-SH/NANO3@drugs-SH, with AZM or CTX+AZM. As shown in Table 1, a significant increase in the co-functionalization of CTX and AZM occurred in the dual-loaded NANO3 systems when compared to non-loaded NANO3 systems. This might have resulted from a higher availability of surface thiol groups, which are derived from an increased surface charge provided by the dual loading of OFLO and DOX (35 ± 0 mV for the dual loaded nanosystem, and 21.5 ± 1.0 mV for the non-loaded NANO3-SH). A similar trend is seen for NANO3@D-SH which has a ζ = 20 ± 0 mV, com- parable to that of the empty NANO3-SH. This will result in fewer cross-linking phenomena Int. J. Mol. Sci. 2023, 24, 6612 12 of 23 between surface thiols and, subsequently, in a higher biofunctionalization rate for AZM and CTX. When regarding OFLO-containing NANO3 systems, there is also a variation in the surface charge (26.6 ± 0.9 mV); however, it is not as significant as that provided by the dual loading. This results in a more efficient biofunctionalization of the CTX and AZM molecules than that presented by the non-loaded system, but not as effective as that provided by the dual loaded system. Table 1. Summary of all final NANO3 formulations, with drug and targeting species content. Drug Loading Biofunctionalization NANO3 DOX OFLOX CTX AZM-SH CTX AZM-SH Sample (mg) (mg/gNANO ) (mg/gNANO) (mg/gNANO ) (mg/gNANO ) (#/NANO) (#/NANO) NANO3-CTX 5.0 - - 13.65 - 5.54 - NANO3-AZM 5.0 - - - 0.27 - 58.91 NANO3-CTX+AZM 5.0 - - 5.62 0.33 2.28 72.39 NANO3@D-CTX 5.0 278.5 - 14.12 - 5.73 - NANO3@D-AZM 5.0 278.5 - - 0.32 - 69.85 NANO3@D- 5.0 278.5 - 7.05 0.22 2.86 47.64 CTX+AZM NANO3@DO-CTX 5.0 274.0 140.0 8.38 - 3.40 - NANO3@DO-AZM 5.0 274.0 140.0 - 0.48 - 106.87 NANO3@DO- 5.0 274.0 140.0 11.18 0.61 4.54 133.80 CTX+AZM NANO3@O-CTX 5.0 - 182.8 9.76 - 3.96 - NANO3@O-AZM 5.0 - 182.8 - 0.42 - 93.41 NANO3@O- 5.0 - 182.8 6.13 0.35 2.49 77.97 CTX+AZM All samples were resuspended in 1 mL PBS 0.01 M (pH 7.4) and then dried under vacuum. #/NANO is equivalent to the number of molecules per nanoparticle. The occurring variations in surface charge are a response to the presence of one or two drugs and are influenced by the loading mechanisms of the drugs to the particles. These are modulated by their water solubility and electrostatic interactions between drugs and parti- cles. The presence of ofloxacin always results in a higher surface charge, possibly due to the presence of a fluor atom in the molecule, which increases stereochemical impediment in the porous matrix, but is also modulated by ofloxacin solubility in water (1.4 mg/mL), which is higher than that presented by doxorubicin (0.3 mg/mL) [63,78]. With DOX presenting thus a lower water solubility, a loading into a more internal section of the mesoporous matrix is favoured, which is detrimental to a more external OFLO encapsulation. 2.6. Cytotoxicity, Antitumor Activity, Cellular Uptake, and Targetability The cytotoxicity of the biofunctionalized NANO3 systems was assessed against HCT- 116 and HT-29 cell lines, as representative CRC models (Figure 6b). These cells were selected due to their hCA IX and EGFR overexpression, which was confirmed by Western blot, where high intensity bands are visible for anti-EGFR and anti-CA IX in the HCT-116 cell line and HT-29 cell line, respectively. These results indicate an EGFR overexpression in HCT-116 cells, whereas for the HT-29 cell line, there is an overexpression of the hCA IX receptor. The housekeeping α-tubulin protein was used as a control to normalize and ensure the presence of a similar cell concentration between the two lines (Figure 6a). As per Figure 6b, the absence of the toxicity of the free OFLO supports the non- toxic role of the antibiotic in its nanometric formulations, which is further confirmed by the absence of toxicity from NANO3@O systems, regardless of the chosen CRC cell line. An equal lack of cytotoxicity was also observed for the empty NANO3 systems, which demonstrates the systems’ biocompatibility and possible usage in a human directed therapy. This is aligned with the cytotoxic profiles reported in the literature for similar Int. J. Mol. Sci. 2023, 24, 6612 13 of 23 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 15 of 25 composite nanomaterials, whose constituents have shown non-cytotoxicity to virtually null cytotoxicity [8,79,80]. Figure Figure 6. 6. (a) (a) Western Western blot blot for for the the overexpression overexpression levels levels of of EGFR EGFR and and CA CA IXIX in in HCT-166 HCT-166 and and HT-29 HT-29 CRC cell lines; (b) IC50 values of biofunctionalized NANO3 and NANO3@drugs against HCT-116 CRC cell lines; (b) IC50 values of biofunctionalized NANO3 and NANO3@drugs against HCT-116 and and HT29 CRC cell lines, for 72 h. Concentrations are given as equivalent DOX concentrations (in HT29 CRC cell lines, for 72 h. Concentrations are given as equivalent DOX concentrations (in µM); µM); (c) internalization rates of different biofunctionalized NANO3@D systems, at different (c) internalization equivalent rates of different DOX concentrations (5,biofunctionalized 10, and 25 µM)NANO3@Din HCT-116 systems, at different and HT-29 CRC equivalent cell lines. DOX concentrations Internalization (5, 10,asand rates given the25 µM) influorescence relative HCT-116 and HT-29 of the CRC cell samples lines. against theInternalization fluorescence ofrates the given as the relative nanoparticles fluorescence in the medium of the before samples treatment against the (excitation at fluorescence of thecollected 480 nm, emission nanoparticles at 550in the nm); (d) LCSMbefore medium images (images (excitation treatment scale = 10 at µm) 480ofnm, HCT-116 emissionandcollected HT-29 cell lines at 550 treated nm); for 3 images (d) LCSM h with NANO3@D-CTX (images scale = 10 andµm) of NANO3@D-AZM, HCT-116 and HT-29 according cell linesto theirfor treated specific 3 h withoverexpressed NANO3@D-CTX targets and (untreated cells were used as negative controls). NANO3@D-AZM, according to their specific overexpressed targets (untreated cells were used as negative controls). 3. Materials and Methods The toxicity/antitumor activity of the prepared materials derives thus only from 3.1. Chemicals DOX present in their formulation, having a more accentuated action against the HCT-116 N-hydroxysuccinimide (NHS. 98+%), ethylenediaminetetraacetic acid tetrasodium line than the HT-29 line. The obtained IC50 for the biofunctionalized NANO3 systems salt hydrate (EDTA. 98%), and tetraethyl orthosilicate (TEOS. ≥ 99.999% metals basis) were account for the total released DOX after 72 h and are in line with those obtained for the bought from AlfaAesar (Haverhill, MA, USA). N-3-dimethylaminopropyl-n- free drug. This demonstrates that the introduction of a protective nanometric material, ethylcarbodiimide hydrochloride (EDC. ≥ 97%), acetone (≥99.5% GC), 4- capable of shielding DOX from harsh external conditions and in-body pharmacokinetics morpholineethanesulfonic acid (MES. ≥ 99%). thioglycolic acid (TGAc. 98%), 5.5′- (i.e., absorption, distribution, metabolism, and excretion) [81–83], does not hinder the dithiobis(2-nitrobenzoic acid) (Ellman’s Reagent. DTNB. ≥ 98%), 2-2-iminothiolane efficacy of the encapsulated drug and drug delivery. hydrochloride (≥98% (TLC)). 5-acetamino-1.3.4-thiadiazole-2-sulfonamide To better understand the possible influence of the different biofunctionalizations (CTX, (Acetazolamide. AZM. ≥ 99%), 3-mercaptopropionic AZM and CTX+AZM) in the NANO3 systems, internalization acid assays (99%),were anhydrous conducted N.N- for dimethylformamide (DMF. both cell lines. Exposure 99.8%), periods were anhydrous tetrahydrofuran predetermined (THF. the at 3 h to evaluate 99.9%), short TRIS time (hydroxymethyl)aminomethane effects of each biofunctionalization hydrochloride (TRISand in cellular uptake HCl), doxorubicin at 72 h to assess hydrochloride the effects of a (DOX. 98.0–102.0% (HPLC)), DL-Dithiothreitol (DTT), β-mercaptoethanol maximum cumulative uptake. After the predetermined exposure times, cells were (molecular stained biology grade), oxalyl chloride (>99%), and phosphate-buffered saline and visualized by laser scanning confocal microscopy (LSCM). As depicted in Figure(PBS tablets) were 6d, purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl. three distinct fluorescent signals were collected, referring to cellular membrane staining 37%) and sodium (green), hydroxide (NaOH) nuclei position (blue),were andbought at Scharlab.(red). DOX distribution SL (Barcelona, Spain).localized DOX was mostly Ofloxacin in (OFLO. 98%) was bought from ThermoFisher (Waltham, MA, USA). Deuterated the cytoplasmic environment of both cell lines, confirming the successful internalization of reagents as dimethyl sulfoxide-D NANO3@D-CTX 6 (DMSO-d6). Acetone-D and NANO3@D-AZM nanosystems.6 and acetonitrile-D3 (CD3CN) were purchased from Acros Organics (Geel, Belgium). Anti-EGFR A quantitative assessment of the uptake profiles was thus Cetuximab evaluated(CTX through5.2mg/mL. cellular 100%) was acquired accumulation from assays, Selleckchem in which (Houston, an cells underwent TX,exposure USA). Eudragit-L100 ® period for each and Eudragit- nanoformu- S100® were supplied by Evonik Operations GmbH (Essen, Germany). All reagents and solutions were used as acquired without any further purification. Int. J. Mol. Sci. 2023, 24, 6612 14 of 23 lation of 3 h, similar to those presented in the aforementioned uptake assays with LSCM. Results depicted in Figure 6c show similar internalization percentages in regard to single and dual functionalized nanovectors, indicating a comparable targetability activity from the novel AZM derivative and the commercially available CTX, regardless of the type of biofunctionalization performed. Slight differences can be perceived when assessing internalization values such as a marginal increase in cellular uptake from NANO3@D-AZM (10.26 ± 1.96%; 10.05 ± 0.77%) in comparison to NANO3@D-CTX in the same respec- tive internalization percentages (9.27 ± 0.51%; 8.84 ± 0.59%) for HCT-116 and HT-29 cell lines, respectively, in response to a concentration of 5 µM for both employed nanovectors. Although not excessively different, these alterations point to the possibility of a higher internalization degree in the presence of AZM derivatives as targeting agents. 3. Materials and Methods 3.1. Chemicals N-hydroxysuccinimide (NHS. 98+%), ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA. 98%), and tetraethyl orthosilicate (TEOS. ≥ 99.999% metals basis) were bought from AlfaAesar (Haverhill, MA, USA). N-3-dimethylaminopropyl-n-ethylcar- bodiimide hydrochloride (EDC. ≥ 97%), acetone (≥99.5% GC), 4-morpholineethanesulfonic acid (MES. ≥ 99%). thioglycolic acid (TGAc. 98%), 5.50 -dithiobis(2-nitrobenzoic acid) (Ellman’s Reagent. DTNB. ≥ 98%), 2-2-iminothiolane hydrochloride (≥98% (TLC)). 5- acetamino-1.3.4-thiadiazole-2-sulfonamide (Acetazolamide. AZM. ≥ 99%), 3-mercapto- propionic acid (99%), anhydrous N.N-dimethylformamide (DMF. 99.8%), anhydrous tetrahy- drofuran (THF. 99.9%), TRIS (hydroxymethyl)aminomethane hydrochloride (TRIS HCl), doxorubicin hydrochloride (DOX. 98.0–102.0% (HPLC)), DL-Dithiothreitol (DTT), β-mer- captoethanol (molecular biology grade), oxalyl chloride (>99%), and phosphate-buffered saline (PBS tablets) were purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrochlo- ric acid (HCl. 37%) and sodium hydroxide (NaOH) were bought at Scharlab. SL (Barcelona, Spain). Ofloxacin (OFLO. 98%) was bought from ThermoFisher (Waltham, MA, USA). Deuterated reagents as dimethyl sulfoxide-D6 (DMSO-d6 ). Acetone-D6 and acetonitrile-D3 (CD3 CN) were purchased from Acros Organics (Geel, Belgium). Anti-EGFR Cetuximab (CTX 5.2 mg/mL. 100%) was acquired from Selleckchem (Houston, TX, USA). Eudragit- L100® and Eudragit-S100® were supplied by Evonik Operations GmbH (Essen, Germany). All reagents and solutions were used as acquired without any further purification. 3.2. Instrumentation ATR-infrared spectra were collected in a Tensor 27 (Bruker, Billerica MA, USA) and 1 H/13 C nuclear magnetic resonance spectroscopy (NMR) performed in a Bruker FT-NMR Avance 400 (Ettlingen, Germany) spectrometer at 300 K from the Departamento de Química, Universidad Politécnica de Valencia. Mass spectroscopy analysis was performed by ESI+QTOF MS in a SCIEX 6600 plus TripleTOF of the SCSIE-UVEG of the University of Valencia. Melting point was determined in a Gallenkamp apparatus (England) from the Bioscope Group LAQV-NOVAFCT. Elemental analysis was performed by CHNS method in an Elemental analyzer Thermo Finnigan-CE Instruments Flash EA 1112 CHNS series from the Analysis Laboratory at LAQV-NOVAFCT. Ninety-six-well plate sampling analysis was performed in an EnSpireTM multilabel reader (Perkin Elmer. Singapore Pte. Ltd., Singapore) from Departamento de Química, Universidad Politécnica de Valencia. Temperature controlled incubations and release studies were performed in an HC24 PCMT Thermo-shaker (Grant Instruments, Cambridge, UK). DLS experiments were per- formed in a Malvern Nano ZS Zetasizer (633 nm laser diode). UV–vis absorption spectra were obtained in a Jasco V-650 spectrophotometer and fluorescence spectroscopy mea- surements in a HORIBA Scientific FLUOROMAX-4 spectrofluorometer from the Bioscope Group LAQV- FCT NOVA and a JASCO FP-8300 spectrophotometer from UPV. Cell images were taken using a laser scanning confocal microscope (LSCM) SP5 (Leica), and cell viability was measured in a multilabel plate reader (Victor 3 PerkinElmer). Int. J. Mol. Sci. 2023, 24, 6612 15 of 23 3.3. Synthesis and Characterization 3.3.1. N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)-3-sulfanylpropanamide (AZM-SH) Synthesis The first step for the synthesis of the novel N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)- 3-sulfanylpropanamide (AZM-SH) was the production of both starting materials: 5- amino-1,3,4-thiadiazole-2-sulfonamide (AZM-NH2 ) and 3-sulfanylpropanoyl chloride. A schematic overview of the complete synthetic route is depicted in Scheme 1. AZM-NH2 was produced following a procedure patented by Kandula [84]. Briefly, 1.5 g (6.75 mmol) of 5-acetamino-1,3,4-thiadiazole-2-sulfonamide (AZM) was thoroughly dissolved in a mixture of ethanol (10.0 mL) and HCl 37% (3.0 mL, 12M) and refluxed for 4.5 h. Refluxing was followed by cooling and solvent evaporation under vacuum. The resulting solid residue was redissolved in MilliQ H2 O (7.5 mL) and neutralized to pH 7.0 with a 5 M aqueous solution of NaOH. Upon neutralization, a white precipitate was formed, which was collected by filtration and recrystallized in water overnight. AZM-NH2 was then collected in the form of white crystals (yield of 40% wt.), ESI+QTOF MS: [C2 H4 N4 O2 S2 +H]+ 180.9848 m/z (error—3.8 ppm). 1 H NMR spectrum (DMSO-d6, 400 MHz) δ (ppm): 7.81 (s, 2H, H11-NH2), 8.05 (s, 2H, H1-NH2). 13 C NMR spectrum (DMSO-d6, 400 MHz) δ (ppm): 158.61 (C2), 172.05 (C5). The product was also characterized by ATR-FTIR. The chlorination of 3-mercaptopropionic acid, for 3-sulfanylpropanoyl chloride forma- tion, was accomplished by a typical Vilsmeier mechanism, as detailed by Mohapatra et al. [85]. A solution of 3-mercaptopropionic acid (0.87 mL, 10.0 mmol), in DCM (5 mL), was firstly cooled to 0 ◦ C, under N2 atmosphere. To this mix, catalytic amounts of anhydrous DMF were added, followed by a dropwise addition of oxalyl chloride (2.0 mL, 20 mmol). The resulting mixture was then slowly stirred for 4.5 h at room temperature. Solvents and excess oxalyl chloride were removed by evaporation under vacuum. The product, 3- sulfanylpropanoyl chloride, was obtained in the form of a dark-brown liquid and charac- terized by 1 H-NMR and 13 C-NMR. For the production of the final product, N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-3-sulfany- lpropanamide, both starting materials were combined in an adapted protocol from those described by de Simone et al. [26] and Bolla and Nangia [86]. Ninety milligrams (0.5 mmol) of AZM-NH2 was dissolved in THF (5 mL) and mixed with 50 µL (1 mmol) of 3-sulfanyl- propanoyl chloride, at room temperature and N2 atmosphere. After 12 h of continuous stirring, the solvent was evaporated under vacuum and the obtained green precipitate was left to dry for 48 h under vacuum. The dry pellet was then redissolved in MilliQ H2 O, tuning from its original green colouration to a brownish-white colour and filtered under vacuum with five consecutive washes in water, dichloromethane, and diethyl ether. The resulting product, N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)-3-sulfanylpropanamide (AZM-SH, FW = 268.34), was dried at 37 ◦ C and collected in the form of brownish- white powder (70.4% wt. yield, mp 203.6 ± 1.0 ◦ C), ESI+QTOF MS: [C5 H8 N4 O3 S3 +H]+ 268.9831 m/z (error—2.5 ppm) and [C5 H8 N4 O3 S3 -H]+ 266.9853 m/z (error—2.2 ppm). El- emental analysis of C5 H8 N4 O3 S3 was found (calculated): C 21.64% (22.38%), H 2.28% (3.00%); N 17.41% (20.88%), S 29.66% (35.85%). 1 H NMR spectrum (CD CN, 400 MHz) δ (ppm): 1.30 (s, 1H, H1 -SH) 2.70–2.97 (dt, 2H, 3 H2 -CH2), 3.23–3.37 (dt, 2H, H3-CH2 ), 6.43 (d, 2H, H13-NH2 , J = 33.8 Hz), 10.62 (br s, H, H6-NH-). 13 C NMR spectrum (DMSO-d6, 400 MHz) δ (ppm): 24.04 (C1), 34.56 (C2), 161.29 (C3), 164.94 (C4), 170.78 (C5). The product was also characterized using ATR-FTIR. 3.3.2. Synthesis of NANO3-SH NANO3 nanoparticles previously synthesized in [34] were obtained using EDC/NHS cross-linkage of NANO2-COOH with the NH2 groups of the synthesized SiQDs, in a SiQD excess to avoid inter-NANO3 cross-linkage. The same strategy was used to thi- olate NANO3 surface NH2 groups with thioglycolic acid (TGA), producing NANO3- SH (see Scheme 2). NANO3-SH activation of its surface thiol moieties was attained us- ing Ellman’s reagent (DTNB), whose colourimetric change from transparent to yellow (412 nm) allows for a simultaneous estimation of the present -SH groups and degree of NANO3 nanoparticles previously synthesized in [34] were obtained using EDC/NHS cross-linkage of NANO2-COOH with the NH2 groups of the synthesized SiQDs, in a SiQD excess to avoid inter-NANO3 cross-linkage. The same strategy was used to thiolate NANO3 surface NH2 groups with thioglycolic acid (TGA), producing NANO3-SH (see Int. J. Mol. Sci. 2023, 24, 6612 Scheme 2). NANO3-SH activation of its surface thiol moieties was attained using Ellman’s 16 of 23 reagent (DTNB), whose colourimetric change from transparent to yellow (412 nm) allows for a simultaneous estimation of the present -SH groups and degree of activation. All materials, at each step, were thoroughly characterized by TEM, DLS, and activation. All materials, at each step, were thoroughly characterized by TEM, DLS, and absorption/fluorescence spectroscopy. absorption/fluorescence spectroscopy. Structures of nanoparticles NANO2-COOH, NANO3, and NANO3-SH. Scheme 2. Structures 3.4. Modelling 3.4. Modelling Studies Studies and and Molecular Molecular Docking Docking Simulations Simulations Docking simulations Docking simulations were were accomplished accomplished in in AutoDock4, AutoDock4, using using the the hCA hCA IXIX crystal crystal structure and active site, from the 3IAI protein complex, available at Protein Data structure and active site, from the 3IAI protein complex, available at Protein Data Bank Bank [53]. Docking parameters were defined according to AutoDock4 [53]. Docking parameters were defined according to AutoDock4 Zn Zn forcefield, adaptedthe forcefield, adapted to to docking the of small docking molecules of small to zinctometalloproteins molecules [54]. Ligand zinc metalloproteins [54]. (AZM-SH) and receptor Ligand (AZM-SH) and (hCA IX) were prepared separately. Ligand coordinates were processed with Openbabel [87] receptor (hCA IX) were prepared separately. Ligand coordinates were processed with software to add hydrogen at pH 7.0. AutoDockTools v.1.5.6 [88] script prepare_ligand4.py Openbabel [87] software to add hydrogen at pH 7.0. AutoDockTools v.1.5.6 [88] script with default settings was used to add Gasteiger–Marsili charges, merge non-polar hy- prepare_ligand4.py with default settings was used to add Gasteiger–Marsili charges, merge drogens, and assign atom types. The number of torsions was set to 7 to include its ro- non-polar hydrogens, and assign atom types. The number of torsions was set to 7 to tatable amide bond. The receptor structure was prepared using AutoDockTools script prepare_receptor4.py with default settings, except the removal of water molecules, to assign Gasteiger–Marsili charges, merge non-polar hydrogens, add polar hydrogens, and assign atom types. The charge of the zinc ion was left to its default value (0.0) when using the Autodock4Zn forcefield. The TZ pseudo-atom responsible for the attractive interaction in the tetrahedral coordination arrangement of zinc was added to the receptor molecule and to the standard forcefield table (Figure S6). Docking search parameters followed a Genetic algorithm, with 50 runs and a population size of 300. Three-dimensional representations of AZM, hCA IX, and docked complexes were performed with UCSF Chimera [89]. Modula- tions with the normal AutoDock4 forcefield, without the presence of the TZ pseudo-atom, were also run as comparison. Best-fitting models were chosen for predictions with the lowest inhibition constant (Ki), the lowest binding energy and RSMD (<2.0 Å), and the highest binding efficiency. 3.5. CA In Vitro Inhibition Assay Experimental in vitro AZM-SH inhibition activity was assessed using the CA Inhibitor Screening Kit (Colourimetric), by BioVision Inc., following the manufacturer’s instruc- tions. The kit uses the esterase activity of an active CA on an ester substrate, producing a chromogenic product easily quantified by absorbance, at 405 nm. In the presence of a CA specific inhibitor, enzymatic activity decreases, accompanied by a reduction in the product’s absorbance. AZM-SH inhibitor 2 mM stock solution was prepared in 0.01 M PBS pH 7.4, and dilutions up to 2 pM were performed in the same buffer. The assay was prepared in a 96-well plate for AZM-SH concentrations of 2 mM, 1 mM, 0.5 mM, 0.2 mM, 20 µM, 2 µM, 20 nM, 2 nM, 0.2 nM, and 2 pM, and the absorbance was collected in a mi- croplate reader at 405 nm, over the course of 60 min for 30 time points. Inhibition constant (Ki) was obtained as in vitro IC50 and compared to that of the standard AZM. Int. J. Mol. Sci. 2023, 24, 6612 17 of 23 3.6. NANO3 Biofunctionalization with CTX-SH and AZM-SH For each of the four 5 mg NANO3-SH samples, 400 µL of a cetuximab (CTX) 1 mg/mL stock solution in 0.01 M PBS pH 7.4 was mixed with 8 µL of a 1.93 mg/mL solution of 2-iminothiolane hydrochloride in 0.01 M PBS (pH 8.5), with 0.1 mM EDTA, for 1 h at 4 ◦ C (in the dark). Each batch of the resulting thiolated cetuximab (CTX-SH) was 4 times centrifuged in a 10 K MWCO Vivacon® membrane at 13.500 rpm and 10 ◦ C for 5 min. The CTX-SH was solvent-exchanged into a final volume of 100 µL of 0.01 M HEPES, with 0.1 mM EDTA ([CTX-SH]final = 4.0 µg/µL). For the biofunctionalization of the already activated NANO3-SH, 5 mg batches were used for each type of modification (i.e., single or dual modification). The 4 µg/µL (27.4 mM) CTX-SH solutions, in 0.01 M HEPES buffer, and a 1 mg/mL (3.73 mM) solution of AZM-SH, in 0.1 M Tris-HCl with 0.1 mM EDTA, were used as stock solutions. Briefly, 5 mg of NANO3 was resuspended in 0.1 M Tris-HCl (pH 8.05) to a total volume of 1.7 mL and mixed for 3 h at 4 ◦ C (in the dark) with (1) 25 µL of CTX-SH stock solution; (2) 53 µL of AZM-SH stock solution; or (3) 25 µL and 53 µL of CTX-SH and AZM-SH stock solution. All samples were recovered by centrifugation, washed twice with 0.01 M PBS (V = 1 mL, pH 7.4), dried under vacuum, and sterilized by UV for 30 min (253.7 nm, 200 V, 50 Hz). 3.7. Drug Loading and Release Assays Ofloxacin (OFLO) and doxorubicin (DOX) were used as antimicrobial and anti-tumoral model drugs and were loaded either alone or conjugated in a 1:1 wt. ratio. A 2 mg/mL dox- orubicin stock solution was prepared in milliQ H2 O and a 2 mg/mL ofloxacin stock solution in 0.01 M PBS (pH 7.0). Loadings were performed in batches of 20 mg of template-free NANO2-COOH. Briefly, 20 mg of NANO2 were resuspended in 3.2 mL of the correspond- ing drug stock solution and diluted in the same volume of the complementary solvent (miliQ H2 O for ofloxacin and 0.01 M PBS for doxorubicin). Co-loading of OFLO and DOX was performed similarly, with 20 mg of NANO2-COOH being resuspended in 3.2 mL of OFLO and 3.2 mL of DOX stock solutions. Each sample was collected by centrifugation (10,000 rpm for 10 min) and washed 4 times with 1 mL of PBS 0.01 M (pH 7.0). All supernatants were isolated and used to estimate, using a mass balance, the loading capacity of NANO2-COOH for each drug, via UV-vis spectroscopy at 330 (OFLO) and 480 (DOX) nm. The loaded nanoparticles were then submitted to the same surface modifications described above, yielding NANO3@D, NANO3@O, and NANO3@DO, and their DOX and OFLO content was assessed by the same procedure. Each product was further modified with CTX-SH and AZM-SH, accordingly. pH-dependent release studies were conducted in PBS 0.01 M, with pH of 7.4, and pH 5.0, at 37 ◦ C. Briefly, 1 mg of each loaded NANO3 was resuspended in 2 mL of buffer solution and shaken for up to 72 h. After centrifugation (12,000 rpm for 5 min), the released drug content in the supernatant was measured for the 5, 15, 30, 45, 60, 90 min, and 2, 3, 4, 24, 72 h time points, by fluorescence: OFLO at pH 7.4 (λexc = 300 nm, λexc = 460 nm), OFLO at pH 5.0 (λexc = 300 nm, λexc = 490 nm) and DOX (λexc = 480 nm, λexc = 560 nm). 3.8. Enteric Coatings and Simulated Digestive Track Release The enteric coating of the final formulations was achieved using Eudragit® polymers, Eudragit® L100 (E-L100) and Eudragit® S100 (E-S100), as model enteric coatings. Two types of coatings were prepared, one with only E-S100 and another with E-S100 followed by E-L100. Then, 0.2% w/v stock solutions of each polymer were prepared in ethanol. Briefly, 1 mg of each nanoparticle formulation was resuspended 1 mL of milliQ H2 O and mixed with 250 µL of E-S100 stock solution overnight, at room temperature (in the dark). With all ethanol evaporated, all NANO3-S100 formulations were recovered by centrifugation (10,000 rpm, 15 min) and kept away from humidity in a desiccator. For further modification with E-L100, the selected products were treated with the same approach, but this time using E-L100 stock solution, yielding NANO3-SL100 formulations. All surface modifications were followed by DLS/Zeta characterization. Int. J. Mol. Sci. 2023, 24, 6612 18 of 23 For a simulated gastric release, all formulations were suspended in the appropriate medium under continuous stirring at 37 ◦ C, for the respective passage time of the simulated gastric section. All samples were collected (11,000 rpm, 3 min) after the appointed time, and the release amount of drugs was spectroscopically quantified. Firstly, the prepared formulations (1 mg) were resuspended in 2 mL of 0.025 M HCl (pH 1.75) for 2 h to simulate stomach conditions, followed by solvent exchange into another 2 mL of 0.01 M PBS (pH 6.5) for 5 h to simulate small intestine conditions and, lastly, by another solvent exchange into 2 mL of 0.01 M PBS (pH 7.0) for 15 h to simulate colonic conditions. Note that in this last part, the samples were quantified after 5 h and 15 h of incubation to assess the release of the drugs at ascending colon conditions and later at the end of the colon/rectum, respectively. 3.9. CA-IX and EGFR Expression in CRC Cell Lines Human colorectal carcinoma cells (HCT 116) and human colorectal adenocarcinoma cells (HT-29) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT116 and HT-29 cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) containing 10% heat-inactivated foetal bovine serum and 1% antibiotic–antimycotic solution (Gibco) at 37 ◦ C in a humidified 10% CO2 atmosphere. HCT-116 and HT-29 cells in exponential growth were collected. 106 cells of each cell line were pelleted, washed twice with PBS, and lysed using M-PER Extraction Reagent (Thermo Scientific). The total protein content was determined via a BCA protein assay (Thermo Scientific). Proteins from extracts from both cell lines were separated by size using SDS-PAGE and transferred into a PVDF membrane for Western Blotting. Epidermal Growth Factor Receptor (EGFR) and Carbonic Anhydrase IX (CA IX) levels were quantified after immunoblotting with anti-EGFR (Sc-03, Santa Cruz Biotechnology) and anti-CAIX (ref. 10107-R003, Sino Biological), respectively. 3.10. Cytotoxicity and Cellular Uptake The cytotoxicity of biofunctionalized NANO3-SH systems (i.e., NANO3-CTX, NANO3- AZM, and NANO3-CTX+AZM) and their loaded counterparts (with DOX and OLFO) was tested in HT-29 and HCT-116 human CRC cell lines using the PrestoBlue assay. Cells were seeded into a 96-well plate at a cell density of 3.0 × 103 cells/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 was added to each well, and the plates were incubated for 4 h at 37 ◦ C. The effect on cell viability was measured using a multilabel plate reader (Victor 3 PerkinElmer). Its fluorescence was quantified at λ = 615 nm (after excitation at λ = 531 nm), and 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 three times, and each concentration was tested in triplicate. For cellular accumulation determination, HT-29 and HCT-116 cells on exponential growth were plated in 24-well plates at a density of 20,000 cells per well. Cells with exponential growth were plated 24-well plates, at a density of 20,000 cells per dish. Cells were grown overnight and treated with 5, 10, and 25 µg/mL of each nanoparticle for 3 h (untreated cells were used as negative controls). After treatment, cells were washed twice with PBS, detached with trypsin, resuspended, and lysed with PBS buffer with 1% of SDS. Fluorescence from lysates was measured in a Cary Eclipse fluorometer (samples were excited at 480 nm, and emission light was collected at 554 nm). The relative fluorescence of the samples was compared to the fluorescence of the nanoparticles in the medium before the treatment. Cellular uptake experiments were also performed on HT-29 and HCT-116 cell lines. Cells with exponential growth were plated in MatTek® 35 mm dishes, at a density of 40,000 cells per dish. Cells were grown overnight and treated with 25 µg/mL of NANO3@D- CTX, NANO3@D-AZM, and NANO3@D-CTX+AZM for 3 h (untreated cells were used as Int. J. Mol. Sci. 2023, 24, 6612 19 of 23 negative controls). After treatment, cells were counter-stained with Hoechst and CellMask and observed in a Confocal Microscope Olympus FV1000. 4. Conclusions In light of the current need for new non-invasive and targeting therapies against CRC, herein, we detail the successful synthesis of a new acetazolamide derivative (AZM-SH), with anti-hCA IX inhibitory activity, dual-cargo, and dual-targeting magneto fluorescent nanosystem (NANO3) as a possible vehicle for a new orally administered anti-CRC ther- apy. Beyond their already confirmed antimicrobial activity, through the inclusion of an OFLO-containing formulation and hyperthermia properties, the NANO3 systems showed consistent and competitive anti-tumoral activity against both tested CRC cell lines. The inclusion of CTX and AZM derivatives as targeting agents at the surface of NANO3 showed benefits for their uptake in HCT-116 and HT-29 cell lines, particularly those functionalized with AZM-SH. Moreover, an oral delivery approach was confirmed through a proof-of- concept modification of the drug-loaded nanoparticles, with the inclusion of a combinatory enteric coating of E-S100 and E-L100 successfully delaying the release in simulated gastric and small-intestine conditions and favouring it in colonic conditions. Supplementary Materials: The supporting information can be downloaded at https://www.mdpi. com/article/10.3390/ijms24076612/s1. Author Contributions: Conceptualization, G.A.M. and E.O.; methodology, G.A.M., D.M., R.M.-M., J.L., C.L. and E.O.; validation, G.A.M., D.M., J.G., E.O. and J.L.; formal analysis, G.A.M., D.M., R.M.-M., J.L., C.L. and E.O.; investigation, G.A.M., D.M. and E.O.; resources, R.M.-M., J.L.C.-M., J.L., C.L. and E.O.; data curation, G.A.M., D.M., J.G., E.O. and J.L.; writing—original draft preparation, G.A.M., J.G. and E.O.; writing—review and editing, all authors; visualization, G.A.M. and E.O.; super- vision, C.L. and E.O.; project administration, C.L. and E.O.; funding acquisition, R.M.-M., J.L.C.-M., J.L., C.L. and E.O. All authors have read and agreed to the published version of the manuscript. Funding: This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the projects UIDB/50006/2020 and UIDP/50006/2020. G.M. acknowledges the financial support 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), as well as the Scientific Society PROTEOMASS (Portugal) for funding support (General Funding Grant). G.M. and J.G. thank FCT/MEC (Portugal) for their doctoral grant PD/BD/142865/2018 and 2022.09495.BD, respectively. E.O. thanks FCT (Fundação para a Ciência e Tecnologia) for funding through the Scientific Employment Stimulus-Individual Call (Ref. CEECIND/00648/2017). J.L. acknowledges the funding received from the Ministerio de Ciencia, Innovación y Universidades, Spain, grant PID2021- 127983OB-C22 funded by MCIN/AEI/10.13039/501100011033. RMM acknowledges the funding received from project PID2021-126304OB-C41 funded by MCIN/AEI /10.13039/501100011033/ and by European Regional Development Fund—A way of doing Europe and Generalitat Valenciana (CIPROM/2021/007). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available online within this article or in the Supplementary Materials. Conflicts of Interest: The authors declare no conflict of interest. References 1. Xi, Y.; Xu, P. Global Colorectal Cancer Burden in 2020 and Projections to 2040. Transl. Oncol. 2021, 14, 101174. [CrossRef] [PubMed] 2. Read, B.; Sylla, P. Aggressive Colorectal Cancer in the Young. Clin. Colon Rectal Surg. 2020, 33, 298–304. [CrossRef] [PubMed] 3. Van der Jeught, K.; Xu, H.C.; Li, Y.J.; Lu, X.B.; Ji, G. Drug Resistance and New Therapies in Colorectal Cancer. World J. Gastroenterol. 2018, 24, 3834–3848. [CrossRef] [PubMed] Int. J. Mol. 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