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Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy

Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy molecules Article Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy 1 1 2 2 3 , 4 João Nogueira , Sofia F. Soares , Carlos O. Amorim , João S. Amaral , Cláudia Silva , 3 , 4 1 1 , Fátima Martel , Tito Trindade and Ana L. Daniel-da-Silva * CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; jh.nogueira@ua.pt (J.N.); sofiafsoares@ua.pt (S.F.S.); tito@ua.pt (T.T.) CICECO-Aveiro Institute of Materials, Department of Physics, University of Aveiro, 3810-193 Aveiro, Portugal; amorim5@ua.pt (C.O.A.); jamaral@ua.pt (J.S.A.) Unit of Biochemistry, Department of Biomedicine, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal; rakelclaudia@hotmail.com (C.S.); fmartel@med.up.pt (F.M.) i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal * Correspondence: ana.luisa@ua.pt; Tel.: +351-234-370-368 Received: 11 December 2019; Accepted: 10 January 2020; Published: 14 January 2020 Abstract: Doxorubicin is one of the most widely used anti-cancer drugs, but side e ects and selectivity problems create a demand for alternative drug delivery systems. Herein we describe a hybrid magnetic nanomaterial as a pH-dependent doxorubicin release carrier. This nanocarrier comprises magnetic iron oxide cores with a diameter of 10 nm, enveloped in a hybrid material made of siliceous shells and k-carrageenan. The hybrid shells possess high drug loading capacity and a favorable drug release profile, while the iron oxide cores allows easy manipulation via an external magnetic field. The pH responsiveness was assessed in phosphate bu ers at pH levels equivalent to those of blood (pH 7.4) and tumor microenvironment (pH 4.2 and 5). The nanoparticles have a loading capacity of up to 12.3 wt.% and a release profile of 80% in 5 h at acidic pH versus 25% at blood pH. In vitro drug delivery tests on human breast cancer and non-cancer cellular cultures have shown that, compared to the free drug, the loaded nanocarriers have comparable antiproliferative e ect but a less intense cytotoxic e ect, especially in the non-cancer cell line. The results show a clear potential for these new hybrid nanomaterials as alternative drug carriers for doxorubicin. Keywords: doxorubicin; magnetic nanoparticles; iron oxide; -carrageenan; drug carrier; pH-responsive 1. Introduction Doxorubicin (DOX, Scheme 1) is an anthracycline antitumor agent, one of the most common and e ective anticancer drugs used in the treatment of various forms of cancer such as lymphomas, sarcomas and breast cancers [1]. The latter in particular is the most common cancer in women, and doxorubicin is one of the main treatments for early and advanced breast cancers [2]. However, doxorubicin presents a number of drawbacks. About 40% of the drug and its metabolites are excreted, and thus, only a small amount of drug actually reaches and acts on the tumor target site [3]. It also causes a notable, well documented cardiac toxicity [4–6]. Another major complication is the possible development of drug resistance mechanisms. Tumor drug resistance contributes to a 90% failure rate of chemotherapy treatments of metastatic disease [7]. Drug resistance can occur via over-expression of cell surface pumps (such as the ATP-binding cassette superfamily) [8], or via changes in DNA repair and cellular apoptosis mechanisms [9]. Molecules 2020, 25, 333; doi:10.3390/molecules25020333 www.mdpi.com/journal/molecules Molecules 2020, 25, 333 3 of 20 3,6-anhydrogalactose units that bears one sulfate group per disaccharide unit [43,44]. Carrageenan-based micro- and nano-scale delivery systems have been reported, often taking advantage of the sulfate content, which results in a negative surface charge that promotes an uptake of cationic target molecules or facilitates functionalization with positively charged components for complex drug carriers [43,45]. Examples of systems reported in the literature include pH-responsive hydrogels for intestinal drug delivery [46], hybrid calcium-biopolymer microparticles for DOX delivery [47,48] and ĸ-carrageenan-coated γ-Fe2O3 nanoparticles with chemotherapeutic potential Molecules 2020, 25, 333 2 of 19 [49]. While there has been research on the combination of magnetic particles with carrageenan for drug delivery [27], there are no reported works combining such systems with DOX. Scheme 1. Chemical structure of doxorubicin (left) and disaccharide unit of κ-carrageenan (right). Scheme 1. Chemical structure of doxorubicin (left) and disaccharide unit of -carrageenan (right). To overcome these chemotherapy issues, one of the promising avenues is the use of nanostructured In the past few years, our research group has developed a new strategy for the preparation of platforms as drug delivery systems [10–12]. Nanosized drug delivery systems can carry drugs to core/shell spheroidal nanoparticles, comprising magnetite cores (Fe3O4) coated with amorphous tumor cells, then penetrate physiological barriers, thus protecting the drug from biological clearance, silica shells [50], or with siliceous shells enriched in polymers from renewable sources including bypass drug resistance mechanisms and finally release the drug specifically on tumor, namely after starch [51], alginate [52], chitosan [53,54] and carrageenan [51,55]. We aimed at creating magnetic triggering by a specific stimulus [3,13]. A drug-loaded carrier can accumulate on tumor sites by bionanocomposites whose ability to interact with target molecular species depends on the chemical exploiting the enhanced permeability and retention (EPR) e ect, first described in 1986 [14], where functionalities of the shells. In this work, κ-carrageenan based magnetic hybrid nanoparticles were tumor vascular permeability is greatly increased compared with healthy tissue [15]. This e ect investigated in the controlled release of DOX. The hybrid particles were tested for pH-responsive occurs in solid tumors leading to enhanced di usion of macromolecules and nanoparticles into DOX release and for the cytotoxic activity against human breast cancer and non-cancer cell lines. The the tumor. Owing to this e ect, it is possible to explore passive tumor targeting by drug-loaded loaded hybrid particles have successfully shown to favor drug release at acidic conditions, as those nanoparticles, having a particle size range between 10 and 100 nm, which has been reported to be found in tumor microenvironments, and to cause cellular damage to human cell lines. optimal for in vivo targeting delivery [13,16]. Additional features can be added to the carrier aiming 2. Results and Discussion to enhance drug delivery performance. For example, the use of pH-responsive biopolymers such as chitosan led to carriers that provide enhanced drug release in the tumor acidic microenvironment 2.1. Characterization of the Magnetic Nanocarriers (due to anaerobic glucose metabolism) and in acidic intracellular organelles (such as endosomes and lysosomes), over release in blood [3,17–21]. Magnetic nanocarriers, either individual or as components The powder XRD pattern of the magnetic cores (Figure 1a) matched the diffraction patterns of hybrid nanomaterials [16,22–26], have also been recognized as drug release vectors having with reported for cubic magnetite (Fe3O4) [56]. The FTIR spectrum of Fe3O4 (Figure 1b) shows a strong several advantages [10,27]. Indeed, magnetic nanocarriers can be guided towards target tissues under −1 band at 552 cm characteristic of the stretching vibration of the magnetite lattice [57]. This a magnetic gradient and eventually be used as contrast agents for magnetic resonance imaging (MRI), −1 vibrational mode is also observed as a band slightly shifted 559 cm , in the FTIR spectrum of the thus allowing for simultaneous cancer treatment and monitoring [24,26]. Furthermore, internalized coated Fe3O4 particles. Two other vibrational are observed in the FTIR spectrum of Fe3O4, one due to magnetic nanocarriers, when exposed to an alternative magnetic field, increase locally the temperature, −1 the stretching of –CO groups at 1349 cm and the other one due to the asymmetric stretching of – which allows us to exploit in a single platform a therapeutic action (magnetic hyperthermia) combined − −1 COO groups at 1569 cm , both owed to the surface citrate ions that stabilize the colloidal magnetic with drug delivery [28,29]. nanoparticles [50]. The FTIR spectrum of Fe3O4@SiĸCRG particles displays the characteristic In the context of doxorubicin based therapies, and in particular its use in breast cancer treatment, −1 vibrational bands of ĸ-carrageenan in the region 1000–1100 cm (C–O and C–OH stretching several studies have reported the use of nanocarriers to enhance doxorubicin-based treatment [2]. −1 −1 vibrations), at 847 cm (α(1-3)-D-galactose C–O–S stretching vibration) and at 1184 cm (O=S=O Hence, liposomal doxorubicin, a system where DOX is encapsulated in a spherical lipid-based −1 asymmetric stretching of sulfonate groups) [58]. The strong band at 449 cm , is ascribed to the O–Si– vesicle, has several variants, either in the market or in clinical stages [30]. Doxil is a polyethylene O deformation [59], which is in agreement with the formation of a siliceous network coating the iron glycol (PEG) based form of liposomal DOX. Known also as Caelyx , was the first market-approved oxide cores. Despite overlapping with vibrational bands from the biopolymer, the bands assigned to nanoformulation containing doxorubicin. As compared to free DOX, it shows comparable eciency −1 amorphous silica [51,60] are observed at 797 and 1036 cm due respectively to symmetric and against metastatic breast cancer, but presents a lower risk of cardiac damage. However, Doxil −1 −1 asymmetric Si–O–Si stretching and at 946 cm due to Si–OH stretching. The band at 1558 cm is presents other side e ects, notably palmar-plantar erythrodysteria (PPE), also known as foot-and-hand ascribed to N–H bending modes coupled to C–N stretching, which confirms the covalent bonding syndrome, characterized by redness, tenderness and peeling of the skin [31]. Another possible side between the carrageenan and the siliceous network through a urethane linkage [61,62]. The band at e ect is an often mild, but possibly severe, allergic reaction caused by some of the formulation’s −1 1645 cm can be either ascribed to adsorbed water (H2O bending mode) or due to the stretching compounds [32]. A commercialized, non-PEGylated but still liposomal alternative is Myocet , using a phosphatidylcholine and cholesterol membrane. While having a lower incidence of PPE, the overall survival rate was not improved, and the formulation has worse hematological toxicity and a higher risk of neutropenia [33]. The final liposomal DOX formulation in the market is Lipo-Dox , which includes PEGylated liposomes as well; however, it fails to show improvements over Doxil regarding ecacy and toxicity [34]. Owing to the drawbacks above mentioned, there is a demand for more e ective nanocarriers for doxorubicin. In the context of breast cancer treatment some attempts have been reported using nanoscale Molecules 2020, 25, 333 3 of 19 systems, including chitosan nanoparticles [35], organosilicon nanoparticles [36,37] and magnetic iron oxide nanocarriers [38]. Magnetic carriers comprising iron oxide nanoparticles functionalized with organic coatings, including azo [23], chitosan [17], polymethacrylic acid [39] and carboxymethyl cellulose [40] are also being investigated. To the best of our knowledge, magnetic nanocarriers based on k-Carrageenan shells have not been explored for DOX release in cancer therapies. Carrageenans are a family of linear sulfated polysaccharides (100–1000 kDa) extracted from marine red algae (Rhodophyceae), widely used as an emulsifier, stabilizers and thickeners in the food and pharmaceutical industries. In human nutrition, they are a good source of dietary fiber and can reduce serum cholesterol and triglyceride levels [41]. Carrageenans have very low toxicity in humans but are known for an inflammatory e ect in some rodents, being frequently used for inflammatory induction in toxicology studies in vivo and have also shown anticoagulant, antiviral and antitumor activities [42]. k-Carrageenan (Scheme 1), composed of d-galactose and 3,6-anhydrogalactose units that bears one sulfate group per disaccharide unit [43,44]. Carrageenan-based micro- and nano-scale delivery systems have been reported, often taking advantage of the sulfate content, which results in a negative surface charge that promotes an uptake of cationic target molecules or facilitates functionalization with positively charged components for complex drug carriers [43,45]. Examples of systems reported in the literature include pH-responsive hydrogels for intestinal drug delivery [46], hybrid calcium-biopolymer microparticles for DOX delivery [47,48] and k-carrageenan-coated -Fe O 2 3 nanoparticles with chemotherapeutic potential [49]. While there has been research on the combination of magnetic particles with carrageenan for drug delivery [27], there are no reported works combining such systems with DOX. In the past few years, our research group has developed a new strategy for the preparation of core/shell spheroidal nanoparticles, comprising magnetite cores (Fe O ) coated with amorphous 3 4 silica shells [50], or with siliceous shells enriched in polymers from renewable sources including starch [51], alginate [52], chitosan [53,54] and carrageenan [51,55]. We aimed at creating magnetic bionanocomposites whose ability to interact with target molecular species depends on the chemical functionalities of the shells. In this work, -carrageenan based magnetic hybrid nanoparticles were investigated in the controlled release of DOX. The hybrid particles were tested for pH-responsive DOX release and for the cytotoxic activity against human breast cancer and non-cancer cell lines. The loaded hybrid particles have successfully shown to favor drug release at acidic conditions, as those found in tumor microenvironments, and to cause cellular damage to human cell lines. 2. Results and Discussion 2.1. Characterization of the Magnetic Nanocarriers The powder XRD pattern of the magnetic cores (Figure 1a) matched the di raction patterns reported for cubic magnetite (Fe O ) [56]. The FTIR spectrum of Fe O (Figure 1b) shows a strong band 3 4 3 4 at 552 cm characteristic of the stretching vibration of the magnetite lattice [57]. This vibrational mode is also observed as a band slightly shifted 559 cm , in the FTIR spectrum of the coated Fe O particles. 3 4 Two other vibrational are observed in the FTIR spectrum of Fe O , one due to the stretching of –CO 3 4 1 1 groups at 1349 cm and the other one due to the asymmetric stretching of –COO groups at 1569 cm , both owed to the surface citrate ions that stabilize the colloidal magnetic nanoparticles [50]. The FTIR spectrum of Fe O @SikCRG particles displays the characteristic vibrational bands of k-carrageenan 3 4 1 1 in the region 1000–1100 cm (C–O and C–OH stretching vibrations), at 847 cm ( (1-3)-d-galactose C–O–S stretching vibration) and at 1184 cm (O=S=O asymmetric stretching of sulfonate groups) [58]. The strong band at 449 cm , is ascribed to the O–Si–O deformation [59], which is in agreement with the formation of a siliceous network coating the iron oxide cores. Despite overlapping with vibrational bands from the biopolymer, the bands assigned to amorphous silica [51,60] are observed at 797 and 1 1 1036 cm due respectively to symmetric and asymmetric Si–O–Si stretching and at 946 cm due to Si–OH stretching. The band at 1558 cm is ascribed to N–H bending modes coupled to C–N Molecules 2020, 25, 333 4 of 19 stretching, which confirms the covalent bonding between the carrageenan and the siliceous network through a urethane linkage [61,62]. The band at 1645 cm can be either ascribed to adsorbed water (H O bending mode) or due to the stretching vibration of hydrogen-bonded C=O in urea, [63] thus indicating the coexistence of urea and urethane groups in the hybrid shells [50,51,55,60]. Molecules 2020, 25, 333 5 of 20 (a) (b) 15 20 25 30 35 40 45 50 55 60 65 70 °2θ (c) (d) (e) Figure Figure 1. 1. Physicochemical Physicochemical characterization characterization of of nanoparticle nanoparticles. s. (a (a ) XRD ) XRDdiffraction pattern, w di raction pattern, iwith th Bragg Bragg reflections identified. (b) ATR-FTIR spectra, with main bands identified. TEM images of Fe3O4 (c) reflections identified. (b) ATR-FTIR spectra, with main bands identified. TEM images of Fe O (c) and 3 4 and Fe3O4@SiκCRG (d) nanoparticles. (e) Histogram for size distribution of Fe3O4 core nanoparticles. Fe O @SiCRG (d) nanoparticles. (e) Histogram for size distribution of Fe O core nanoparticles. 3 4 3 4 Figur e 1c,d show the morphological characteristics of the nanoparticles as assessed by transmission electron microscopy (TEM). The TEM image of the Fe O particles shows spheroidal nanoparticles 3 4 with an average size of 10  2.5 nm, as shown in the particle size histogram (Figure 1e). The TEM Intensity (220) (311) (400) (511) (440) Molecules 2020, 25, 333 5 of 19 images of the coated particles show a thin (5.2 2.1 nm) outer-layer corresponding to the siliceous shell encapsulating the magnetic cores. Elemental microanalysis of the Fe O @SikCRG particles (Table 1) 3 4 indicates the presence of sulphur, which originates from the sulfate groups of the biopolymer that forms the coating. Furthermore, the content of carbon, which in the bare Fe O is due to citrate employed for 3 4 colloidal stabilization, markedly increased after coating the particles. These results are in agreement with the presence of the sulfated polysaccharide k-carrageenan in the coating shells. Note that functionalized used here yielded a marked higher sulphur content (1.27 wt.%) when compared with the results obtained via the conventional multi-step functionalization by covalent grafting of k-carrageenan to amine functionalized silica coated Fe O nanoparticles (0.08 wt.%) [64]. These results are consistent 3 4 with higher biopolymer content at the surface of the nanoparticles using a surface modification approach based on the hydrolytic condensation of the k-carrageenan Si-alkoxide derivative. Table 1. Elemental analysis and textural properties of citrate-capped Fe O and Fe O @SiCRG 3 4 3 4 nanoparticles (D—core diameter, T—coating thickness, S —specific surface area, C,H,S—carbon, BET hydrogen and sulphur content). Sample D (nm) T (nm) C (%) H (%) S (%) S (m g ) BET Fe O 10 2.5 6.55 1.28 152.00 3 4 Fe O @SiCRG 10 2.5 5.2 2.1 13.85 2.27 1.27 13.94 3 4 The BET specific surface area was determined by N adsorption data in the relative pressure (p/p ) 2 1 range 0.01–0.20, and decreased from 151.95 to 13.94 m g after coating (Table 1). This decrease can be explained by the increase in overall particle size after biopolymer incorporation. [51] The surface charge of the Fe O @SikCRG particles was assessed by zeta potential measurements 3 4 at variable pH. The zeta potential was markedly negative, ranging from 34.4  2.1 at pH = 4.2 to 42.2  2.4 mV at pH = 7.4 (Figure 2), which is consistent with the presence of sulfonate groups from carrageenan at the surface of the coated magnetic particles. It is worth noting that in this case the negatively charged surface are highly desirable because they will favor electrostatic interactions with the protonated primary amine groups of doxorubicin, at acidic pH, this promoting the loading of DOX. Furthermore, it should be stressed that at pH 7.4 (physiological medium) the colloidal particles still show a negative zeta potential value that imparts colloidal stability due to interparticle electr Molecules ostatic 2020, 25 repulsions. , 333 6 of 20 Figure 2. Zeta potential titration for Fe O @SiCRG particles. Figure 2. Zeta potential titration for Fe3 3O4 4@SiκCRG particles. Magnetic measurements were performed in a Quantum Design MPMS3 magnetometer for Magnetic measurements were performed in a Quantum Design MPMS3 magnetometer for both both magnetite and the corresponding coated particles. The field dependence of the magnetization magnetite and the corresponding coated particles. The field dependence of the magnetization normalized with the iron oxide content of the nanoparticles measured at 300 K and 5 K is displayed in normalized with the iron oxide content of the nanoparticles measured at 300 K and 5 K is displayed in Figure 3a,b (non-normalized curves are shown in Figure S2, supporting information). The parameters coercivity (Hc), remanent magnetization (Mr) and saturation magnetization (Ms) values obtained from the magnetization curves at 300 K are listed in Table 2. Both bare and coated particles exhibited superparamagnetic-like behavior at room temperature with a hysteresis loop, residual remnant magnetization and coercive field values compatible with the effect of a remnant field of the superconducting coils. This behavior is expected due to the small particle size of the nanoparticles. The observed magnetization saturation was of 25 and 27 emu/g Fe3O4 at 300 K; 32 and 34 emu/g Fe3O4 at 5 K, for bare and coated nanoparticles, respectively. Though magnetic properties of magnetite nanoparticles are dependent on particle size, these values are lower than those typically found in high purity magnetite nanoparticles of similar sizes [65]. These differences may be explained by incomplete crystallization of magnetite, or irregularities in the spheroidal shapes of the particles [66]. The Fe3O4@SiĸCRG particles show an almost identical magnetic saturation when compared to bare nanoparticles. Magnetization versus applied magnetic field data at 5 K show coercivity values around 250 Oe. This suggests a blocking temperature above this temperature, as confirmed via zero-field cooled and field-cooled measurements, shown in Figure 3c,d. The lower blocking temperatures of the Fe3O4@SiĸCRG particles hint at the effect of the coating on the kinetics of the particles during magnetic relaxation. Due to their superparamagnetic quality, the small particles have not only the potential to be a drug release system but also to be used for magnetic resonance imaging (MRI), which translates to a possible theranostic application. It is thus of high interest that these particles show an adequate drug loading and release profile. Molecules 2020, 25, 333 6 of 19 Figure 3a,b (non-normalized curves are shown in Figure S2, Supporting Information). The parameters coercivity (H ), remanent magnetization (M ) and saturation magnetization (M ) values obtained c r s from the magnetization curves at 300 K are listed in Table 2. Both bare and coated particles exhibited superparamagnetic-like behavior at room temperature with a hysteresis loop, residual remnant magnetization and coercive field values compatible with the e ect of a remnant field of the superconducting coils. This behavior is expected due to the small particle size of the nanoparticles. The observed magnetization saturation was of 25 and 27 emu/g Fe O at 300 K; 32 and 34 emu/g Fe O 3 4 3 4 at 5 K, for bare and coated nanoparticles, respectively. Though magnetic properties of magnetite nanoparticles are dependent on particle size, these values are lower than those typically found in high purity magnetite nanoparticles of similar sizes [65]. These di erences may be explained by incomplete crystallization of magnetite, or irregularities in the spheroidal shapes of the particles [66]. The Fe O @SikCRG particles show an almost identical magnetic saturation when compared to bare 3 4 nanoparticles. Magnetization versus applied magnetic field data at 5 K show coercivity values around 250 Oe. This suggests a blocking temperature above this temperature, as confirmed via zero-field cooled and field-cooled measurements, shown in Figure 3c,d. The lower blocking temperatures of the Fe O @SikCRG particles hint at the e ect of the coating on the kinetics of the particles during magnetic 3 4 relaxation. Due to their superparamagnetic quality, the small particles have not only the potential to be a drug release system but also to be used for magnetic resonance imaging (MRI), which translates to a possible theranostic application. It is thus of high interest that these particles show an adequate drug loading and release profile. Molecules 2020, 25, 333 7 of 20 (b) (a) (d) (c) Figure 3. Field dependent magnetization curves (normalized for iron oxide content) of Fe3O4 Figure 3. Field dependent magnetization curves (normalized for iron oxide content) of Fe O 3 4 nanoparticles (a) and Fe3O4@SiκCRG nanoparticles (b). Temperature dependent magnetization nanoparticles (a) and Fe O @SiCRG nanoparticles (b). Temperature dependent magnetization curves 3 4 curves of Fe3O4 nanoparticles (c) and Fe3O4@SiĸCRG nanoparticles (d). of Fe O nanoparticles (c) and Fe O @SikCRG nanoparticles (d). 3 4 3 4 Table 2. Magnetic parameters for Fe3O4 and Fe3O4@SiκCRG particles, at 300 K. Material Ms (emu/gsample) Ms (emu/gFe3O4) Mr (emu/gFe3O4) Hc (Oe) Fe3O4 23 25 1.2 31 Fe3O4@SiĸCRG 15 27 1.9 32 2.2. Doxorubicin Loading The nanoparticles Fe3O4@SiĸCRG were loaded with DOX using a buffer solution at pH = 6, DOX concentration ranging from 110 to 350 μg/mL. The results of loading efficiency and capacity are shown in Table S1. The loading efficiency ranged from 37% to 55% and the loading capacity from 33 to 123 μg/mg (3.3–12.3 wt.%) and has increased with initial DOX concentration in the aqueous solution (Figure 4). These results were similar to values obtained in previous studies using other magnetic carriers, such as chitosan-coated magnetite nanoparticles (18 wt.%) [17] and magnetic polymersomes (12 wt.%) [67]. Molecules 2020, 25, 333 7 of 19 Table 2. Magnetic parameters for Fe O and Fe O @SiCRG particles, at 300 K. 3 4 3 4 Material Ms (emu/g ) Ms (emu/g ) Mr (emu/g ) Hc (Oe) sample Fe3O4 Fe3O4 Fe O 23 25 1.2 31 3 4 Fe O @SikCRG 15 27 1.9 32 3 4 2.2. Doxorubicin Loading The nanoparticles Fe O @SikCRG were loaded with DOX using a bu er solution at pH = 6, DOX 3 4 concentration ranging from 110 to 350 g/mL. The results of loading eciency and capacity are shown in Table S1. The loading eciency ranged from 37% to 55% and the loading capacity from 33 to 123 g/mg (3.3–12.3 wt.%) and has increased with initial DOX concentration in the aqueous solution (Figure 4). These results were similar to values obtained in previous studies using other magnetic carriers, such as chitosan-coated magnetite nanoparticles (18 wt.%) [17] and magnetic polymersomes (12 wt.%) [67]. Molecules 2020, 25, 333 8 of 20 100 140 Efficiency Capacity 0 0 100 150 200 250 300 350 400 [DOX] (μg/mL) initial Figure 4. Uptake curve for doxorubicin, expressed in loading eciency (%) and nanoparticle capacity Figure 4. Uptake curve for doxorubicin, expressed in loading efficiency (%) and nanoparticle (g DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. capacity (μg DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. In aqueous solution, DOX molecules adopt di erent conformations depending on the solution pH In aqueous solution, DOX molecules adopt different conformations depending on the solution (Figure S3, Supporting Information) [68]. At pH 6, it is expected that drug loading is promoted by pH (Figure S3, supporting information) [68]. At pH 6, it is expected that drug loading is promoted by electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the carrageenan. Moreover, hydrogen-bonding interactions between OH groups of carrageenan/siliceous carrageenan. Moreover, hydrogen-bonding interactions between OH groups of network and hydroxyl groups of the anthraquinone ring in DOX will also favor drug loading at this carrageenan/siliceous network and hydroxyl groups of the anthraquinone ring in DOX will also pH. Previous studies have indicated this type of interactions is of uttermost relevance in the formation favor drug loading at this pH. Previous studies have indicated this type of interactions is of of complexes between DOX and dextran sulfate, which is also a sulfated polysaccharide [69]. There uttermost relevance in the formation of complexes between DOX and dextran sulfate, which is also a are also – stacking interactions between the aromatic groups of DOX molecules being brought sulfated polysaccharide [69]. There are also π–π stacking interactions between the aromatic groups close together, which also promotes drug loading [69,70]. As expected, with DOX loading the surface of DOX molecules being brought close together, which also promotes drug loading [69,70]. As of the nanoparticles becomes less negative. Still, the zeta potential of loaded particles ranged from expected, with DOX loading the surface of the nanoparticles becomes less negative. Still, the zeta 17.9 0.9 mV at pH = 4.2 to34.2 2.0 mV at pH = 7.4 (Figure 5), which is indicative of moderate potential of loaded particles ranged from −17.9 ± 0.9 mV at pH = 4.2 to −34.2 ± 2.0 mV at pH = 7.4 colloidal stability [71]. It is worth noting that the zeta potential was assessed in bu ers. In biological (Figure 5), which is indicative of moderate colloidal stability [71]. It is worth noting that the zeta environment and in cell cultures medium the adsorption of proteins may alter the surface properties of potential was assessed in buffers. In biological environment and in cell cultures medium the the nanoparticles and influence the colloidal stability. adsorption of proteins may alter the surface properties of the nanoparticles and influence the colloidal stability. Figure 5. Zeta potential titration for Fe3O4@SiκCRG particles loaded with doxorubicin (loading capacity was 3.2 wt.%). Efficiency (%) Capacity (μg DOX/mg NP) Molecules 2020, 25, 333 8 of 20 100 140 Efficiency Capacity 0 0 100 150 200 250 300 350 400 [DOX] (μg/mL) initial Figure 4. Uptake curve for doxorubicin, expressed in loading efficiency (%) and nanoparticle capacity (μg DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. In aqueous solution, DOX molecules adopt different conformations depending on the solution pH (Figure S3, supporting information) [68]. At pH 6, it is expected that drug loading is promoted by electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the carrageenan. Moreover, hydrogen-bonding interactions between OH groups of carrageenan/siliceous network and hydroxyl groups of the anthraquinone ring in DOX will also favor drug loading at this pH. Previous studies have indicated this type of interactions is of uttermost relevance in the formation of complexes between DOX and dextran sulfate, which is also a sulfated polysaccharide [69]. There are also π–π stacking interactions between the aromatic groups of DOX molecules being brought close together, which also promotes drug loading [69,70]. As expected, with DOX loading the surface of the nanoparticles becomes less negative. Still, the zeta potential of loaded particles ranged from −17.9 ± 0.9 mV at pH = 4.2 to −34.2 ± 2.0 mV at pH = 7.4 (Figure 5), which is indicative of moderate colloidal stability [71]. It is worth noting that the zeta potential was assessed in buffers. In biological environment and in cell cultures medium the adsorption of proteins may alter the surface properties of the nanoparticles and influence the Molecules 2020, 25, 333 8 of 19 colloidal stability. Figure 5. Zeta potential titration for Fe O @SiCRG particles loaded with doxorubicin (loading capacity Figure 5. Zeta potential titration for Fe3O4@SiκCRG particles loaded with doxorubicin (loading 3 4 was 3.2 wt.%). capacity was 3.2 wt.%). Molecules 2020, 25, 333 9 of 20 2.3. Doxorubicin pH Mediated Release Studies 2.3. Doxorubicin pH Mediated Release Studies Figure 6 shows the cumulative release of DOX from the Fe O @Si CRG nanocarriers at pH 4.2, 5.0 3 4 Figure 6 shows the cumulative release of DOX from the Fe3O4@SiCRG nanocarriers at pH 4.2, and 7.4, in bu ered solutions. After 1 h, a burst release occurs in all formulations and the equilibrium is 5.0 and 7.4, in buffered solutions. After 1 h, a burst release occurs in all formulations and the reached after 5 h, at pH 7.4 and pH 5. At pH 4.2 the release of DOX was gradually prolonged up to 48 h. equilibrium is reached after 5 h, at pH 7.4 and pH 5. At pH 4.2 the release of DOX was gradually The DOX release was pH-dependent being markedly faster in acidic conditions. Thus, after 5 h the prolonged up to 48 h. The DOX release was pH-dependent being markedly faster in acidic DOX release was approximately 73% and 80% in acidic conditions (pH 4.2 and 5, respectively) and 25% conditions. Thus, after 5 h the DOX release was approximately 73% and 80% in acidic conditions (pH at pH 7.4. Similar behavior has been observed for pH-responsive DOX nanocarriers based on chitosan 4.2 and 5, respectively) and 25% at pH 7.4. Similar behavior has been observed for pH-responsive coated magnetic nanoparticles [17]. This pH-sensitive behavior of Fe O @SikCRG nanocarriers is of 3 4 DOX nanocarriers based on chitosan coated magnetic nanoparticles [17]. This pH-sensitive behavior high interest for the controlled release of may benefit the release of doxorubicin in tumor cells because of Fe3O4@SiĸCRG nanocarriers is of high interest for the controlled release of may benefit the release the intracellular pH is lower than in the healthy cells [72]. of doxorubicin in tumor cells because the intracellular pH is lower than in the healthy cells [72]. Figure Figure 6. 6. Doxor Doxorubicin release ubicin release over over t time ime f fr rom om l loaded oaded F Fe e3O O 4@@kCRG ĸCRG carriers, carriers, at 37 °C and va at 37 C and riable pH variable pH 3 4 (loading (loading capacity capacity was was 12 12.3 .3 wt. wt.%). %). F Full ull prof profile ile for 48 h for 48 h (( left left ) )and first and first 5 h ( 5 hright (right ). ). It is likely that the release of DOX occurs through a cation exchange mechanism (Figure 7) It is likely that the release of DOX occurs through a cation exchange mechanism (Figure 7) because surface charge of the nanocarriers is negative, not varying significantly across the work pH because surface charge of the nanocarriers is negative, not varying significantly across the work range (Figure 2), and DOX is cationic in acidic conditions. The acidic environment promotes the pH range (Figure 2), and DOX is cationic in acidic conditions. The acidic environment promotes exchange of protons between the primary amine groups of DOX and the sulfonate groups of the exchange of protons between the primary amine groups of DOX and the sulfonate groups of κ-carrageenan [73]. The DOX release was slightly pronounced at pH 5 than at pH 4.2 for the first five -carrageenan [73]. The DOX release was slightly pronounced at pH 5 than at pH 4.2 for the first hours but became equalized within a 24 h period. We interpreted the faster initial release at pH 5 as five hours but became equalized within a 24 h period. We interpreted the faster initial release at pH 5 due to a polymer swelling effect. The outer shell of the nanocarriers contain ĸ-carrageenan in its composition, which is well known to form hydrogels with a pH-dependent swelling behavior [74]. Although in these nanocarriers the ĸ-carrageenan was grafted to the siliceous network forming the outer shells, there were still domains enriched in ĸ-carrageenan that can undergo swelling in an aqueous medium. Carrageenan hydrogels experience increased swelling when the pH increased from acidic to neutral conditions. This swelling effect can enhance the drug release by favoring the diffusion of DOX molecules within the hybrid network [74,75]. Efficiency (%) Capacity (μg DOX/mg NP) Molecules 2020, 25, 333 9 of 19 as due to a polymer swelling e ect. The outer shell of the nanocarriers contain k-carrageenan in its composition, which is well known to form hydrogels with a pH-dependent swelling behavior [74]. Although in these nanocarriers the k-carrageenan was grafted to the siliceous network forming the outer shells, there were still domains enriched in k-carrageenan that can undergo swelling in an aqueous medium. Carrageenan hydrogels experience increased swelling when the pH increased from acidic to neutral conditions. This swelling e ect can enhance the drug release by favoring the di usion of DOX molecules within the hybrid network [74,75]. Molecules 2020, 25, 333 10 of 20 Figure 7. Schematic representation of the proposed cation exchange mechanism behind DOX release Figure 7. Schematic representation of the proposed cation exchange mechanism behind DOX release from the nanocarriers in acidic conditions, with the equation showing the equilibrium shift. from the nanocarriers in acidic conditions, with the equation showing the equilibrium shift. It is the strong swelling behavior experienced by carrageenan at neutral pH that also contributes It is the strong swelling behavior experienced by carrageenan at neutral pH that also to DOX release at pH 7.4. In this case, the release of DOX was slower due to a less extent of contributes to DOX release at pH 7.4. In this case, the release of DOX was slower due to a less extent protonation, which results in lower competition of the protons for the sulphonated sites of k-carrageenan. of protonation, which results in lower competition of the protons for the sulphonated sites of Nevertheless, there is still a 25% drug release because at this pH there will be a number of neutral DOX ĸ-carrageenan. Nevertheless, there is still a 25% drug release because at this pH there will be a molecules less prone to interact with sulfonate groups at the surface of the magnetic nanocarriers. number of neutral DOX molecules less prone to interact with sulfonate groups at the surface of the The release kinetic data were reasonably described by the Weibull model (equation S1) with magnetic nanocarriers. a coecient of determination (R ) in the range 0.926–0.958 (Supporting Information, Figure S4 and The release kinetic data were reasonably described by the Weibull model (equation S1) with a Table S2). This is a general empirical equation that has been successfully applied to several drug release coefficient of determination (R ) in the range 0.926–0.958 (Supporting Information, Figure S4 and systems [76,77]. Table S2). This is a general empirical equation that has been successfully applied to several drug release systems [76,77]. 2.4. Antiproliferative and Cytotoxicity In Vitro Evaluation The proliferation and viability of the cells after their exposure to free doxorubicin (DOX), unloaded 2.4. Antiproliferative and Cytotoxicity In Vitro Evaluation nanoparticles (NP) and nanoparticles loaded with doxorubicin (NP + DOX) are shown in Figures 8 The proliferation and viability of the cells after their exposure to free doxorubicin (DOX), and 9, respectively. The tests were performed in MCF-7 and MDA-MB-231 human breast cancer cells unloaded nanoparticles (NP) and nanoparticles loaded with doxorubicin (NP + DOX) are shown in and in MCF12A non-cancerous human breast epithelial cells. Figures 8 and 9, respectively. The tests were performed in MCF-7 and MDA-MB-231 human breast As expected, free DOX has a concentration-dependent antiproliferative e ect on all cell cancer cells and in MCF12A non-cancerous human breast epithelial cells. lines (Figure 8a). Loaded nanoparticles also present a concentration-dependent antiproliferative e ect As expected, free DOX has a concentration-dependent antiproliferative effect on all cell lines on all cell lines, and this e ect was more marked than the e ect of unloaded nanoparticles (Figure 8b). (Figure 8a). Loaded nanoparticles also present a concentration-dependent antiproliferative effect on The antiproliferative e ect of DOX-loaded NPs is similar to that of free DOX at DOX concentrations of all cell lines, and this effect was more marked than the effect of unloaded nanoparticles (Figure 8b). 50 M, in all cell lines (Figure 8). The antiproliferative effect of DOX-loaded NPs is similar to that of free DOX at DOX concentrations As expected, free DOX shows a concentration-dependent cytotoxic e ect on all cell lines (Figure 9a). of 50 μM, in all cell lines (Figure 8). Both cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous cell line (MCF12A) su er As expected, free DOX shows a concentration-dependent cytotoxic effect on all cell lines (Figure deleterious cytotoxic e ects when exposed to loaded nanoparticles, and again, this e ect is much 9a). Both cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous cell line (MCF12A) more marked than the e ect observed with unloaded nanoparticles (Figure 9b). The cytotoxic e ect of suffer deleterious cytotoxic effects when exposed to loaded nanoparticles, and again, this effect is much more marked than the effect observed with unloaded nanoparticles (Figure 9b). The cytotoxic effect of DOX-NPs is lower than that of DOX itself in all the three cell lines, but this is especially evident in the non-cancerous cell line (Figure 9). Molecules 2020, 25, 333 10 of 19 DOX-NPs is lower than that of DOX itself in all the three cell lines, but this is especially evident in the Molecules 2020, 25, 333 11 of 20 non-cancerous cell line (Figure 9). (a) (b) Figure 8. Cell proliferation after exposure for 24 h to (a) free doxorubicin (M) and (b) unloaded Figure 8. Cell proliferation after exposure for 24 h to (a) free doxorubicin (μM) and (b) unloaded nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; M), for all cell lines. Shown are nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; μM), for all cell lines. Shown are arithmetic means  SEM (n = 6). (a) * significantly di erent from control (p < 0.05; Student’s t test). arithmetic means ± SEM (n = 6). (a) * significantly different from control (p < 0.05; Student’s t test). (b) (b) * significantly di erent from control; significantly di erent from each other (p < 0.05; ANOVA + ns * significantly different from control; significantly different from each other (p < 0.05; ANOVA + Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). ns Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). When comparing the e ect of NP + DOX in the di erent cell lines, we observed that its antiprWhen comparing the effe oliferative e ect was equally ct of NP potent in + the DOX in th three celle different cell lines, we o lines (Figure 8b), but its cytotoxic bserved that e ect was its less potent in MDA-MB-231 cells than in the other cell lines (Figure 9b). So, the antiproliferative and antiproliferative effect was equally potent in the three cell lines (Figure 8b), but its cytotoxic effect cytotoxic was less pot e ect ent in of MDA NP-DOX -MB do -23not 1 ceappear lls than to in t be he ot cellhe type-specific. r cell lines (FThis igureis 9b not ). So surprising , the antiprol as the iferNP ativ+ e DOX e ect was not meant to be selective according to cell type since all cells possess acidic endosomes and cytotoxic effect of NP-DOX do not appear to be cell type-specific. This is not surprising as the that NP + DOX e can provide ffect was not conditionsmeant to be selective for DOX release once the acco particles rding to cell type since are taken up, butall rather cells posse its selectivity ss acidis ic due to local tumor vascularity and acidic tumor microenvironment when in physiological conditions. endosomes that can provide conditions for DOX release once the particles are taken up, but rather its The select loaded ivity nanoparticles is due to loc arael t expected umor v toascul leave arit the y bloodstr and acid eam ic tand umor microen enter in contact vironment with cancer when in cells via the EPR e ect and then begin releasing DOX in their vicinity due to a local decrease in pH. physiological conditions. The loaded nanoparticles are expected to leave the bloodstream and enter in contact wit When comparing h cancerthe cells e via the E ect of DOX PR e inffect the thr and ee cell then begi lines used, n rele we asing DOX i concluded nthat theithe r vinon-cancer cinity due to a ous cell line (MCF12A) appeared to be more susceptible to DOX-induced cytotoxicity, but not more local decrease in pH. susceptible When comparin to the antipr g the e oliferative ffect of DOX e ect than the in the three cancer cell cell lines lines (MCF-7 used, we and MDA-MB-231; concluded that Figuresthe 8 and 9). Moreover, of the cancer cell lines, MDA-MB-231 appears more resistant to the cytotoxic non-cancerous cell line (MCF12A) appeared to be more susceptible to DOX-induced cytotoxicity, but enot more ect of DOX suscepti than bl MCF-7 e to the cells,an but tiprol both ifera cell tive lines effect tha are equally n the ca vulnerable ncer cell li to itsnes (MCF- antiproliferative 7 and e ect. In other words, susceptibility to the cytotoxic e ect of DOX varies in the distinct cell lines, MDA-MB-231; Figures 8 and 9). Moreover, of the cancer cell lines, MDA-MB-231 appears more but resista not nvulnerability t to the cytotoxi toc effect of its antipr DOX tha oliferative n MCF- e ect.7 This cells,is bu in t both cell line with lidoxor nes arubicin’s e equally vul mechanism nerable to of action: it disrupts topoisomerase, preventing cellular replication and ultimately inducing programmed its antiproliferative effect. In other words, susceptibility to the cytotoxic effect of DOX varies in the cellular distinct cel death. l lines, The but specific not vu cytotoxic lnerabilit damage y to its ant and ipro resulting liferative form effect of cell . Thi death s is in induced line with by doxor doxor ubicin’s ubicin varies mechawith nism of concentration, action: it di treatment srupts topoi duration, somerase, specific preventi form of ng cellu cancerla and r repl drug icartion a esistance nd ul [78tima ]. Both tely inducing programmed cellular death. The specific cytotoxic damage and resulting form of cell death induced by doxorubicin varies with concentration, treatment duration, specific form of cancer and drug resistance [78]. Both MDA-MB-231 [79] and MCF-7 [78] cell lines have shown the ability to gain resistance to doxorubicin. Nevertheless, the opposite can happen and some studies have shown a Molecules 2020, 25, 333 11 of 19 Molecules 2020, 25, 333 12 of 20 MDA-MB-231 [79] and MCF-7 [78] cell lines have shown the ability to gain resistance to doxorubicin. lower IC50 for DOX in MDA-MB-231 than in MCF-7 cells [80,81]. Different rates of nanoparticles Nevertheless, the opposite can happen and some studies have shown a lower IC for DOX in uptake, depending on cell type, may also be at play to explain this. Nonetheless, it is clearly shown MDA-MB-231 than in MCF-7 cells [80,81]. Di erent rates of nanoparticles uptake, depending on cell that DOX-NPs disrupts cellular division and reduces cell viability to a comparable level to free type, may also be at play to explain this. Nonetheless, it is clearly shown that DOX-NPs disrupts cellular doxorubicin, and that, differently from what is found with DOX, its antiproliferative effect is not division and reduces cell viability to a comparable level to free doxorubicin, and that, di erently from more marked in non-cancerous cells. Our results thus show the potential of the hybrid nanoparticles what is found with DOX, its antiproliferative e ect is not more marked in non-cancerous cells. Our as a novel anticancer drug delivery system. results thus show the potential of the hybrid nanoparticles as a novel anticancer drug delivery system. (a) (b) Figure 9. Cell viability after exposure for 24 h to (a) free doxorubicin (M) and to (b) unloaded Figure 9. Cell viability after exposure for 24 h to (a) free doxorubicin (μM) and to (b) unloaded nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; M), for all cellular lines. Shown nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; μM), for all cellular lines. Shown are arithmetic means SEM (n = 8). (a) * significantly di erent from control (p < 0.05) (Student’s t test). are arithmetic means ± SEM (n = 8). (a) * significantly different from control (p < 0.05) (Student’s t (b) * significantly di erent from control; significantly di erent from each other (p < 0.05; ANOVA + test). (b) * significantly different from control; significantly different from each other (p < 0.05; ns Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). ns ANOVA + Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman– Keuls test). 3. Materials and Methods 3.1. Materials 3. Materials and Methods Doxorubicin hydrochloride (95%; DOX HCl) was purchased from Thermo Fisher (Waltham, MA, 3.1. Materials USA). -Carrageenan (CRG), 3-(triethoxysilyl)propyl isocyanate (ICPTES) (95%), ethanol (CH CH OH; 3 2 absolute) Doxorubic and i acetone n hydrochloride (C H O; ( 100%) ≥95%; D wer Oe X H pur Cchased l) was p fr u om rcha Honeywell sed from TFluka hermo(Seelze, Fisher (Germany). Waltham, 3 6 MA, U Dimethylformamide SA). κ-Carrage (DMF) enan ( [HCON(CH κCRG), 3-(triet ) ] hoxys was pur ilychased l)propyfr l isoc om Carlo yanate Erba (ICPTES Reagents ) (95 (Chauss %), etha éenol du 3 2 (CH Vexin, 3CH France). 2OH; ab Tso etraethyl lute) anorthosilicate d acetone (C(TEOS; 3H6O; 100% 99%), ) ir were pur on (III) chloride chased from Hon hexahydrate eywell F (FeClluka 6H (See O; 99%), lze, 3 2 Germany). iron (II) chloride Dimethylformamide tetrahydrate (FeCl (DM4H F) O; [HCON 99%),(sodium CH3)2] w citr asa p teu tribasic rchased dihydrate from Carlo (Na E Crba H Re O agent 2H O; s 2 2 3 6 5 7 2 (Chaussée 99%), nitric du Vexin, Franc acid (HNO ; 65%), e). Tetr sodium aethyl orthosilicate (TEO phosphate dibasic anhydr S; 99%ous ), iron (Na (III) chloride hex HPO ; 99%) and ahydrate sodium 3 2 4 (FeCl phosphate 3·6H2Omonobasic ; 99%), iro(NaH n (II) c POhlo ; 99%) ride t wer etra ehpur ydrchased ate (Fefr Com l2·4H Sigma-Aldrich 2O; 99%), sodi (St. um ci Louis, tratMO, e triUSA). basic 2 4 dihydr Sodium ate hydr (Na oxide 3C6H(NaOH; 5O7·2H2O; 99%) 99% and ), n potassium itric acid ( hydr HNO oxide 3; 65% (KOH; ), sodi >86%) um pwer hosp e pur hate chased dibasfr icom anh Labchem ydrous (Na2HPO4; 99%) and sodium phosphate monobasic (NaH2PO4; 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH; 99%) and potassium hydroxide (KOH; >86%) were purchased from Labchem (Zelienople, PA, USA). Ammonia (NH4; 25%) and NP 10 NP 50 NP+Dox 10 NP+Dox 50 NP 10 NP 50 P D x N + o 10 NP+Dox 50 P 10 NP 50 NP+Dox 10 NP+Dox 50 Extracellular LDH activity (% control) Molecules 2020, 25, 333 12 of 19 Molecules 2020, 25, 333 13 of 20 methanol (CH3OH; solvent) were purchased from VWR (Radnor, PA, USA). Milli-Q water was (Zelienople, PA, USA). Ammonia (NH ; 25%) and methanol (CH OH; solvent) were purchased from 4 3 obtained from the Synergy equipment from Millipore (Burlington, MA, USA) with a 0.22 μm filter. VWR (Radnor, PA, USA). Milli-Q water was obtained from the Synergy equipment from Millipore H-thymidine ([methyl-3H]thymidine; specific activity 79.0 Ci/mmol) was purchased from GE (Burlington, MA, USA) with a 0.22 m filter. Healthcare GmbH (Freiburg, Germany). DMEM:Ham’s F12 medium (1:1; catalogue #FG4815) was H-thymidine ([methyl-3H]thymidine; specific activity 79.0 Ci/mmol) was purchased from GE purchased from Biochrom (Berlin, Germany). Human epidermal growth factor, MEM (catalogue Healthcare GmbH (Freiburg, Germany). DMEM:Ham’s F12 medium (1:1; catalogue #FG4815) was #31095-052) was purchased from Gibco (Thermo Fisher, Waltham, MA, USA). Cholera toxin (95%), purchased from Biochrom (Berlin, Germany). Human epidermal growth factor, MEM (catalogue heat-inactivated horse serum, bovine insulin, hydrocortisone (98%), nicotinamide adenine #31095-052) was purchased from Gibco (Thermo Fisher, Waltham, MA, USA). Cholera toxin (95%), dinucleotide (NADH; ≥95%), sodium pyruvate (99%), trichloroacetic acid (TCA; 99%), heat-inactivated horse serum, bovine insulin, hydrocortisone (98%), nicotinamide adenine dinucleotide −1 −1 −1 antibiotic/antimycotic solution (100 U mL penicillin, 100 μg mL streptomycin and 0.25 μg mL , (NADH; 95%), sodium pyruvate (99%), trichloroacetic acid (TCA; 99%), antibiotic/antimycotic 1 1 1 amphotericin B), FBS (fetal bovine serum), L-glutamine, HEPES solution (100 U mL penicillin, 100 g mL streptomycin and 0.25 g mL , amphotericin B), FBS (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (9 0 9.5%), (fetal bovine serum), l-glutamine, HEPES (N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid) methylthiazolyldiphenyl-tetrazolium bromide (MTT; 98%), RPMI 1640 (catalogue #R6504), trypsin– (99.5%), methylthiazolyldiphenyl-tetrazolium bromide (MTT; 98%), RPMI 1640 (catalogue #R6504), EDTA solution (0.25%), Triton X-100 and PBS (phosphate buffered saline) were purchased from trypsin–EDTA solution (0.25%), Triton X-100 and PBS (phosphate bu ered saline) were purchased Sigma (St. Louis, MO, USA). The compounds to be tested were dissolved in water; controls were run from Sigma (St. Louis, MO, USA). The compounds to be tested were dissolved in water; controls were in the presence of the respective solvent. run in the presence of the respective solvent. 3.2. Synthesis of the Magnetic Nanocarriers 3.2. Synthesis of the Magnetic Nanocarriers The synthesis of magnetic carriers (Fe3O4@SiĸCRG) comprises two distinct steps. The first stage The synthesis of magnetic carriers (Fe O @SikCRG) comprises two distinct steps. The first stage 3 4 consists of the synthesis of the magnetic core (magnetite—Fe3O4) by the co-precipitation method. consists of the synthesis of the magnetic core (magnetite—Fe O ) by the co-precipitation method. Then 3 4 Then Fe3O4 particles were encapsulated in shells of a siliceous hybrid material containing Fe O particles were encapsulated in shells of a siliceous hybrid material containing -carrageenan 3 4 κ-carrageenan covalently linked to the network, with an adaptation of the Stöber method. The covalently linked to the network, with an adaptation of the Stöber method. The procedure is procedure is summarized in Scheme 2. summarized in Scheme 2. Scheme 2. Preparation of siliceous carrageenan coated iron oxide nanoparticles. Scheme 2. Preparation of siliceous carrageenan coated iron oxide nanoparticles. Spherical superparamagnetic magnetite nanoparticles with an average size of 10 nm were Spherical superparamagnetic magnetite nanoparticles with an average size of 10 nm were 3+ 2+ synthesized by co-precipitation of ferric (Fe ) and ferrous (Fe ) ions and then stabilized with citrate 3+ 2+ synthesized by co-precipitation of ferric (Fe ) and ferrous (Fe ) ions and then stabilized with citrate ions [50]. In a typical procedure, 4.43 g of FeCl .6H O and 1.625 g of FeCl .4H O was dissolved in 3 2 2 2 ions [50]. In a typical procedure, 4.43 g of FeCl3.6H2O and 1.625 g of FeCl2.4H2O was dissolved in 190 190 mL of deoxygenated ultrapure water under a N atmosphere with mechanical stirring (650 rpm). mL of deoxygenated ultrapure water under a N2 atmosphere with mechanical stirring (650 rpm). To To start the reaction, 10 mL of ammonia was added and stirred for 10 min. The nanoparticles were start the reaction, 10 mL of ammonia was added and stirred for 10 min. The nanoparticles were magnetically separated and washed with distilled water, then washed twice with HNO (2 M), with magnetically separated and washed with distilled water, then washed twice with HNO3 (2 M), with magnetic separation. The particles were dispersed in 200 mL water, and the pH was adjusted to 2.5 magnetic separation. The particles were dispersed in 200 mL water, and the pH was adjusted to 2.5 with a few drops of NaOH (1 M). Sodium citrate (5 mL, 0.5 M) was added to the suspension, which was with a few drops of NaOH (1 M). Sodium citrate (5 mL, 0.5 M) was added to the suspension, which mechanically stirred for 1 h. The nanoparticles were magnetically recovered and then freeze-dried. was mechanically stirred for 1 h. The nanoparticles were magnetically recovered and then freeze-dried. Molecules 2020, 25, 333 13 of 19 For the encapsulation, 100 mg of Fe O particles were dispersed in 18 mL of ultra-pure water, 3 4 with sonication for 10 min. The biopolymer k-carrageenan was modified with alkoxysilyl groups by reaction with ICPTES [55]. A mixture comprising the silicon derivative of carrageenan (0.4 g), TEOS (0.4 mL) and ethanol (3 mL) was added to the dispersion. Triethylamine (0.1 mL) was added as catalysts. The mixture was placed in an ice bath and was sonicated (Vibracell, Sonics & Materials, Newtown, CT, USA) for 15 min. The resulting particles were collected magnetically, washed with acetone and ethanol and dried at room temperature. 3.3. Nanoparticle Characterization The textural properties of the hybrids were assessed by nitrogen physisorption performed with a Gemini V2.0 Micromeritics Instruments (Micromeritics, Norcross, GA, USA). The specific surface area of the particles was assessed by N adsorption isotherm measurements performed with a Gemini V2.0 Micromeritics instruments at 196 C. The specific surface area was determined using the Brunauer–Emmett–Teller (BET) equation for relative pressures (p/p ) up to 0.3 [82]. Prior to BET measurements, the samples were degassed at 80 C under nitrogen flow overnight. Elemental analysis of carbon, sulphur and hydrogen was obtained on a Leco Truspec-Micro CHNS 630-200-200. Fourier transform infrared (FTIR) spectra of the materials were collected using a Bruker Optics Tensor 27 spectrometer (Bruker, Billerica, MA, USA) coupled to a horizontal attenuated total reflectance (ATR) cell, using 256 scans at a resolution of 4 cm . The nanomaterials were analyzed by powder X-ray di raction (XRD) using a Rigaku Geigerflex Dmax-C di ractometer (Rigaku, Tokyo, Japan) equipped with a CuK monochromatic radiation source with a step size of 0.026 and time per step of 350 s. The morphology and size of the particles were analyzed by electron microscopy using a Hitachi HD-2700 scanning transmission electron microscope (Hitachi, Tokyo, Japan) operating at 200 kV. Samples for electron microscopy analysis were prepared by evaporating the diluted suspensions of the particles on a copper grid coated with an amorphous carbon film. The surface charge of the nanoparticles was assessed by zeta potential measurements through electrophoretic light scattering performed using a Zetasizer Nano ZS instrument equipped with a HeNe laser operating at 633 nm and a scattering detector at 173 degrees, from Malvern Instruments (Malvern, UK). The measurements were performed in aqueous suspensions of the particles using a disposable folded capillary cell. The magnetic measurements were performed using the Quantum Design SQUID MPMS3 (Quantum Design, San Diego, CA, USA) both in VSM and DC modes, as a function of the applied magnetic field (from +50 to 50 kOe), at 300 K and 5 K. The magnetic dc susceptibility was recorded at increasing temperatures (from 10 to 300 K) with an applied H = 100 Oe after initial cooling in the absence of the field (ZFC procedure) and in app the presence of H . To estimate the Fe O content of the coated particles, 10 mg of particles were app 3 4 digested in 20 mL of hydrochloride acid (37%). Afterwards, the solutions were analyzed using atomic absorption spectrophotometry (AAS) in a Perkin Elmer Analyst 100 apparatus (PerkinElmer, Waltham, MA, USA) to assess the Fe content. 3.4. Doxorubicin Loading and Release Studies Doxorubicin hydrochloride solutions of known concentration were prepared in sodium phosphate bu er (0.2 M, pH 6). Nanoparticles were accurately weighed and incubated in 2 mL of the solution (1.25 mg NPs/mL bu er) and the mixtures were vertically stirred (Heidolph Reax 2, Heidolph Instruments, Schwabach, Germany, 30 rpm) for 24 h at room temperature in dark. The nanoparticles were separated magnetically and the solution’s remaining doxorubicin content ([DOX] ) was final determined via UV-Vis spectrophotometry at 480 nm (Cintra 303 GBC Scientific Equipment, Melbourne, Australia). The loading eciency and the nanoparticle capacity were calculated using equations 1 and 2, respectively [17]. Control experiments using loading solutions without nanoparticles were also carried out in parallel to exclude adsorption or degradation phenomena as the cause for the decrease of DOX concentration in the solutions. A quantification curve for doxorubicin was drawn Molecules 2020, 25, 333 14 of 19 from several standards—5, 10, 20, 30, 40 and 50 g/mL, on the mentioned bu er (Figure S1—Supporting Information). [DOX] [DOX] initial f inal Loading E f f iciency (%) =  100 (1) [DOX] initial loaded doxorubicin Nanoparticle Capacity (m /m ) = (2) DOX NP nanoparticles To obtain the doxorubicin release profiles, the loaded nanoparticles (2.5 mg NP) were transferred to 10 mL of sodium phosphate bu er (0.2 M) with pH value of 4.2, 5.0 and 7.4, representing the pH of intracellular acidic endosomes, the pH of the tumor microenvironment, and the pH of blood, respectively. The dispersion was vertically stirred (Heidolph Reax 2; 30 rpm) at 37 C for 48 h [17]. At specific time intervals an aliquot (1 mL) of the solution was taken and replaced by equal volume of fresh bu er. The nanoparticles were magnetically separated to the bottom before taking the supernatant sample. The sample was then analyzed using UV-Vis spectrophotometry (480 nm) to assess DOX concentration. 3.5. Cell Studies 3.5.1. Cell Culture and Treatment The breast cell lines used were: MCF7 (an estrogen receptor (ER)-positive human breast epithelial adenocarcinoma cell line; ATCC HTB-22; passage numbers 79–82), MDA-MB-231 (a triple negative human breast adenocarcinoma cell line; ATCC HTB-26; passage numbers 47–50) and MCF12A (a non-tumorigenic epithelial cell line; ATCC CRL-10782; passage numbers 29–32). Cells were maintained in a humidified atmosphere of 5% CO –95% air and were grown in RPMI 1640 medium supplemented with 2 mM l-glutamine, 10 mM sodium bicarbonate, 15% heat-inactivated FBS and 1% antibiotic/antimycotic (MCF-7 and MDA-MB-231 cell lines) or DMEM:Ham’s F12 medium (1:1) supplemented with 20 ng/mL human epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL bovine insulin, 500 ng/mL hydrocortisone, 5% heat-inactivated horse serum and 1% antibiotic/antimycotic (MCF12A cell line). For the assays, cells were seeded in 24-well culture dishes and used at 90% confluence. Cells were exposed to the treatment (or to the solvent—water) for 24 h in FBS-free culture medium. 3.5.2. Cytotoxicity and Cell Proliferation Determination Cytotoxicity and cell proliferation tests aimed at assessing the nanoparticles’ viability as a drug carrier towards cancer cells. Unloaded nanoparticles and doxorubicin were used in separate tests to determine the e ectiveness of the loaded carrier compared to just the carrier itself and the free drug. MCF-7 and MDA-MB-231 human breast cancer cells and MCF12A human breast cells were exposed for 24 h to doxorubicin (free, or carrier loaded) at concentrations between 1 and 50 M, and equivalent concentrations of unloaded nanoparticles. Cell proliferation was determined through thymidine incorporation assay. Briefly, cells were 3 3 incubated with H-thymidine ( HT) during the last 5 h of their 24 h exposure to the drug or nanoparticles, 3 3 where HT is incorporated in new strands of DNA during cell division. Excess HT was removed with 300 mL of 10% TCA for 1 h at 4 C, followed by drying for 30 min, and addition of 1 mol/L NaOH (280 M /well), and the lysate was neutralized with 5 mol/L HCl prior to the addition of scintillation fluid. The radioactivity of the samples was then quantified by liquid scintillometry and expressed as incorporation of HT in mCi/mg protein (compared to a control). Cellular viability was determined through l-lactic dehydrogenase (LDH) activity assay, expressed as the percentage of extracellular activity in relation to total cellular LDH activity [83]. After a 24 h exposure of the cells to drug or nanoparticles, cellular leakage of LDH into the extracellular culture medium was determined spectrophotometrically by measuring the decrease in absorbance of NADH Molecules 2020, 25, 333 15 of 19 (340 nm) during the reduction of pyruvate to lactate. To determine total cellular LDH activity, control cells were exposed to 0.1% (v/v) Triton X-100 for 30 min at 37 C. 4. Conclusions In conclusion, we reported here a promising drug delivery system comprising DOX loaded nanocarriers for anti-cancer therapy. These nanocarriers (Fe O @SikCRG) are composed of siliceous 3 4 and carrageenan hybrid shells that coat superparamagnetic magnetite nanoparticles. It was found that the Fe O @SikCRG nanocarriers have a loading capacity for DOX between 3.3 and 12.3 wt%, which is 3 4 comparable to other reported magnetic hybrid systems, and an optimal drug release profile in aqueous solutions—80% (tumor microenvironment pH) versus 25% (blood pH) in over 6 h. The underlying mechanism for the pH-dependent DOX release behavior relies on a cation exchange process that takes into account the presence of ammonium groups in the drug and the sulfonated moieties of k-carrageenan. Therefore, by controlling the surface chemistry of the Fe O @SikCRG nanocarriers, it 3 4 provides a way to adjust the DOX release upon exposure of the nanocarriers to biological environments of distinct pH. Since this is of acute relevance for DOX based cancer therapies, the drug loaded nanocarriers were also tested in cellular cultures (cancer and non-cancer cell lines) and showed clear cytotoxic and antiproliferative activity. Future work should include clinical tests to confirm the enhanced permeability and retention e ect and optimal pH-responsiveness apply in physiological conditions. Finally, the superparamagnetic quality of the Fe O @SikCRG nanocarriers opens the way 3 4 towards the development of theranostic agents, namely by assessing their MRI-responsiveness. Supplementary Materials: The following are available online, Figure S1: Calibration curve to determine the DOX concentration using UV-Vis spectroscopy at 480 nm, Table S1: Doxorubicin loading eciency and nanoparticle capacity at variable DOX concentration (pH = 6, C = 1.25 mg/mL), Figure S2: Field Dependent Magnetization NP Curves (without normalization) of Fe O nanoparticles (left) and Fe O @SiCRG nanoparticles (right), Figure S3: 3 4 3 4 Speciation of DOX, Figure S4: Doxorubicin release profiles over 48 h, with corresponding fitting using the Weibull model, Table S2: Parameters and , as estimated from the application of the Weibull model to the DOX release data, and coecient of determination (R ). Author Contributions: A.L.D.-d.-S. and T.T. conceived the topic and supervised the experimental work. J.N. and A.L.D.-d.-S. outlined the manuscript and mainly wrote it. S.F.S. assisted in the synthesis and characterization studies of the nanocarriers. C.O.A. and J.S.A. performed the study of the nanocarriers’ magnetic properties. C.S. and F.M. performed the cellular studies. All authors have read and agreed to the published version of the manuscript. Funding: This work was developed within the scope of the exploratory project IF/00405/2014 and the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. A.L.Daniel-da-Silva thanks FCT for funding in the frame of the program IF2014. J.S.Amaral thanks FCT for the IF/01089/2015 grant. S.F.Soares thanks FCT for the Ph.D. grant SFRH/BD/121366/2016. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. 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In Methods of Enzymatic Analysis; Academic Press: New York, NY, USA, 1974; pp. 574–579. Sample Availability: Not available. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecules Unpaywall

Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy

Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy

Abstract

molecules Article Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy 1 1 2 2 3 , 4 João Nogueira , Sofia F. Soares , Carlos O. Amorim , João S. Amaral , Cláudia Silva , 3 , 4 1 1 , Fátima Martel , Tito Trindade and Ana L. Daniel-da-Silva * CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; jh.nogueira@ua.pt (J.N.); sofiafsoares@ua.pt (S.F.S.); tito@ua.pt (T.T.) CICECO-Aveiro Institute of Materials, Department of Physics, University of Aveiro, 3810-193 Aveiro, Portugal; amorim5@ua.pt (C.O.A.); jamaral@ua.pt (J.S.A.) Unit of Biochemistry, Department of Biomedicine, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal; rakelclaudia@hotmail.com (C.S.); fmartel@med.up.pt (F.M.) i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal * Correspondence: ana.luisa@ua.pt; Tel.: +351-234-370-368 Received: 11 December 2019; Accepted: 10 January 2020; Published: 14 January 2020 Abstract: Doxorubicin is one of the most widely used anti-cancer drugs, but side e ects and selectivity problems create a demand for alternative drug delivery systems. Herein we describe a hybrid magnetic nanomaterial as a pH-dependent doxorubicin release carrier. This nanocarrier comprises magnetic iron oxide cores with a diameter of 10 nm, enveloped in a hybrid material made of siliceous shells

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1420-3049
DOI
10.3390/molecules25020333
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molecules Article Magnetic Driven Nanocarriers for pH-Responsive Doxorubicin Release in Cancer Therapy 1 1 2 2 3 , 4 João Nogueira , Sofia F. Soares , Carlos O. Amorim , João S. Amaral , Cláudia Silva , 3 , 4 1 1 , Fátima Martel , Tito Trindade and Ana L. Daniel-da-Silva * CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal; jh.nogueira@ua.pt (J.N.); sofiafsoares@ua.pt (S.F.S.); tito@ua.pt (T.T.) CICECO-Aveiro Institute of Materials, Department of Physics, University of Aveiro, 3810-193 Aveiro, Portugal; amorim5@ua.pt (C.O.A.); jamaral@ua.pt (J.S.A.) Unit of Biochemistry, Department of Biomedicine, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal; rakelclaudia@hotmail.com (C.S.); fmartel@med.up.pt (F.M.) i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal * Correspondence: ana.luisa@ua.pt; Tel.: +351-234-370-368 Received: 11 December 2019; Accepted: 10 January 2020; Published: 14 January 2020 Abstract: Doxorubicin is one of the most widely used anti-cancer drugs, but side e ects and selectivity problems create a demand for alternative drug delivery systems. Herein we describe a hybrid magnetic nanomaterial as a pH-dependent doxorubicin release carrier. This nanocarrier comprises magnetic iron oxide cores with a diameter of 10 nm, enveloped in a hybrid material made of siliceous shells and k-carrageenan. The hybrid shells possess high drug loading capacity and a favorable drug release profile, while the iron oxide cores allows easy manipulation via an external magnetic field. The pH responsiveness was assessed in phosphate bu ers at pH levels equivalent to those of blood (pH 7.4) and tumor microenvironment (pH 4.2 and 5). The nanoparticles have a loading capacity of up to 12.3 wt.% and a release profile of 80% in 5 h at acidic pH versus 25% at blood pH. In vitro drug delivery tests on human breast cancer and non-cancer cellular cultures have shown that, compared to the free drug, the loaded nanocarriers have comparable antiproliferative e ect but a less intense cytotoxic e ect, especially in the non-cancer cell line. The results show a clear potential for these new hybrid nanomaterials as alternative drug carriers for doxorubicin. Keywords: doxorubicin; magnetic nanoparticles; iron oxide; -carrageenan; drug carrier; pH-responsive 1. Introduction Doxorubicin (DOX, Scheme 1) is an anthracycline antitumor agent, one of the most common and e ective anticancer drugs used in the treatment of various forms of cancer such as lymphomas, sarcomas and breast cancers [1]. The latter in particular is the most common cancer in women, and doxorubicin is one of the main treatments for early and advanced breast cancers [2]. However, doxorubicin presents a number of drawbacks. About 40% of the drug and its metabolites are excreted, and thus, only a small amount of drug actually reaches and acts on the tumor target site [3]. It also causes a notable, well documented cardiac toxicity [4–6]. Another major complication is the possible development of drug resistance mechanisms. Tumor drug resistance contributes to a 90% failure rate of chemotherapy treatments of metastatic disease [7]. Drug resistance can occur via over-expression of cell surface pumps (such as the ATP-binding cassette superfamily) [8], or via changes in DNA repair and cellular apoptosis mechanisms [9]. Molecules 2020, 25, 333; doi:10.3390/molecules25020333 www.mdpi.com/journal/molecules Molecules 2020, 25, 333 3 of 20 3,6-anhydrogalactose units that bears one sulfate group per disaccharide unit [43,44]. Carrageenan-based micro- and nano-scale delivery systems have been reported, often taking advantage of the sulfate content, which results in a negative surface charge that promotes an uptake of cationic target molecules or facilitates functionalization with positively charged components for complex drug carriers [43,45]. Examples of systems reported in the literature include pH-responsive hydrogels for intestinal drug delivery [46], hybrid calcium-biopolymer microparticles for DOX delivery [47,48] and ĸ-carrageenan-coated γ-Fe2O3 nanoparticles with chemotherapeutic potential Molecules 2020, 25, 333 2 of 19 [49]. While there has been research on the combination of magnetic particles with carrageenan for drug delivery [27], there are no reported works combining such systems with DOX. Scheme 1. Chemical structure of doxorubicin (left) and disaccharide unit of κ-carrageenan (right). Scheme 1. Chemical structure of doxorubicin (left) and disaccharide unit of -carrageenan (right). To overcome these chemotherapy issues, one of the promising avenues is the use of nanostructured In the past few years, our research group has developed a new strategy for the preparation of platforms as drug delivery systems [10–12]. Nanosized drug delivery systems can carry drugs to core/shell spheroidal nanoparticles, comprising magnetite cores (Fe3O4) coated with amorphous tumor cells, then penetrate physiological barriers, thus protecting the drug from biological clearance, silica shells [50], or with siliceous shells enriched in polymers from renewable sources including bypass drug resistance mechanisms and finally release the drug specifically on tumor, namely after starch [51], alginate [52], chitosan [53,54] and carrageenan [51,55]. We aimed at creating magnetic triggering by a specific stimulus [3,13]. A drug-loaded carrier can accumulate on tumor sites by bionanocomposites whose ability to interact with target molecular species depends on the chemical exploiting the enhanced permeability and retention (EPR) e ect, first described in 1986 [14], where functionalities of the shells. In this work, κ-carrageenan based magnetic hybrid nanoparticles were tumor vascular permeability is greatly increased compared with healthy tissue [15]. This e ect investigated in the controlled release of DOX. The hybrid particles were tested for pH-responsive occurs in solid tumors leading to enhanced di usion of macromolecules and nanoparticles into DOX release and for the cytotoxic activity against human breast cancer and non-cancer cell lines. The the tumor. Owing to this e ect, it is possible to explore passive tumor targeting by drug-loaded loaded hybrid particles have successfully shown to favor drug release at acidic conditions, as those nanoparticles, having a particle size range between 10 and 100 nm, which has been reported to be found in tumor microenvironments, and to cause cellular damage to human cell lines. optimal for in vivo targeting delivery [13,16]. Additional features can be added to the carrier aiming 2. Results and Discussion to enhance drug delivery performance. For example, the use of pH-responsive biopolymers such as chitosan led to carriers that provide enhanced drug release in the tumor acidic microenvironment 2.1. Characterization of the Magnetic Nanocarriers (due to anaerobic glucose metabolism) and in acidic intracellular organelles (such as endosomes and lysosomes), over release in blood [3,17–21]. Magnetic nanocarriers, either individual or as components The powder XRD pattern of the magnetic cores (Figure 1a) matched the diffraction patterns of hybrid nanomaterials [16,22–26], have also been recognized as drug release vectors having with reported for cubic magnetite (Fe3O4) [56]. The FTIR spectrum of Fe3O4 (Figure 1b) shows a strong several advantages [10,27]. Indeed, magnetic nanocarriers can be guided towards target tissues under −1 band at 552 cm characteristic of the stretching vibration of the magnetite lattice [57]. This a magnetic gradient and eventually be used as contrast agents for magnetic resonance imaging (MRI), −1 vibrational mode is also observed as a band slightly shifted 559 cm , in the FTIR spectrum of the thus allowing for simultaneous cancer treatment and monitoring [24,26]. Furthermore, internalized coated Fe3O4 particles. Two other vibrational are observed in the FTIR spectrum of Fe3O4, one due to magnetic nanocarriers, when exposed to an alternative magnetic field, increase locally the temperature, −1 the stretching of –CO groups at 1349 cm and the other one due to the asymmetric stretching of – which allows us to exploit in a single platform a therapeutic action (magnetic hyperthermia) combined − −1 COO groups at 1569 cm , both owed to the surface citrate ions that stabilize the colloidal magnetic with drug delivery [28,29]. nanoparticles [50]. The FTIR spectrum of Fe3O4@SiĸCRG particles displays the characteristic In the context of doxorubicin based therapies, and in particular its use in breast cancer treatment, −1 vibrational bands of ĸ-carrageenan in the region 1000–1100 cm (C–O and C–OH stretching several studies have reported the use of nanocarriers to enhance doxorubicin-based treatment [2]. −1 −1 vibrations), at 847 cm (α(1-3)-D-galactose C–O–S stretching vibration) and at 1184 cm (O=S=O Hence, liposomal doxorubicin, a system where DOX is encapsulated in a spherical lipid-based −1 asymmetric stretching of sulfonate groups) [58]. The strong band at 449 cm , is ascribed to the O–Si– vesicle, has several variants, either in the market or in clinical stages [30]. Doxil is a polyethylene O deformation [59], which is in agreement with the formation of a siliceous network coating the iron glycol (PEG) based form of liposomal DOX. Known also as Caelyx , was the first market-approved oxide cores. Despite overlapping with vibrational bands from the biopolymer, the bands assigned to nanoformulation containing doxorubicin. As compared to free DOX, it shows comparable eciency −1 amorphous silica [51,60] are observed at 797 and 1036 cm due respectively to symmetric and against metastatic breast cancer, but presents a lower risk of cardiac damage. However, Doxil −1 −1 asymmetric Si–O–Si stretching and at 946 cm due to Si–OH stretching. The band at 1558 cm is presents other side e ects, notably palmar-plantar erythrodysteria (PPE), also known as foot-and-hand ascribed to N–H bending modes coupled to C–N stretching, which confirms the covalent bonding syndrome, characterized by redness, tenderness and peeling of the skin [31]. Another possible side between the carrageenan and the siliceous network through a urethane linkage [61,62]. The band at e ect is an often mild, but possibly severe, allergic reaction caused by some of the formulation’s −1 1645 cm can be either ascribed to adsorbed water (H2O bending mode) or due to the stretching compounds [32]. A commercialized, non-PEGylated but still liposomal alternative is Myocet , using a phosphatidylcholine and cholesterol membrane. While having a lower incidence of PPE, the overall survival rate was not improved, and the formulation has worse hematological toxicity and a higher risk of neutropenia [33]. The final liposomal DOX formulation in the market is Lipo-Dox , which includes PEGylated liposomes as well; however, it fails to show improvements over Doxil regarding ecacy and toxicity [34]. Owing to the drawbacks above mentioned, there is a demand for more e ective nanocarriers for doxorubicin. In the context of breast cancer treatment some attempts have been reported using nanoscale Molecules 2020, 25, 333 3 of 19 systems, including chitosan nanoparticles [35], organosilicon nanoparticles [36,37] and magnetic iron oxide nanocarriers [38]. Magnetic carriers comprising iron oxide nanoparticles functionalized with organic coatings, including azo [23], chitosan [17], polymethacrylic acid [39] and carboxymethyl cellulose [40] are also being investigated. To the best of our knowledge, magnetic nanocarriers based on k-Carrageenan shells have not been explored for DOX release in cancer therapies. Carrageenans are a family of linear sulfated polysaccharides (100–1000 kDa) extracted from marine red algae (Rhodophyceae), widely used as an emulsifier, stabilizers and thickeners in the food and pharmaceutical industries. In human nutrition, they are a good source of dietary fiber and can reduce serum cholesterol and triglyceride levels [41]. Carrageenans have very low toxicity in humans but are known for an inflammatory e ect in some rodents, being frequently used for inflammatory induction in toxicology studies in vivo and have also shown anticoagulant, antiviral and antitumor activities [42]. k-Carrageenan (Scheme 1), composed of d-galactose and 3,6-anhydrogalactose units that bears one sulfate group per disaccharide unit [43,44]. Carrageenan-based micro- and nano-scale delivery systems have been reported, often taking advantage of the sulfate content, which results in a negative surface charge that promotes an uptake of cationic target molecules or facilitates functionalization with positively charged components for complex drug carriers [43,45]. Examples of systems reported in the literature include pH-responsive hydrogels for intestinal drug delivery [46], hybrid calcium-biopolymer microparticles for DOX delivery [47,48] and k-carrageenan-coated -Fe O 2 3 nanoparticles with chemotherapeutic potential [49]. While there has been research on the combination of magnetic particles with carrageenan for drug delivery [27], there are no reported works combining such systems with DOX. In the past few years, our research group has developed a new strategy for the preparation of core/shell spheroidal nanoparticles, comprising magnetite cores (Fe O ) coated with amorphous 3 4 silica shells [50], or with siliceous shells enriched in polymers from renewable sources including starch [51], alginate [52], chitosan [53,54] and carrageenan [51,55]. We aimed at creating magnetic bionanocomposites whose ability to interact with target molecular species depends on the chemical functionalities of the shells. In this work, -carrageenan based magnetic hybrid nanoparticles were investigated in the controlled release of DOX. The hybrid particles were tested for pH-responsive DOX release and for the cytotoxic activity against human breast cancer and non-cancer cell lines. The loaded hybrid particles have successfully shown to favor drug release at acidic conditions, as those found in tumor microenvironments, and to cause cellular damage to human cell lines. 2. Results and Discussion 2.1. Characterization of the Magnetic Nanocarriers The powder XRD pattern of the magnetic cores (Figure 1a) matched the di raction patterns reported for cubic magnetite (Fe O ) [56]. The FTIR spectrum of Fe O (Figure 1b) shows a strong band 3 4 3 4 at 552 cm characteristic of the stretching vibration of the magnetite lattice [57]. This vibrational mode is also observed as a band slightly shifted 559 cm , in the FTIR spectrum of the coated Fe O particles. 3 4 Two other vibrational are observed in the FTIR spectrum of Fe O , one due to the stretching of –CO 3 4 1 1 groups at 1349 cm and the other one due to the asymmetric stretching of –COO groups at 1569 cm , both owed to the surface citrate ions that stabilize the colloidal magnetic nanoparticles [50]. The FTIR spectrum of Fe O @SikCRG particles displays the characteristic vibrational bands of k-carrageenan 3 4 1 1 in the region 1000–1100 cm (C–O and C–OH stretching vibrations), at 847 cm ( (1-3)-d-galactose C–O–S stretching vibration) and at 1184 cm (O=S=O asymmetric stretching of sulfonate groups) [58]. The strong band at 449 cm , is ascribed to the O–Si–O deformation [59], which is in agreement with the formation of a siliceous network coating the iron oxide cores. Despite overlapping with vibrational bands from the biopolymer, the bands assigned to amorphous silica [51,60] are observed at 797 and 1 1 1036 cm due respectively to symmetric and asymmetric Si–O–Si stretching and at 946 cm due to Si–OH stretching. The band at 1558 cm is ascribed to N–H bending modes coupled to C–N Molecules 2020, 25, 333 4 of 19 stretching, which confirms the covalent bonding between the carrageenan and the siliceous network through a urethane linkage [61,62]. The band at 1645 cm can be either ascribed to adsorbed water (H O bending mode) or due to the stretching vibration of hydrogen-bonded C=O in urea, [63] thus indicating the coexistence of urea and urethane groups in the hybrid shells [50,51,55,60]. Molecules 2020, 25, 333 5 of 20 (a) (b) 15 20 25 30 35 40 45 50 55 60 65 70 °2θ (c) (d) (e) Figure Figure 1. 1. Physicochemical Physicochemical characterization characterization of of nanoparticle nanoparticles. s. (a (a ) XRD ) XRDdiffraction pattern, w di raction pattern, iwith th Bragg Bragg reflections identified. (b) ATR-FTIR spectra, with main bands identified. TEM images of Fe3O4 (c) reflections identified. (b) ATR-FTIR spectra, with main bands identified. TEM images of Fe O (c) and 3 4 and Fe3O4@SiκCRG (d) nanoparticles. (e) Histogram for size distribution of Fe3O4 core nanoparticles. Fe O @SiCRG (d) nanoparticles. (e) Histogram for size distribution of Fe O core nanoparticles. 3 4 3 4 Figur e 1c,d show the morphological characteristics of the nanoparticles as assessed by transmission electron microscopy (TEM). The TEM image of the Fe O particles shows spheroidal nanoparticles 3 4 with an average size of 10  2.5 nm, as shown in the particle size histogram (Figure 1e). The TEM Intensity (220) (311) (400) (511) (440) Molecules 2020, 25, 333 5 of 19 images of the coated particles show a thin (5.2 2.1 nm) outer-layer corresponding to the siliceous shell encapsulating the magnetic cores. Elemental microanalysis of the Fe O @SikCRG particles (Table 1) 3 4 indicates the presence of sulphur, which originates from the sulfate groups of the biopolymer that forms the coating. Furthermore, the content of carbon, which in the bare Fe O is due to citrate employed for 3 4 colloidal stabilization, markedly increased after coating the particles. These results are in agreement with the presence of the sulfated polysaccharide k-carrageenan in the coating shells. Note that functionalized used here yielded a marked higher sulphur content (1.27 wt.%) when compared with the results obtained via the conventional multi-step functionalization by covalent grafting of k-carrageenan to amine functionalized silica coated Fe O nanoparticles (0.08 wt.%) [64]. These results are consistent 3 4 with higher biopolymer content at the surface of the nanoparticles using a surface modification approach based on the hydrolytic condensation of the k-carrageenan Si-alkoxide derivative. Table 1. Elemental analysis and textural properties of citrate-capped Fe O and Fe O @SiCRG 3 4 3 4 nanoparticles (D—core diameter, T—coating thickness, S —specific surface area, C,H,S—carbon, BET hydrogen and sulphur content). Sample D (nm) T (nm) C (%) H (%) S (%) S (m g ) BET Fe O 10 2.5 6.55 1.28 152.00 3 4 Fe O @SiCRG 10 2.5 5.2 2.1 13.85 2.27 1.27 13.94 3 4 The BET specific surface area was determined by N adsorption data in the relative pressure (p/p ) 2 1 range 0.01–0.20, and decreased from 151.95 to 13.94 m g after coating (Table 1). This decrease can be explained by the increase in overall particle size after biopolymer incorporation. [51] The surface charge of the Fe O @SikCRG particles was assessed by zeta potential measurements 3 4 at variable pH. The zeta potential was markedly negative, ranging from 34.4  2.1 at pH = 4.2 to 42.2  2.4 mV at pH = 7.4 (Figure 2), which is consistent with the presence of sulfonate groups from carrageenan at the surface of the coated magnetic particles. It is worth noting that in this case the negatively charged surface are highly desirable because they will favor electrostatic interactions with the protonated primary amine groups of doxorubicin, at acidic pH, this promoting the loading of DOX. Furthermore, it should be stressed that at pH 7.4 (physiological medium) the colloidal particles still show a negative zeta potential value that imparts colloidal stability due to interparticle electr Molecules ostatic 2020, 25 repulsions. , 333 6 of 20 Figure 2. Zeta potential titration for Fe O @SiCRG particles. Figure 2. Zeta potential titration for Fe3 3O4 4@SiκCRG particles. Magnetic measurements were performed in a Quantum Design MPMS3 magnetometer for Magnetic measurements were performed in a Quantum Design MPMS3 magnetometer for both both magnetite and the corresponding coated particles. The field dependence of the magnetization magnetite and the corresponding coated particles. The field dependence of the magnetization normalized with the iron oxide content of the nanoparticles measured at 300 K and 5 K is displayed in normalized with the iron oxide content of the nanoparticles measured at 300 K and 5 K is displayed in Figure 3a,b (non-normalized curves are shown in Figure S2, supporting information). The parameters coercivity (Hc), remanent magnetization (Mr) and saturation magnetization (Ms) values obtained from the magnetization curves at 300 K are listed in Table 2. Both bare and coated particles exhibited superparamagnetic-like behavior at room temperature with a hysteresis loop, residual remnant magnetization and coercive field values compatible with the effect of a remnant field of the superconducting coils. This behavior is expected due to the small particle size of the nanoparticles. The observed magnetization saturation was of 25 and 27 emu/g Fe3O4 at 300 K; 32 and 34 emu/g Fe3O4 at 5 K, for bare and coated nanoparticles, respectively. Though magnetic properties of magnetite nanoparticles are dependent on particle size, these values are lower than those typically found in high purity magnetite nanoparticles of similar sizes [65]. These differences may be explained by incomplete crystallization of magnetite, or irregularities in the spheroidal shapes of the particles [66]. The Fe3O4@SiĸCRG particles show an almost identical magnetic saturation when compared to bare nanoparticles. Magnetization versus applied magnetic field data at 5 K show coercivity values around 250 Oe. This suggests a blocking temperature above this temperature, as confirmed via zero-field cooled and field-cooled measurements, shown in Figure 3c,d. The lower blocking temperatures of the Fe3O4@SiĸCRG particles hint at the effect of the coating on the kinetics of the particles during magnetic relaxation. Due to their superparamagnetic quality, the small particles have not only the potential to be a drug release system but also to be used for magnetic resonance imaging (MRI), which translates to a possible theranostic application. It is thus of high interest that these particles show an adequate drug loading and release profile. Molecules 2020, 25, 333 6 of 19 Figure 3a,b (non-normalized curves are shown in Figure S2, Supporting Information). The parameters coercivity (H ), remanent magnetization (M ) and saturation magnetization (M ) values obtained c r s from the magnetization curves at 300 K are listed in Table 2. Both bare and coated particles exhibited superparamagnetic-like behavior at room temperature with a hysteresis loop, residual remnant magnetization and coercive field values compatible with the e ect of a remnant field of the superconducting coils. This behavior is expected due to the small particle size of the nanoparticles. The observed magnetization saturation was of 25 and 27 emu/g Fe O at 300 K; 32 and 34 emu/g Fe O 3 4 3 4 at 5 K, for bare and coated nanoparticles, respectively. Though magnetic properties of magnetite nanoparticles are dependent on particle size, these values are lower than those typically found in high purity magnetite nanoparticles of similar sizes [65]. These di erences may be explained by incomplete crystallization of magnetite, or irregularities in the spheroidal shapes of the particles [66]. The Fe O @SikCRG particles show an almost identical magnetic saturation when compared to bare 3 4 nanoparticles. Magnetization versus applied magnetic field data at 5 K show coercivity values around 250 Oe. This suggests a blocking temperature above this temperature, as confirmed via zero-field cooled and field-cooled measurements, shown in Figure 3c,d. The lower blocking temperatures of the Fe O @SikCRG particles hint at the e ect of the coating on the kinetics of the particles during magnetic 3 4 relaxation. Due to their superparamagnetic quality, the small particles have not only the potential to be a drug release system but also to be used for magnetic resonance imaging (MRI), which translates to a possible theranostic application. It is thus of high interest that these particles show an adequate drug loading and release profile. Molecules 2020, 25, 333 7 of 20 (b) (a) (d) (c) Figure 3. Field dependent magnetization curves (normalized for iron oxide content) of Fe3O4 Figure 3. Field dependent magnetization curves (normalized for iron oxide content) of Fe O 3 4 nanoparticles (a) and Fe3O4@SiκCRG nanoparticles (b). Temperature dependent magnetization nanoparticles (a) and Fe O @SiCRG nanoparticles (b). Temperature dependent magnetization curves 3 4 curves of Fe3O4 nanoparticles (c) and Fe3O4@SiĸCRG nanoparticles (d). of Fe O nanoparticles (c) and Fe O @SikCRG nanoparticles (d). 3 4 3 4 Table 2. Magnetic parameters for Fe3O4 and Fe3O4@SiκCRG particles, at 300 K. Material Ms (emu/gsample) Ms (emu/gFe3O4) Mr (emu/gFe3O4) Hc (Oe) Fe3O4 23 25 1.2 31 Fe3O4@SiĸCRG 15 27 1.9 32 2.2. Doxorubicin Loading The nanoparticles Fe3O4@SiĸCRG were loaded with DOX using a buffer solution at pH = 6, DOX concentration ranging from 110 to 350 μg/mL. The results of loading efficiency and capacity are shown in Table S1. The loading efficiency ranged from 37% to 55% and the loading capacity from 33 to 123 μg/mg (3.3–12.3 wt.%) and has increased with initial DOX concentration in the aqueous solution (Figure 4). These results were similar to values obtained in previous studies using other magnetic carriers, such as chitosan-coated magnetite nanoparticles (18 wt.%) [17] and magnetic polymersomes (12 wt.%) [67]. Molecules 2020, 25, 333 7 of 19 Table 2. Magnetic parameters for Fe O and Fe O @SiCRG particles, at 300 K. 3 4 3 4 Material Ms (emu/g ) Ms (emu/g ) Mr (emu/g ) Hc (Oe) sample Fe3O4 Fe3O4 Fe O 23 25 1.2 31 3 4 Fe O @SikCRG 15 27 1.9 32 3 4 2.2. Doxorubicin Loading The nanoparticles Fe O @SikCRG were loaded with DOX using a bu er solution at pH = 6, DOX 3 4 concentration ranging from 110 to 350 g/mL. The results of loading eciency and capacity are shown in Table S1. The loading eciency ranged from 37% to 55% and the loading capacity from 33 to 123 g/mg (3.3–12.3 wt.%) and has increased with initial DOX concentration in the aqueous solution (Figure 4). These results were similar to values obtained in previous studies using other magnetic carriers, such as chitosan-coated magnetite nanoparticles (18 wt.%) [17] and magnetic polymersomes (12 wt.%) [67]. Molecules 2020, 25, 333 8 of 20 100 140 Efficiency Capacity 0 0 100 150 200 250 300 350 400 [DOX] (μg/mL) initial Figure 4. Uptake curve for doxorubicin, expressed in loading eciency (%) and nanoparticle capacity Figure 4. Uptake curve for doxorubicin, expressed in loading efficiency (%) and nanoparticle (g DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. capacity (μg DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. In aqueous solution, DOX molecules adopt di erent conformations depending on the solution pH In aqueous solution, DOX molecules adopt different conformations depending on the solution (Figure S3, Supporting Information) [68]. At pH 6, it is expected that drug loading is promoted by pH (Figure S3, supporting information) [68]. At pH 6, it is expected that drug loading is promoted by electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the carrageenan. Moreover, hydrogen-bonding interactions between OH groups of carrageenan/siliceous carrageenan. Moreover, hydrogen-bonding interactions between OH groups of network and hydroxyl groups of the anthraquinone ring in DOX will also favor drug loading at this carrageenan/siliceous network and hydroxyl groups of the anthraquinone ring in DOX will also pH. Previous studies have indicated this type of interactions is of uttermost relevance in the formation favor drug loading at this pH. Previous studies have indicated this type of interactions is of of complexes between DOX and dextran sulfate, which is also a sulfated polysaccharide [69]. There uttermost relevance in the formation of complexes between DOX and dextran sulfate, which is also a are also – stacking interactions between the aromatic groups of DOX molecules being brought sulfated polysaccharide [69]. There are also π–π stacking interactions between the aromatic groups close together, which also promotes drug loading [69,70]. As expected, with DOX loading the surface of DOX molecules being brought close together, which also promotes drug loading [69,70]. As of the nanoparticles becomes less negative. Still, the zeta potential of loaded particles ranged from expected, with DOX loading the surface of the nanoparticles becomes less negative. Still, the zeta 17.9 0.9 mV at pH = 4.2 to34.2 2.0 mV at pH = 7.4 (Figure 5), which is indicative of moderate potential of loaded particles ranged from −17.9 ± 0.9 mV at pH = 4.2 to −34.2 ± 2.0 mV at pH = 7.4 colloidal stability [71]. It is worth noting that the zeta potential was assessed in bu ers. In biological (Figure 5), which is indicative of moderate colloidal stability [71]. It is worth noting that the zeta environment and in cell cultures medium the adsorption of proteins may alter the surface properties of potential was assessed in buffers. In biological environment and in cell cultures medium the the nanoparticles and influence the colloidal stability. adsorption of proteins may alter the surface properties of the nanoparticles and influence the colloidal stability. Figure 5. Zeta potential titration for Fe3O4@SiκCRG particles loaded with doxorubicin (loading capacity was 3.2 wt.%). Efficiency (%) Capacity (μg DOX/mg NP) Molecules 2020, 25, 333 8 of 20 100 140 Efficiency Capacity 0 0 100 150 200 250 300 350 400 [DOX] (μg/mL) initial Figure 4. Uptake curve for doxorubicin, expressed in loading efficiency (%) and nanoparticle capacity (μg DOX/mg NP). pH = 6, [NP] = 1.25 mg/mL. In aqueous solution, DOX molecules adopt different conformations depending on the solution pH (Figure S3, supporting information) [68]. At pH 6, it is expected that drug loading is promoted by electrostatic interactions between the primary amine groups of DOX and the sulfonate groups of the carrageenan. Moreover, hydrogen-bonding interactions between OH groups of carrageenan/siliceous network and hydroxyl groups of the anthraquinone ring in DOX will also favor drug loading at this pH. Previous studies have indicated this type of interactions is of uttermost relevance in the formation of complexes between DOX and dextran sulfate, which is also a sulfated polysaccharide [69]. There are also π–π stacking interactions between the aromatic groups of DOX molecules being brought close together, which also promotes drug loading [69,70]. As expected, with DOX loading the surface of the nanoparticles becomes less negative. Still, the zeta potential of loaded particles ranged from −17.9 ± 0.9 mV at pH = 4.2 to −34.2 ± 2.0 mV at pH = 7.4 (Figure 5), which is indicative of moderate colloidal stability [71]. It is worth noting that the zeta potential was assessed in buffers. In biological environment and in cell cultures medium the adsorption of proteins may alter the surface properties of the nanoparticles and influence the Molecules 2020, 25, 333 8 of 19 colloidal stability. Figure 5. Zeta potential titration for Fe O @SiCRG particles loaded with doxorubicin (loading capacity Figure 5. Zeta potential titration for Fe3O4@SiκCRG particles loaded with doxorubicin (loading 3 4 was 3.2 wt.%). capacity was 3.2 wt.%). Molecules 2020, 25, 333 9 of 20 2.3. Doxorubicin pH Mediated Release Studies 2.3. Doxorubicin pH Mediated Release Studies Figure 6 shows the cumulative release of DOX from the Fe O @Si CRG nanocarriers at pH 4.2, 5.0 3 4 Figure 6 shows the cumulative release of DOX from the Fe3O4@SiCRG nanocarriers at pH 4.2, and 7.4, in bu ered solutions. After 1 h, a burst release occurs in all formulations and the equilibrium is 5.0 and 7.4, in buffered solutions. After 1 h, a burst release occurs in all formulations and the reached after 5 h, at pH 7.4 and pH 5. At pH 4.2 the release of DOX was gradually prolonged up to 48 h. equilibrium is reached after 5 h, at pH 7.4 and pH 5. At pH 4.2 the release of DOX was gradually The DOX release was pH-dependent being markedly faster in acidic conditions. Thus, after 5 h the prolonged up to 48 h. The DOX release was pH-dependent being markedly faster in acidic DOX release was approximately 73% and 80% in acidic conditions (pH 4.2 and 5, respectively) and 25% conditions. Thus, after 5 h the DOX release was approximately 73% and 80% in acidic conditions (pH at pH 7.4. Similar behavior has been observed for pH-responsive DOX nanocarriers based on chitosan 4.2 and 5, respectively) and 25% at pH 7.4. Similar behavior has been observed for pH-responsive coated magnetic nanoparticles [17]. This pH-sensitive behavior of Fe O @SikCRG nanocarriers is of 3 4 DOX nanocarriers based on chitosan coated magnetic nanoparticles [17]. This pH-sensitive behavior high interest for the controlled release of may benefit the release of doxorubicin in tumor cells because of Fe3O4@SiĸCRG nanocarriers is of high interest for the controlled release of may benefit the release the intracellular pH is lower than in the healthy cells [72]. of doxorubicin in tumor cells because the intracellular pH is lower than in the healthy cells [72]. Figure Figure 6. 6. Doxor Doxorubicin release ubicin release over over t time ime f fr rom om l loaded oaded F Fe e3O O 4@@kCRG ĸCRG carriers, carriers, at 37 °C and va at 37 C and riable pH variable pH 3 4 (loading (loading capacity capacity was was 12 12.3 .3 wt. wt.%). %). F Full ull prof profile ile for 48 h for 48 h (( left left ) )and first and first 5 h ( 5 hright (right ). ). It is likely that the release of DOX occurs through a cation exchange mechanism (Figure 7) It is likely that the release of DOX occurs through a cation exchange mechanism (Figure 7) because surface charge of the nanocarriers is negative, not varying significantly across the work pH because surface charge of the nanocarriers is negative, not varying significantly across the work range (Figure 2), and DOX is cationic in acidic conditions. The acidic environment promotes the pH range (Figure 2), and DOX is cationic in acidic conditions. The acidic environment promotes exchange of protons between the primary amine groups of DOX and the sulfonate groups of the exchange of protons between the primary amine groups of DOX and the sulfonate groups of κ-carrageenan [73]. The DOX release was slightly pronounced at pH 5 than at pH 4.2 for the first five -carrageenan [73]. The DOX release was slightly pronounced at pH 5 than at pH 4.2 for the first hours but became equalized within a 24 h period. We interpreted the faster initial release at pH 5 as five hours but became equalized within a 24 h period. We interpreted the faster initial release at pH 5 due to a polymer swelling effect. The outer shell of the nanocarriers contain ĸ-carrageenan in its composition, which is well known to form hydrogels with a pH-dependent swelling behavior [74]. Although in these nanocarriers the ĸ-carrageenan was grafted to the siliceous network forming the outer shells, there were still domains enriched in ĸ-carrageenan that can undergo swelling in an aqueous medium. Carrageenan hydrogels experience increased swelling when the pH increased from acidic to neutral conditions. This swelling effect can enhance the drug release by favoring the diffusion of DOX molecules within the hybrid network [74,75]. Efficiency (%) Capacity (μg DOX/mg NP) Molecules 2020, 25, 333 9 of 19 as due to a polymer swelling e ect. The outer shell of the nanocarriers contain k-carrageenan in its composition, which is well known to form hydrogels with a pH-dependent swelling behavior [74]. Although in these nanocarriers the k-carrageenan was grafted to the siliceous network forming the outer shells, there were still domains enriched in k-carrageenan that can undergo swelling in an aqueous medium. Carrageenan hydrogels experience increased swelling when the pH increased from acidic to neutral conditions. This swelling e ect can enhance the drug release by favoring the di usion of DOX molecules within the hybrid network [74,75]. Molecules 2020, 25, 333 10 of 20 Figure 7. Schematic representation of the proposed cation exchange mechanism behind DOX release Figure 7. Schematic representation of the proposed cation exchange mechanism behind DOX release from the nanocarriers in acidic conditions, with the equation showing the equilibrium shift. from the nanocarriers in acidic conditions, with the equation showing the equilibrium shift. It is the strong swelling behavior experienced by carrageenan at neutral pH that also contributes It is the strong swelling behavior experienced by carrageenan at neutral pH that also to DOX release at pH 7.4. In this case, the release of DOX was slower due to a less extent of contributes to DOX release at pH 7.4. In this case, the release of DOX was slower due to a less extent protonation, which results in lower competition of the protons for the sulphonated sites of k-carrageenan. of protonation, which results in lower competition of the protons for the sulphonated sites of Nevertheless, there is still a 25% drug release because at this pH there will be a number of neutral DOX ĸ-carrageenan. Nevertheless, there is still a 25% drug release because at this pH there will be a molecules less prone to interact with sulfonate groups at the surface of the magnetic nanocarriers. number of neutral DOX molecules less prone to interact with sulfonate groups at the surface of the The release kinetic data were reasonably described by the Weibull model (equation S1) with magnetic nanocarriers. a coecient of determination (R ) in the range 0.926–0.958 (Supporting Information, Figure S4 and The release kinetic data were reasonably described by the Weibull model (equation S1) with a Table S2). This is a general empirical equation that has been successfully applied to several drug release coefficient of determination (R ) in the range 0.926–0.958 (Supporting Information, Figure S4 and systems [76,77]. Table S2). This is a general empirical equation that has been successfully applied to several drug release systems [76,77]. 2.4. Antiproliferative and Cytotoxicity In Vitro Evaluation The proliferation and viability of the cells after their exposure to free doxorubicin (DOX), unloaded 2.4. Antiproliferative and Cytotoxicity In Vitro Evaluation nanoparticles (NP) and nanoparticles loaded with doxorubicin (NP + DOX) are shown in Figures 8 The proliferation and viability of the cells after their exposure to free doxorubicin (DOX), and 9, respectively. The tests were performed in MCF-7 and MDA-MB-231 human breast cancer cells unloaded nanoparticles (NP) and nanoparticles loaded with doxorubicin (NP + DOX) are shown in and in MCF12A non-cancerous human breast epithelial cells. Figures 8 and 9, respectively. The tests were performed in MCF-7 and MDA-MB-231 human breast As expected, free DOX has a concentration-dependent antiproliferative e ect on all cell cancer cells and in MCF12A non-cancerous human breast epithelial cells. lines (Figure 8a). Loaded nanoparticles also present a concentration-dependent antiproliferative e ect As expected, free DOX has a concentration-dependent antiproliferative effect on all cell lines on all cell lines, and this e ect was more marked than the e ect of unloaded nanoparticles (Figure 8b). (Figure 8a). Loaded nanoparticles also present a concentration-dependent antiproliferative effect on The antiproliferative e ect of DOX-loaded NPs is similar to that of free DOX at DOX concentrations of all cell lines, and this effect was more marked than the effect of unloaded nanoparticles (Figure 8b). 50 M, in all cell lines (Figure 8). The antiproliferative effect of DOX-loaded NPs is similar to that of free DOX at DOX concentrations As expected, free DOX shows a concentration-dependent cytotoxic e ect on all cell lines (Figure 9a). of 50 μM, in all cell lines (Figure 8). Both cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous cell line (MCF12A) su er As expected, free DOX shows a concentration-dependent cytotoxic effect on all cell lines (Figure deleterious cytotoxic e ects when exposed to loaded nanoparticles, and again, this e ect is much 9a). Both cancer cell lines (MCF-7 and MDA-MB-231) and the non-cancerous cell line (MCF12A) more marked than the e ect observed with unloaded nanoparticles (Figure 9b). The cytotoxic e ect of suffer deleterious cytotoxic effects when exposed to loaded nanoparticles, and again, this effect is much more marked than the effect observed with unloaded nanoparticles (Figure 9b). The cytotoxic effect of DOX-NPs is lower than that of DOX itself in all the three cell lines, but this is especially evident in the non-cancerous cell line (Figure 9). Molecules 2020, 25, 333 10 of 19 DOX-NPs is lower than that of DOX itself in all the three cell lines, but this is especially evident in the Molecules 2020, 25, 333 11 of 20 non-cancerous cell line (Figure 9). (a) (b) Figure 8. Cell proliferation after exposure for 24 h to (a) free doxorubicin (M) and (b) unloaded Figure 8. Cell proliferation after exposure for 24 h to (a) free doxorubicin (μM) and (b) unloaded nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; M), for all cell lines. Shown are nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; μM), for all cell lines. Shown are arithmetic means  SEM (n = 6). (a) * significantly di erent from control (p < 0.05; Student’s t test). arithmetic means ± SEM (n = 6). (a) * significantly different from control (p < 0.05; Student’s t test). (b) (b) * significantly di erent from control; significantly di erent from each other (p < 0.05; ANOVA + ns * significantly different from control; significantly different from each other (p < 0.05; ANOVA + Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). ns Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). When comparing the e ect of NP + DOX in the di erent cell lines, we observed that its antiprWhen comparing the effe oliferative e ect was equally ct of NP potent in + the DOX in th three celle different cell lines, we o lines (Figure 8b), but its cytotoxic bserved that e ect was its less potent in MDA-MB-231 cells than in the other cell lines (Figure 9b). So, the antiproliferative and antiproliferative effect was equally potent in the three cell lines (Figure 8b), but its cytotoxic effect cytotoxic was less pot e ect ent in of MDA NP-DOX -MB do -23not 1 ceappear lls than to in t be he ot cellhe type-specific. r cell lines (FThis igureis 9b not ). So surprising , the antiprol as the iferNP ativ+ e DOX e ect was not meant to be selective according to cell type since all cells possess acidic endosomes and cytotoxic effect of NP-DOX do not appear to be cell type-specific. This is not surprising as the that NP + DOX e can provide ffect was not conditionsmeant to be selective for DOX release once the acco particles rding to cell type since are taken up, butall rather cells posse its selectivity ss acidis ic due to local tumor vascularity and acidic tumor microenvironment when in physiological conditions. endosomes that can provide conditions for DOX release once the particles are taken up, but rather its The select loaded ivity nanoparticles is due to loc arael t expected umor v toascul leave arit the y bloodstr and acid eam ic tand umor microen enter in contact vironment with cancer when in cells via the EPR e ect and then begin releasing DOX in their vicinity due to a local decrease in pH. physiological conditions. The loaded nanoparticles are expected to leave the bloodstream and enter in contact wit When comparing h cancerthe cells e via the E ect of DOX PR e inffect the thr and ee cell then begi lines used, n rele we asing DOX i concluded nthat theithe r vinon-cancer cinity due to a ous cell line (MCF12A) appeared to be more susceptible to DOX-induced cytotoxicity, but not more local decrease in pH. susceptible When comparin to the antipr g the e oliferative ffect of DOX e ect than the in the three cancer cell cell lines lines (MCF-7 used, we and MDA-MB-231; concluded that Figuresthe 8 and 9). Moreover, of the cancer cell lines, MDA-MB-231 appears more resistant to the cytotoxic non-cancerous cell line (MCF12A) appeared to be more susceptible to DOX-induced cytotoxicity, but enot more ect of DOX suscepti than bl MCF-7 e to the cells,an but tiprol both ifera cell tive lines effect tha are equally n the ca vulnerable ncer cell li to itsnes (MCF- antiproliferative 7 and e ect. In other words, susceptibility to the cytotoxic e ect of DOX varies in the distinct cell lines, MDA-MB-231; Figures 8 and 9). Moreover, of the cancer cell lines, MDA-MB-231 appears more but resista not nvulnerability t to the cytotoxi toc effect of its antipr DOX tha oliferative n MCF- e ect.7 This cells,is bu in t both cell line with lidoxor nes arubicin’s e equally vul mechanism nerable to of action: it disrupts topoisomerase, preventing cellular replication and ultimately inducing programmed its antiproliferative effect. In other words, susceptibility to the cytotoxic effect of DOX varies in the cellular distinct cel death. l lines, The but specific not vu cytotoxic lnerabilit damage y to its ant and ipro resulting liferative form effect of cell . Thi death s is in induced line with by doxor doxor ubicin’s ubicin varies mechawith nism of concentration, action: it di treatment srupts topoi duration, somerase, specific preventi form of ng cellu cancerla and r repl drug icartion a esistance nd ul [78tima ]. Both tely inducing programmed cellular death. The specific cytotoxic damage and resulting form of cell death induced by doxorubicin varies with concentration, treatment duration, specific form of cancer and drug resistance [78]. Both MDA-MB-231 [79] and MCF-7 [78] cell lines have shown the ability to gain resistance to doxorubicin. Nevertheless, the opposite can happen and some studies have shown a Molecules 2020, 25, 333 11 of 19 Molecules 2020, 25, 333 12 of 20 MDA-MB-231 [79] and MCF-7 [78] cell lines have shown the ability to gain resistance to doxorubicin. lower IC50 for DOX in MDA-MB-231 than in MCF-7 cells [80,81]. Different rates of nanoparticles Nevertheless, the opposite can happen and some studies have shown a lower IC for DOX in uptake, depending on cell type, may also be at play to explain this. Nonetheless, it is clearly shown MDA-MB-231 than in MCF-7 cells [80,81]. Di erent rates of nanoparticles uptake, depending on cell that DOX-NPs disrupts cellular division and reduces cell viability to a comparable level to free type, may also be at play to explain this. Nonetheless, it is clearly shown that DOX-NPs disrupts cellular doxorubicin, and that, differently from what is found with DOX, its antiproliferative effect is not division and reduces cell viability to a comparable level to free doxorubicin, and that, di erently from more marked in non-cancerous cells. Our results thus show the potential of the hybrid nanoparticles what is found with DOX, its antiproliferative e ect is not more marked in non-cancerous cells. Our as a novel anticancer drug delivery system. results thus show the potential of the hybrid nanoparticles as a novel anticancer drug delivery system. (a) (b) Figure 9. Cell viability after exposure for 24 h to (a) free doxorubicin (M) and to (b) unloaded Figure 9. Cell viability after exposure for 24 h to (a) free doxorubicin (μM) and to (b) unloaded nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; M), for all cellular lines. Shown nanoparticles (NP) and nanoparticles loaded with DOX (NP + DOX; μM), for all cellular lines. Shown are arithmetic means SEM (n = 8). (a) * significantly di erent from control (p < 0.05) (Student’s t test). are arithmetic means ± SEM (n = 8). (a) * significantly different from control (p < 0.05) (Student’s t (b) * significantly di erent from control; significantly di erent from each other (p < 0.05; ANOVA + test). (b) * significantly different from control; significantly different from each other (p < 0.05; ns Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman–Keuls test). ns ANOVA + Student-Neuman–Keuls test); not significant (p < 0.05; ANOVA + Student-Neuman– Keuls test). 3. Materials and Methods 3.1. Materials 3. Materials and Methods Doxorubicin hydrochloride (95%; DOX HCl) was purchased from Thermo Fisher (Waltham, MA, 3.1. Materials USA). -Carrageenan (CRG), 3-(triethoxysilyl)propyl isocyanate (ICPTES) (95%), ethanol (CH CH OH; 3 2 absolute) Doxorubic and i acetone n hydrochloride (C H O; ( 100%) ≥95%; D wer Oe X H pur Cchased l) was p fr u om rcha Honeywell sed from TFluka hermo(Seelze, Fisher (Germany). Waltham, 3 6 MA, U Dimethylformamide SA). κ-Carrage (DMF) enan ( [HCON(CH κCRG), 3-(triet ) ] hoxys was pur ilychased l)propyfr l isoc om Carlo yanate Erba (ICPTES Reagents ) (95 (Chauss %), etha éenol du 3 2 (CH Vexin, 3CH France). 2OH; ab Tso etraethyl lute) anorthosilicate d acetone (C(TEOS; 3H6O; 100% 99%), ) ir were pur on (III) chloride chased from Hon hexahydrate eywell F (FeClluka 6H (See O; 99%), lze, 3 2 Germany). iron (II) chloride Dimethylformamide tetrahydrate (FeCl (DM4H F) O; [HCON 99%),(sodium CH3)2] w citr asa p teu tribasic rchased dihydrate from Carlo (Na E Crba H Re O agent 2H O; s 2 2 3 6 5 7 2 (Chaussée 99%), nitric du Vexin, Franc acid (HNO ; 65%), e). Tetr sodium aethyl orthosilicate (TEO phosphate dibasic anhydr S; 99%ous ), iron (Na (III) chloride hex HPO ; 99%) and ahydrate sodium 3 2 4 (FeCl phosphate 3·6H2Omonobasic ; 99%), iro(NaH n (II) c POhlo ; 99%) ride t wer etra ehpur ydrchased ate (Fefr Com l2·4H Sigma-Aldrich 2O; 99%), sodi (St. um ci Louis, tratMO, e triUSA). basic 2 4 dihydr Sodium ate hydr (Na oxide 3C6H(NaOH; 5O7·2H2O; 99%) 99% and ), n potassium itric acid ( hydr HNO oxide 3; 65% (KOH; ), sodi >86%) um pwer hosp e pur hate chased dibasfr icom anh Labchem ydrous (Na2HPO4; 99%) and sodium phosphate monobasic (NaH2PO4; 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH; 99%) and potassium hydroxide (KOH; >86%) were purchased from Labchem (Zelienople, PA, USA). Ammonia (NH4; 25%) and NP 10 NP 50 NP+Dox 10 NP+Dox 50 NP 10 NP 50 P D x N + o 10 NP+Dox 50 P 10 NP 50 NP+Dox 10 NP+Dox 50 Extracellular LDH activity (% control) Molecules 2020, 25, 333 12 of 19 Molecules 2020, 25, 333 13 of 20 methanol (CH3OH; solvent) were purchased from VWR (Radnor, PA, USA). Milli-Q water was (Zelienople, PA, USA). Ammonia (NH ; 25%) and methanol (CH OH; solvent) were purchased from 4 3 obtained from the Synergy equipment from Millipore (Burlington, MA, USA) with a 0.22 μm filter. VWR (Radnor, PA, USA). Milli-Q water was obtained from the Synergy equipment from Millipore H-thymidine ([methyl-3H]thymidine; specific activity 79.0 Ci/mmol) was purchased from GE (Burlington, MA, USA) with a 0.22 m filter. Healthcare GmbH (Freiburg, Germany). DMEM:Ham’s F12 medium (1:1; catalogue #FG4815) was H-thymidine ([methyl-3H]thymidine; specific activity 79.0 Ci/mmol) was purchased from GE purchased from Biochrom (Berlin, Germany). Human epidermal growth factor, MEM (catalogue Healthcare GmbH (Freiburg, Germany). DMEM:Ham’s F12 medium (1:1; catalogue #FG4815) was #31095-052) was purchased from Gibco (Thermo Fisher, Waltham, MA, USA). Cholera toxin (95%), purchased from Biochrom (Berlin, Germany). Human epidermal growth factor, MEM (catalogue heat-inactivated horse serum, bovine insulin, hydrocortisone (98%), nicotinamide adenine #31095-052) was purchased from Gibco (Thermo Fisher, Waltham, MA, USA). Cholera toxin (95%), dinucleotide (NADH; ≥95%), sodium pyruvate (99%), trichloroacetic acid (TCA; 99%), heat-inactivated horse serum, bovine insulin, hydrocortisone (98%), nicotinamide adenine dinucleotide −1 −1 −1 antibiotic/antimycotic solution (100 U mL penicillin, 100 μg mL streptomycin and 0.25 μg mL , (NADH; 95%), sodium pyruvate (99%), trichloroacetic acid (TCA; 99%), antibiotic/antimycotic 1 1 1 amphotericin B), FBS (fetal bovine serum), L-glutamine, HEPES solution (100 U mL penicillin, 100 g mL streptomycin and 0.25 g mL , amphotericin B), FBS (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (9 0 9.5%), (fetal bovine serum), l-glutamine, HEPES (N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid) methylthiazolyldiphenyl-tetrazolium bromide (MTT; 98%), RPMI 1640 (catalogue #R6504), trypsin– (99.5%), methylthiazolyldiphenyl-tetrazolium bromide (MTT; 98%), RPMI 1640 (catalogue #R6504), EDTA solution (0.25%), Triton X-100 and PBS (phosphate buffered saline) were purchased from trypsin–EDTA solution (0.25%), Triton X-100 and PBS (phosphate bu ered saline) were purchased Sigma (St. Louis, MO, USA). The compounds to be tested were dissolved in water; controls were run from Sigma (St. Louis, MO, USA). The compounds to be tested were dissolved in water; controls were in the presence of the respective solvent. run in the presence of the respective solvent. 3.2. Synthesis of the Magnetic Nanocarriers 3.2. Synthesis of the Magnetic Nanocarriers The synthesis of magnetic carriers (Fe3O4@SiĸCRG) comprises two distinct steps. The first stage The synthesis of magnetic carriers (Fe O @SikCRG) comprises two distinct steps. The first stage 3 4 consists of the synthesis of the magnetic core (magnetite—Fe3O4) by the co-precipitation method. consists of the synthesis of the magnetic core (magnetite—Fe O ) by the co-precipitation method. Then 3 4 Then Fe3O4 particles were encapsulated in shells of a siliceous hybrid material containing Fe O particles were encapsulated in shells of a siliceous hybrid material containing -carrageenan 3 4 κ-carrageenan covalently linked to the network, with an adaptation of the Stöber method. The covalently linked to the network, with an adaptation of the Stöber method. The procedure is procedure is summarized in Scheme 2. summarized in Scheme 2. Scheme 2. Preparation of siliceous carrageenan coated iron oxide nanoparticles. Scheme 2. Preparation of siliceous carrageenan coated iron oxide nanoparticles. Spherical superparamagnetic magnetite nanoparticles with an average size of 10 nm were Spherical superparamagnetic magnetite nanoparticles with an average size of 10 nm were 3+ 2+ synthesized by co-precipitation of ferric (Fe ) and ferrous (Fe ) ions and then stabilized with citrate 3+ 2+ synthesized by co-precipitation of ferric (Fe ) and ferrous (Fe ) ions and then stabilized with citrate ions [50]. In a typical procedure, 4.43 g of FeCl .6H O and 1.625 g of FeCl .4H O was dissolved in 3 2 2 2 ions [50]. In a typical procedure, 4.43 g of FeCl3.6H2O and 1.625 g of FeCl2.4H2O was dissolved in 190 190 mL of deoxygenated ultrapure water under a N atmosphere with mechanical stirring (650 rpm). mL of deoxygenated ultrapure water under a N2 atmosphere with mechanical stirring (650 rpm). To To start the reaction, 10 mL of ammonia was added and stirred for 10 min. The nanoparticles were start the reaction, 10 mL of ammonia was added and stirred for 10 min. The nanoparticles were magnetically separated and washed with distilled water, then washed twice with HNO (2 M), with magnetically separated and washed with distilled water, then washed twice with HNO3 (2 M), with magnetic separation. The particles were dispersed in 200 mL water, and the pH was adjusted to 2.5 magnetic separation. The particles were dispersed in 200 mL water, and the pH was adjusted to 2.5 with a few drops of NaOH (1 M). Sodium citrate (5 mL, 0.5 M) was added to the suspension, which was with a few drops of NaOH (1 M). Sodium citrate (5 mL, 0.5 M) was added to the suspension, which mechanically stirred for 1 h. The nanoparticles were magnetically recovered and then freeze-dried. was mechanically stirred for 1 h. The nanoparticles were magnetically recovered and then freeze-dried. Molecules 2020, 25, 333 13 of 19 For the encapsulation, 100 mg of Fe O particles were dispersed in 18 mL of ultra-pure water, 3 4 with sonication for 10 min. The biopolymer k-carrageenan was modified with alkoxysilyl groups by reaction with ICPTES [55]. A mixture comprising the silicon derivative of carrageenan (0.4 g), TEOS (0.4 mL) and ethanol (3 mL) was added to the dispersion. Triethylamine (0.1 mL) was added as catalysts. The mixture was placed in an ice bath and was sonicated (Vibracell, Sonics & Materials, Newtown, CT, USA) for 15 min. The resulting particles were collected magnetically, washed with acetone and ethanol and dried at room temperature. 3.3. Nanoparticle Characterization The textural properties of the hybrids were assessed by nitrogen physisorption performed with a Gemini V2.0 Micromeritics Instruments (Micromeritics, Norcross, GA, USA). The specific surface area of the particles was assessed by N adsorption isotherm measurements performed with a Gemini V2.0 Micromeritics instruments at 196 C. The specific surface area was determined using the Brunauer–Emmett–Teller (BET) equation for relative pressures (p/p ) up to 0.3 [82]. Prior to BET measurements, the samples were degassed at 80 C under nitrogen flow overnight. Elemental analysis of carbon, sulphur and hydrogen was obtained on a Leco Truspec-Micro CHNS 630-200-200. Fourier transform infrared (FTIR) spectra of the materials were collected using a Bruker Optics Tensor 27 spectrometer (Bruker, Billerica, MA, USA) coupled to a horizontal attenuated total reflectance (ATR) cell, using 256 scans at a resolution of 4 cm . The nanomaterials were analyzed by powder X-ray di raction (XRD) using a Rigaku Geigerflex Dmax-C di ractometer (Rigaku, Tokyo, Japan) equipped with a CuK monochromatic radiation source with a step size of 0.026 and time per step of 350 s. The morphology and size of the particles were analyzed by electron microscopy using a Hitachi HD-2700 scanning transmission electron microscope (Hitachi, Tokyo, Japan) operating at 200 kV. Samples for electron microscopy analysis were prepared by evaporating the diluted suspensions of the particles on a copper grid coated with an amorphous carbon film. The surface charge of the nanoparticles was assessed by zeta potential measurements through electrophoretic light scattering performed using a Zetasizer Nano ZS instrument equipped with a HeNe laser operating at 633 nm and a scattering detector at 173 degrees, from Malvern Instruments (Malvern, UK). The measurements were performed in aqueous suspensions of the particles using a disposable folded capillary cell. The magnetic measurements were performed using the Quantum Design SQUID MPMS3 (Quantum Design, San Diego, CA, USA) both in VSM and DC modes, as a function of the applied magnetic field (from +50 to 50 kOe), at 300 K and 5 K. The magnetic dc susceptibility was recorded at increasing temperatures (from 10 to 300 K) with an applied H = 100 Oe after initial cooling in the absence of the field (ZFC procedure) and in app the presence of H . To estimate the Fe O content of the coated particles, 10 mg of particles were app 3 4 digested in 20 mL of hydrochloride acid (37%). Afterwards, the solutions were analyzed using atomic absorption spectrophotometry (AAS) in a Perkin Elmer Analyst 100 apparatus (PerkinElmer, Waltham, MA, USA) to assess the Fe content. 3.4. Doxorubicin Loading and Release Studies Doxorubicin hydrochloride solutions of known concentration were prepared in sodium phosphate bu er (0.2 M, pH 6). Nanoparticles were accurately weighed and incubated in 2 mL of the solution (1.25 mg NPs/mL bu er) and the mixtures were vertically stirred (Heidolph Reax 2, Heidolph Instruments, Schwabach, Germany, 30 rpm) for 24 h at room temperature in dark. The nanoparticles were separated magnetically and the solution’s remaining doxorubicin content ([DOX] ) was final determined via UV-Vis spectrophotometry at 480 nm (Cintra 303 GBC Scientific Equipment, Melbourne, Australia). The loading eciency and the nanoparticle capacity were calculated using equations 1 and 2, respectively [17]. Control experiments using loading solutions without nanoparticles were also carried out in parallel to exclude adsorption or degradation phenomena as the cause for the decrease of DOX concentration in the solutions. A quantification curve for doxorubicin was drawn Molecules 2020, 25, 333 14 of 19 from several standards—5, 10, 20, 30, 40 and 50 g/mL, on the mentioned bu er (Figure S1—Supporting Information). [DOX] [DOX] initial f inal Loading E f f iciency (%) =  100 (1) [DOX] initial loaded doxorubicin Nanoparticle Capacity (m /m ) = (2) DOX NP nanoparticles To obtain the doxorubicin release profiles, the loaded nanoparticles (2.5 mg NP) were transferred to 10 mL of sodium phosphate bu er (0.2 M) with pH value of 4.2, 5.0 and 7.4, representing the pH of intracellular acidic endosomes, the pH of the tumor microenvironment, and the pH of blood, respectively. The dispersion was vertically stirred (Heidolph Reax 2; 30 rpm) at 37 C for 48 h [17]. At specific time intervals an aliquot (1 mL) of the solution was taken and replaced by equal volume of fresh bu er. The nanoparticles were magnetically separated to the bottom before taking the supernatant sample. The sample was then analyzed using UV-Vis spectrophotometry (480 nm) to assess DOX concentration. 3.5. Cell Studies 3.5.1. Cell Culture and Treatment The breast cell lines used were: MCF7 (an estrogen receptor (ER)-positive human breast epithelial adenocarcinoma cell line; ATCC HTB-22; passage numbers 79–82), MDA-MB-231 (a triple negative human breast adenocarcinoma cell line; ATCC HTB-26; passage numbers 47–50) and MCF12A (a non-tumorigenic epithelial cell line; ATCC CRL-10782; passage numbers 29–32). Cells were maintained in a humidified atmosphere of 5% CO –95% air and were grown in RPMI 1640 medium supplemented with 2 mM l-glutamine, 10 mM sodium bicarbonate, 15% heat-inactivated FBS and 1% antibiotic/antimycotic (MCF-7 and MDA-MB-231 cell lines) or DMEM:Ham’s F12 medium (1:1) supplemented with 20 ng/mL human epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL bovine insulin, 500 ng/mL hydrocortisone, 5% heat-inactivated horse serum and 1% antibiotic/antimycotic (MCF12A cell line). For the assays, cells were seeded in 24-well culture dishes and used at 90% confluence. Cells were exposed to the treatment (or to the solvent—water) for 24 h in FBS-free culture medium. 3.5.2. Cytotoxicity and Cell Proliferation Determination Cytotoxicity and cell proliferation tests aimed at assessing the nanoparticles’ viability as a drug carrier towards cancer cells. Unloaded nanoparticles and doxorubicin were used in separate tests to determine the e ectiveness of the loaded carrier compared to just the carrier itself and the free drug. MCF-7 and MDA-MB-231 human breast cancer cells and MCF12A human breast cells were exposed for 24 h to doxorubicin (free, or carrier loaded) at concentrations between 1 and 50 M, and equivalent concentrations of unloaded nanoparticles. Cell proliferation was determined through thymidine incorporation assay. Briefly, cells were 3 3 incubated with H-thymidine ( HT) during the last 5 h of their 24 h exposure to the drug or nanoparticles, 3 3 where HT is incorporated in new strands of DNA during cell division. Excess HT was removed with 300 mL of 10% TCA for 1 h at 4 C, followed by drying for 30 min, and addition of 1 mol/L NaOH (280 M /well), and the lysate was neutralized with 5 mol/L HCl prior to the addition of scintillation fluid. The radioactivity of the samples was then quantified by liquid scintillometry and expressed as incorporation of HT in mCi/mg protein (compared to a control). Cellular viability was determined through l-lactic dehydrogenase (LDH) activity assay, expressed as the percentage of extracellular activity in relation to total cellular LDH activity [83]. After a 24 h exposure of the cells to drug or nanoparticles, cellular leakage of LDH into the extracellular culture medium was determined spectrophotometrically by measuring the decrease in absorbance of NADH Molecules 2020, 25, 333 15 of 19 (340 nm) during the reduction of pyruvate to lactate. To determine total cellular LDH activity, control cells were exposed to 0.1% (v/v) Triton X-100 for 30 min at 37 C. 4. Conclusions In conclusion, we reported here a promising drug delivery system comprising DOX loaded nanocarriers for anti-cancer therapy. These nanocarriers (Fe O @SikCRG) are composed of siliceous 3 4 and carrageenan hybrid shells that coat superparamagnetic magnetite nanoparticles. It was found that the Fe O @SikCRG nanocarriers have a loading capacity for DOX between 3.3 and 12.3 wt%, which is 3 4 comparable to other reported magnetic hybrid systems, and an optimal drug release profile in aqueous solutions—80% (tumor microenvironment pH) versus 25% (blood pH) in over 6 h. The underlying mechanism for the pH-dependent DOX release behavior relies on a cation exchange process that takes into account the presence of ammonium groups in the drug and the sulfonated moieties of k-carrageenan. Therefore, by controlling the surface chemistry of the Fe O @SikCRG nanocarriers, it 3 4 provides a way to adjust the DOX release upon exposure of the nanocarriers to biological environments of distinct pH. Since this is of acute relevance for DOX based cancer therapies, the drug loaded nanocarriers were also tested in cellular cultures (cancer and non-cancer cell lines) and showed clear cytotoxic and antiproliferative activity. Future work should include clinical tests to confirm the enhanced permeability and retention e ect and optimal pH-responsiveness apply in physiological conditions. Finally, the superparamagnetic quality of the Fe O @SikCRG nanocarriers opens the way 3 4 towards the development of theranostic agents, namely by assessing their MRI-responsiveness. Supplementary Materials: The following are available online, Figure S1: Calibration curve to determine the DOX concentration using UV-Vis spectroscopy at 480 nm, Table S1: Doxorubicin loading eciency and nanoparticle capacity at variable DOX concentration (pH = 6, C = 1.25 mg/mL), Figure S2: Field Dependent Magnetization NP Curves (without normalization) of Fe O nanoparticles (left) and Fe O @SiCRG nanoparticles (right), Figure S3: 3 4 3 4 Speciation of DOX, Figure S4: Doxorubicin release profiles over 48 h, with corresponding fitting using the Weibull model, Table S2: Parameters and , as estimated from the application of the Weibull model to the DOX release data, and coecient of determination (R ). Author Contributions: A.L.D.-d.-S. and T.T. conceived the topic and supervised the experimental work. J.N. and A.L.D.-d.-S. outlined the manuscript and mainly wrote it. S.F.S. assisted in the synthesis and characterization studies of the nanocarriers. C.O.A. and J.S.A. performed the study of the nanocarriers’ magnetic properties. C.S. and F.M. performed the cellular studies. All authors have read and agreed to the published version of the manuscript. Funding: This work was developed within the scope of the exploratory project IF/00405/2014 and the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. A.L.Daniel-da-Silva thanks FCT for funding in the frame of the program IF2014. J.S.Amaral thanks FCT for the IF/01089/2015 grant. S.F.Soares thanks FCT for the Ph.D. grant SFRH/BD/121366/2016. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. 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