Metallo-Liposomes Derived from the [Ru(bpy)3]2+ Complex as Nanocarriers of Therapeutic Agents

chemosensors Article Metallo-Liposomes Derived from the [Ru(bpy)3]2+ Complex as Nanocarriers of Therapeutic Agents Maria Luisa Moyá1, Francisco JoséOstos 1, Izamar Moreno 1, Diandra García1, Paula Moreno-Gordillo 2, Ivan V. Rosado 2, Pilar López-Cornejo 1,* , JoséAntonio Lebrón1,* and Manuel López-López 3,*   Citation: Moyá, M.L.; Ostos, F.J.; Moreno, I.; García, D.; Moreno-Gordillo, P.; V. Rosado, I.; López-Cornejo, P.; Lebrón, J.A.; López-López, M. Metallo-Liposomes Derived from the [Ru(bpy)3]2+ Complex as Nanocarriers of Therapeutic Agents. Chemosensors 2021,9, 90. https://doi.org/ 10.3390/chemosensors9050090 Academic Editor: Nicole Jaffrezic-Renault Received: 12 March 2021 Accepted: 19 April 2021 Published: 25 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Physical Chemistry, Faculty of Chemistry, University of Seville, C/Professor García González 1, 41012 Seville, Spain; [email protected] (M.L.M.); [email protected] (F.J.O.); [email protected] (I.M.); [email protected] (D.G.) 2Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocio/CSIC/University of Seville, Avda. Manuel Siurot s/n, 41013 Seville, Spain; paulamor[email protected] (P.M.-G.); ivr[email protected] (I.V.R.) 3Department of Chemical Engineering, Physical Chemistry and Materials Science, Faculty of Experimental Sciences, Campus de El Carmen, Avda. de las Fuerzas Armadas s/n, 21071 Huelva, Spain *Correspondence: [email protected] (P.L.-C.); [email protected] (J.A.L.); [email protected] (M.L.-L.) Abstract: The obtaining of nanocarriers of gene material and small drugs is still an interesting research line. Side-effects produced by the toxicity of several pharmaceutics, the high concentrations needed to get therapeutic effects, or their excessive use by patients have motivated the search for new nanostructures. For these reasons, cationic metallo-liposomes composed by phosphatidylcholine (PC), cholesterol (CHO) and RuC1C19 (a surfactant derived from the metallic complex [Ru(bpy) 3 ] 2+ ) were prepared and characterized by using diverse techniques (zeta potential, dynamic light scattering and electronic transmission microscopy –TEM-). Unimodal or bimodal populations of spherical aggregates with small sizes were obtained depending on the composition of the liposomes. The presence of cholesterol favored the formation of small aggregates. ct-DNA was condensed in the presence of the liposomes investigated. In-vitro assays demonstrated the ability of these nanoaggregates to internalize into different cell lines. A positive gene transfection into human bone osteosarcoma epithelial cells (U2OS) was also observed. The RuC1C19 surfactant was used as sensor to quantify the binding of DNA to the liposomes. Doxorubicin was encapsulated into the metallo-liposomes, demonstrating their ability to be also used as nanocarriers of drugs. A relationship between then encapsulation percentage of the antibiotic and the composition of the aggregates has been established. Keywords: metallo-liposome; nanocarrier; gene therapy; DNA; doxorubicin; TEM; sensor 1. Introduction Metallosurfactants are amphiphilic molecules containing metal ions anchored in their structure. The metal ion is usually located at the head group of the molecule [ 1 , 2 ], although it can also be found in the hydrophobic tail [ 3 , 4 ] or acting as counter ion of the polar head group [ 5 , 6 ]. The presence of a metal ion in the surfactant confers the molecule characteristic properties for applications such as catalyst in heterogeneous processes or electron storage devices in photoredox processes. Metallosurfactants have also been used as potential precursors in new synthetic methods, as antimicrobial and antibactericidal agents, and as contrast agents in diagnostic tests, etc. [7–10]. The amphiphilic character of surfactants favors the formation of diverse nanostructures such as micelles or liposomes. The use of these assemblies as nanovehicles of drugs has become an important work line for numerous researchers in recent decades [ 11 – 14 ]. Genetic material, antibiotics or antineoplastic agents, among others, have been encapsulated into these structures to improve their delivery into the target cells, to reduce their side effects or to enhance their activity [15,16]. The incorporation of metal ions into these nanostructures has improved the transfection processes of several nanocarriers [ 17 ] and has also showed a decrease in the energy Chemosensors 2021,9, 90. https://doi.org/10.3390/chemosensors9050090 https://www.mdpi.com/journal/chemosensors Chemosensors 2021,9, 90 2 of 19 status in tumor cells that provokes the deactivation of the cell functions and their subsequent death [ 18 ]. Metallo-surfactants of Fe(II), Ni(II), Co(II) and Cu(II) are able to provoke changes in the primary and/or secondary structures of the bovine serum albumin protein, BSA, depending on the type of assembly formed [ 19 ]. Recently, building blocks of calix [ 4 ] resorcinol and a lanthanum-based surfactant have been prepared for encapsulating the anticancer drug cisplatin [ 20 ]. Results showed an increase of the toxicity of the drug to HeLa cells and a decrease to normal Chang liver cells. This selective activity of cisplatin towards cancer cells seems to be an important research line to diminish strong side effects provoked by this drug in cancer treatments. Multifunctional metallo-organic fluorescent vesicles have also been prepared as cell imaging agents, nanocarriers of anticancer drugs and metal ions detection [21]. With respect to the use of metallosurfactants in the preparation of liposomes, several metalloliposomes (also known as metallosomes or metallovesicles) have been synthesized. Liposomes containing Cu(II) metallosurfactants of 1-alkyl-1,4,7-triazaclononane with different hydrocarbon tail lengths were prepared to condense pEGFP-N1 plasmids and be used as vectors with a good transfection efficiency [ 22 ]. On the contrary, the use of the bis-(4-pyridylmethyl hexadecanoate)-(1,4,7-triazacyclononane) copper(II) metallosurfactant showed low or even zero transfection efficiency [ 23 ]. Cruz-Campa [ 24 ] et al. studied the transfection process of liposomes with analogues metallosurfactants containing the same hydrocarbon tail, but different metal ions in the polar head groups. Results reported similar values for gene transfection in in-vitro assays; no dependence on the nature of the metal was observed. Recently, unilamellar liposomes of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and metallosurfactant molecules derived from the [Ru(bpy) 3 ] 2+ complex, with two different hydrocarbon tail lengths, were synthesized by our group [ 25 ]. Strong interaction with DNA, low toxicity and good internalization into cells were observed for different cell lines. However, the transfection efficiency obtained was low, although slightly higher for the liposomes containing metallosurfactant molecules with longer hydrocarbon tails. It is important to emphasize the multifunctional ability provided by the liposome structure. The presence of hydrophobic and hydrophilic areas in the same assembly allows for the encapsulation of more than one drug, and also drugs with different hidrophobic character. This can produce synergetic effects that improve treatments of some diseases [26–28]. Despite the known antitumor and antimetastatic properties of some ruthenium complexes [ 29 , 30 ], few studies with liposomes containing ruthenium-based surfactants are found in the literature to date. Bearing this in mind and with the aim of searching for effective non-viral vectors in gene therapy, new metallo-liposomes have been prepared by using lα -phosphatidylcholine (PC) from egg yolk, cholesterol (CHO) and the singlechained metallosurfactant derived from the [Ru(bpy) 3 ] 2+ complex [Ru(2,2 0 -bipy) 2 (4-(CH 3 )- 4 0 -(C 19 H 39 )-2,2 0 -bipy)]Cl 2 (RuC1C19). The [Ru(bpy) 3 ] 2+ complex was chosen as the polar head group due to its photochemical properties, which allows using the metallosurfactant as biosensor to obtain information about the system, including internalization processes into cells, without the addition of another agent that can provoke changes in the structurers of the nanoaggregates and in their interaction with other molecules. The structural characteristics of this Ru metalloliposomes permits encapsulating a diverse pharmaceutical agent. This was exploited to encapsulate doxorubicin and DNA. The structures of both the different lipids and the surfactant used in the preparation of the metallo-liposomes are shown in Figure 1. Chemosensors 2021,9, 90 3 of 19 Chemosensors 2021, 9, x FOR PEER REVIEW 3 of 19 Figure 1. Structures of both the lipids and the surfactant used in the preparation of the metalloliposomes. 2. Materials and Methods 2.1. Materials Calf thymus DNA (ct-DNA), L-α-phosphatidylcholine (PC) from egg yolk, cholesterol (CHO) and doxorubicin (DOX) were purchased from Sigma-Aldrich. The structures of the lipids, the surfactant and the drug used in the preparation of the metallo-liposomes are shown in Figures 1 and 2. All reagents were of analytical grade (P.A.) and used without further purification. Figure 2. Doxorubicin. The concentration of the polynucleotide, given by phosphate groups, was determined spectrophotometrically. A molar absorption data of 6600 mol−1 dm3 cm−1 at 260 nm was used to calculate the DNA concentration. The molar absorbance ratios of the solutions A260 nm/A280 nm observed were in the range of 1.7–1.8. This suggests the absence of proteins in the solutions. An average number of base pairs per DNA molecule of 10,000 bp was obtained by using agarose gel electrophoresis using ethidium bromide. All solutions were prepared with distilled and deionized water from a Millipore Milli-Q system (Darmstadt, Germany) with a conductivity value lower than 10−6 S m−1. The pH of the solutions was maintained constant at a value of 7.4 with a HEPES buffer (I = 0.01 mol·dm−3). All the measurements were done at 298.0 ± 0.1 K. The synthesis of the ruthenium surfactant [Ru(2,2’-bipy)2(4-CH3,4´-(C19H39)-2,2´- bipy)]Cl2 (RuC1C19, see Figure 1) was described in a previous paper [31]. Its characterization was carried out by using IR and NMR spectra, as well as by elemental analysis (C, H, N): 1H NMR (CD3OD): δ 0.80 (t, JHH = 6.9 Hz, 3H,CH2CH2(CH2)16CH3), 1.18 (m Br, Figure 1. Structures of both the lipids and the surfactant used in the preparation of the metallo-liposomes. 2. Materials and Methods 2.1. Materials Calf thymus DNA (ct-DNA), Lα -phosphatidylcholine (PC) from egg yolk, cholesterol (CHO) and doxorubicin (DOX) were purchased from Sigma-Aldrich. The structures of the lipids, the surfactant and the drug used in the preparation of the metallo-liposomes are shown in Figures 1and 2. All reagents were of analytical grade (P.A.) and used without further purification. Chemosensors 2021, 9, x FOR PEER REVIEW 3 of 19 Figure 1. Structures of both the lipids and the surfactant used in the preparation of the metalloliposomes. 2. Materials and Methods 2.1. Materials Calf thymus DNA (ct-DNA), L-α-phosphatidylcholine (PC) from egg yolk, cholesterol (CHO) and doxorubicin (DOX) were purchased from Sigma-Aldrich. The structures of the lipids, the surfactant and the drug used in the preparation of the metallo-liposomes are shown in Figures 1 and 2. All reagents were of analytical grade (P.A.) and used without further purification. Figure 2. Doxorubicin. The concentration of the polynucleotide, given by phosphate groups, was determined spectrophotometrically. A molar absorption data of 6600 mol−1 dm3 cm−1 at 260 nm was used to calculate the DNA concentration. The molar absorbance ratios of the solutions A260 nm/A280 nm observed were in the range of 1.7–1.8. This suggests the absence of proteins in the solutions. An average number of base pairs per DNA molecule of 10,000 bp was obtained by using agarose gel electrophoresis using ethidium bromide. All solutions were prepared with distilled and deionized water from a Millipore Milli-Q system (Darmstadt, Germany) with a conductivity value lower than 10−6 S m−1. The pH of the solutions was maintained constant at a value of 7.4 with a HEPES buffer (I = 0.01 mol·dm−3). All the measurements were done at 298.0 ± 0.1 K. The synthesis of the ruthenium surfactant [Ru(2,2’-bipy)2(4-CH3,4´-(C19H39)-2,2´- bipy)]Cl2 (RuC1C19, see Figure 1) was described in a previous paper [31]. Its characterization was carried out by using IR and NMR spectra, as well as by elemental analysis (C, H, N): 1H NMR (CD3OD): δ 0.80 (t, JHH = 6.9 Hz, 3H,CH2CH2(CH2)16CH3), 1.18 (m Br, Figure 2. Doxorubicin. The concentration of the polynucleotide, given by phosphate groups, was determined spectrophotometrically. A molar absorption data of 6600 mol −1 dm 3 cm −1 at 260 nm was used to calculate the DNA concentration. The molar absorbance ratios of the solutions A260 nm/A280 nm observed were in the range of 1.7–1.8. This suggests the absence of proteins in the solutions. An average number of base pairs per DNA molecule of 10,000 bp was obtained by using agarose gel electrophoresis using ethidium bromide. All solutions were prepared with distilled and deionized water from a Millipore Milli-Q system (Darmstadt, Germany) with a conductivity value lower than 10 −6 S m −1 . The pH of the solutions was maintained constant at a value of 7.4 with a HEPES buffer (I = 0.01 mol·dm−3). All the measurements were done at 298.0 ±0.1 K. The synthesis of the ruthenium surfactant [Ru(2,2 0 -bipy) 2 (4-CH 3 ,4 0 -(C 19 H 39 )-2,2 0 - bipy)]Cl 2 (RuC1C19, see Figure 1) was described in a previous paper [ 31 ]. Its characterization was carried out by using IR and NMR spectra, as well as by elemental analysis Chemosensors 2021,9, 90 4 of 19 (C, H, N): 1H NMR (CD 3 OD): δ 0.80 (t, JHH = 6.9 Hz, 3H,CH 2 CH 2 (CH 2 ) 16 CH 3 ), 1.18 (m Br, 32H, CH 2 CH 2 (CH 2 ) 16 CH 3 ), 1.65 (q, JHH = 7.2 Hz, 2H, CH 2 CH 2 (CH 2 ) 16 CH 3 ), 2.56 (s, 3H, CH 3 ), 2.79 (t, JHH = 7.7 Hz, 2H, CH 2 CH 2 (CH 2 ) 16 CH 3 ), 7.25 (t, JHH = 5.9 Hz, 2H, 5,5 0 -CH of alkyl-substituted bipy), 7.46 (m, 4H, 5,5 0 -CH of bipy), 7.62 (m, 2H, 6,6 0 -CH of alkyl-substituted bipy), 7.64 (t, JHH = 5.3 Hz, 4H, 4,4 0 -CH of bipy), 8.06 (m, 4H, 6,6 0 -CH of bipy), 8.59 (s, 2H, 3,3 0 -CH of alkylsubstituted bipy), 9.07 (d, JHH = 7.9 Hz, 4H, 3,3 0 -CH of bipy). 13C{1H}NMR (CD3OD): δ 14.6 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 20.5 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 22.6 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 32.1–32.5 (several s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 34.2 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 36.5 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 54.3 (s, CH 2 CH 2 CH 2 (CH 2 ) 13 CH 2 CH 2 CH 3 ), 125.0–131.2 (several s, 3,3 0 or 5,5 0 -CH of bipy), 140.9 (s, 4,4 0 -CH of bipy), 153.2–160.1 (several s, 2,2 0 or 6,6 0 -CH of bipy). Elemental analysis for C 50 H 64 Cl 2 N 6 Ru: calc.: C, 49.1; H, 4.71; N, 8.18, Experimental: C, 49.3; H, 4.69; N, 8.14. ESI-MS in positive ion mode: found m/z 851.43 for [RuC1C19+1]+, calculated for C50H64N6Ru, 850.42. All these measurements were done in the Research Services of the University of Seville (CITIUS). Results agree with those previously reported. 2.2. Liposome Formation The lipid thin film hydration method was used in the preparation of the metalloliposomes. Desired amounts of cholesterol, phosphatidylcholine, and the ruthenium-based surfactant RuC1C19 were dissolved in 2 mL of chloroform and sonicated for 120 s. Different quantities of lipids and surfactant were mixed to obtain the required molar fraction. The organic solvent of the mixture was evaporated using a rotary evaporator for 50 min at 310 K (human body temperature). This resulted in a dry lipid film that was stored at 193 K for at least 24 h to avoid degradation of the phospholipid. After 24 h, the lipid film was hydrated with 2 mL of a 10 mM HEPES aqueous solution (pH = 7.4) and subjected to 10 alternating cycles of vortex (3 min/1200 rpm) and sonication (2 min, JP Selecta Ultrasons system 200 W and 50 kHz). Finally, the solution was stirred for 1 h at room temperature. The extrusion of the liposome solutions gave uniform populations of unilamellar liposomes with a homogeneous size distribution (low polydispersity). This procedure was carried out using a mini extruder from Avanti Polar Lipids and polycarbonate membranes of 100 nm diameters from Whatman. The liposome solutions were extruded 10 times. Complete stabilization of the nanosystems was achieved, keeping the solutions in darkness at 277 K for 24 h. The composition of the cationic liposomes was expressed in molar fraction ( α ), which is defined as the molar fraction of cationic surfactant (see Equation (1)). α=n+ n++nPC +nCHO (1) n+ , nPC and nCHO being the mole number of the cationic surfactant (RuC1C19), zwitterionic lipid (PC) and neutral lipid (CHO), respectively. All concentrations are referred to the total volume of solution. Different liposome samples were prepared, varying the mass of both the lipids and the surfactant used. The composition of the different samples is collected in Table 1. As can be seen, the mass of CHO was changing in Series 1, the PC in Series 2 and the RuC1C19 in Series 3. Chemosensors 2021,9, 90 5 of 19 Table 1. Liposome composition (mass ratio and mole ratio) and value of molar ratio ( α ). All values are referred to a mass of 6 ×10−4g (or 6.51 ×10−7mol dm−3) of RuC1C19. SERIES SAMPLE Mass Ratio RuC1C19:PC:CHO Mole Ratio RuC1C19:PC:CHO α A1:0.1:0.083 1:0.11:0.20 0.76 1 B 1:0.1:0.83 1:0.11:1.20 0.43 C1:0.1:2.55 1:0.11:2.00 0.32 D1:0.05:0.17 1:0.058:0.51 0.69 2 E 1:0.3:0.17 1:0.36:0.51 0.57 F1:0.5:0.17 1:0.58:0.51 0.51 G1.16:0.33:0.17 1.16:0.23:0.40 0.65 3 H 0.83:0.33:0.17 0.83:0.23:0.40 0.57 I0.33:0.33:0.17 0.33:0.23:0.40 0.35 2.3. Lipoplex Formation The composition of the lipoplexes (liposome/DNA complexes) can be expressed by the mass ratio L/D, defined as: L D=total lipid mass DNA mass =Ru+mass +PC mass +CHO mass DNA mass (2) where Ru+ mass is the mass of the ruthenium-based surfactant (RuC1C19). The same mass of DNA (1.0 × 10 −4 g) was used in all the measurements, which corresponds to a concentration value in base pairs of 8.1 × 10 −5 mol · dm −3 . Appropriate volumes of liposome and aqueous DNA solution were mixed to obtain the L/D required for each α value studied. All solutions are prepared in HEPES 10 mM (pH = 7.4) 2.4. Dynamic Light-Scattering Measurements (DLS) DLS technique (Malvern, Worcestershire, UK) was used to determine the size and the polydispersity index of the different liposomes. Samples were illuminated with a laser at a fixed detection arrangement of 90 ◦ to the center of the cell area. Fluctuations in the intensities of the scattered light were analyzed. The results obtained were the average of 10 measurements. The total lipid + surfactant concentration used was 150 µg/mL. 2.5. Zeta-Potential Measurements A Zetasizer Nano ZS Malvern Instrument Ltd. (Malver, Worcestershire, UK) was used in Zeta-potential ( ζ ) experiments. The electrophoretic mobility of the sample was measured from the velocity of the particles using a Laser Doppler velocimeter (LDV, (Malver, Worcestershire, UK). A DTS1060 polycarbonate capillary cell was used at 310.1 ±0.1 K . The total lipid + surfactant concentration used was 150 µg/mL. 2.6. Electronic Transsmision Microscopy (TEM) A Zeiss Libra 120 scanning electron microscope at 80 kV was used to record TEM images of lipoplexes. Samples were prepared by impregnation on a 300 mesh copper grid coated with collodion. Images were processed with a bottom-mounted TEM CCD camera and recorded with a resolution of 2048 × 2048 pixels. A concentration of total lipid of 150 µg/mL was used. 2.7. Emission Spectra Emission spectra of liposomal solutions were performed in the presence and absence of DNA to quantify the interaction between DNA and the liposome; that is, to estimate the equilibrium constant of lipoplex formation. Measurements were carried out in a Hitachi F-2500 spectrofluorimeter interfaced to a PC for the recording and handling of the spectra. This apparatus was connected to a flow Lauda thermostat to maintain the temperature at 310.0 ± 0.1 K. A standard fluorescence quartz cell of 10 mm path length Chemosensors 2021,9, 90 6 of 19 was used. Emission intensities of the lipoplexes prepared (PC/CHO/RuC1C19-based liposomes + DNA ) were run at α = 0.32 and different L/D values. DNA concentration used was 8.1 × 10 −5 mol dm −3 . The excitation and emission wavelengths used were 456 nm and 600 nm, respectively. Emission spectra were also run to obtain information about the release of DOX from liposomes in the presence and absence of DNA at different polynucleotide concentrations at human body temperature (310.1 ± 0.1 K). The excitation and emission wavelengths used were 500 nm and 538 nm, respectively. It is important to note that these measurements were carried out at λ exc = 500 nm instead of 490 nm (the maximum absorbance wavelength of DOX in solution) in order to avoid an overlap of this band with that corresponding to the emission of the RuC1C19 surfactant (λem = 600 nm and λexc = 456 nm). 2.8. Circular Dichroism Spectra Electronic circular dichroism (CD) spectra were recorded in a Biologic Mos-450 spectropolarimeter (Barcelona, Spain). Spectra of naked DNA and lipoplexes were collected at a constant α value and different L/D values. A DNA concentration of 8.1 ×10−5mol dm−3 was used. Each spectrum was obtained from an average of 10 runs with a 5 min equilibration before each scan at 310.1 ± 0.1 K by using a standard quartz cell of 10 mm path length. The spectra obtained were expressed in terms of ellipticity, Өobs. 2.9. Encapsulation Efficiency Measurements Drug encapsulation efficiency was measured using a dialysis method. 600 µ L of drug-loaded liposome was added to a Sigma-Aldrich Pur-A-Lyzer Midi 1000 dialysis kit (MWCO 1 kDa). A dialysis tube with a molecular weight limit of 12 kDa (Sigma-Aldrich, St. Louis, MO, USA) was also used, and the results obtained were similar. The dialyzer was plunged into a beaker containing 30 mL of the same buffer used for liposome hydration. The liposome’s stabilization was ensured, carrying out the dialysis process at 277.1 ± 0.1 K throughout all the measurements. An aliquot (1 mL) of the buffer deposited in the beaker was taken every 15 min. The quantification of loaded antibiotics was carried out by UV-vis spectroscopy ( λ = 490 nm for DOX). These aliquots were replaced each time by an equal volume of buffer to keep constant the total volume of buffer in the beaker. Dialysis was continued for a period of at least 24 h. The encapsulation efficiency (EE) was obtained using Equations (3) and (4). EE%=[Drug]L [Drug]total ×100 (3) [Drug]L=[Drug]total −[Drug]buffer (4) [Drug]L , [Drug]buffer and [Drug]total being the drug concentration encapsulated in the liposomes, the concentration in the buffer solution, and the total concentration added to the liposomes, respectively. All concentrations are referred to the same total volume. Each measurement was performed in triplicate. 2.10. In Vitro Assays Cytotoxic activity was carried out using the MTT assay. Cell lines were plated out into 96-well plates at a density of 3000 cells per plate. The cell lines studied were: A549 (adenocarcinomic human alveolar basal epithelial cell line), HepG2 (human liver cancer cell line), LS180 (adenocarcinomic human colonic epithelial cell line), MCF7 (breast cancer cell line), RPE-1 (hTERT-immortalized retinal pigment epithelial cell line, normal cell line) and U2OS (human bone osteosarcoma epithelial cells). The assays were recorded at different doses of liposome. Liposome solutions were added to the wells and the plate returned to the incubator for three more days. Later, they were pulsed with MTS (ROCHE). Cell viability was measured by luminometry according to the manufacturer’s instructions. Each dose point was measured in triplicate. Chemosensors 2021,9, 90 7 of 19 Fluorescence microscopy was used to prove the intake of the liposomes prepared in diverse cancer and normal cell lines, studying the luminescence properties of the [Ru(bpy) 3 ] 2+ complex. In these assays, living cells were exposed to liposome solutions of RuC1C19 for 24 h. After the incubation time, cells were repeatedly washed with a PBS solution and mounted on coverslips using Prolong antifade mounting medium (Invitrogen, Ltd., Inchinnan, Renfrewshire, Scotland). Images were taken with a DP72 camera attached to an Olympus BX61 fluorescence microscope using 40 × magnification lenses, with an excitation filter at 470–490 nm and emission long-pass filter at 520 nm. Images were analysed using cellSens Dimension software. Gene transfection assays were done in the U2OS cell line. 4 × 10 4 cells were seeded in 24-well plates. The plasmid pEGFP (0.5 µ g) was mixed with liposomes at α = 0.32 (L/D = 11 and 110) or α = 0.69 (L/D = 8 and 80), with and without DOPE, in 100 µ L of Optimem and incubated for 20 min at RT before being added to the cell cultures, and incubated for further 24 h. Upon incubation, the transfection medium was replaced by fresh DMEM (Dubelcco’s modified eagle medium) and incubated for further 16 h. Transfected cells were seeded in coverslips (VWR) and incubated for 16 h before being subjected to fluorescent microscopy. Following the incubation, cells were fixed with 4% formaldehyde in PBS at 277 K for 15 min, and further permeabilized using 0.25% Triton X-100 in PBS for 15 min. Then, cells were incubated with 4 0 6-diamidine-2-phenylindole dihydrochloride (DAPI) (1/2000) for 15 min at RT, to stain nuclear DNA. Finally, coverslips were mounted in ProLong®Gold Antifade Reagent (Invitrogen). 3. Results and Discussion 3.1. Characterization of Metallo-Liposomes Metallo-liposomes containing a mixing of lipids (PC and CHO) and the rutheniumbased surfactant RuC1C19 (see Figure 1) were prepared at different α molar fractions. Table 1contains both the mass and the molar ratio of the liposomes formulated. The aggregates were characterized by using different methods. The size, polydispersity index and zeta potential values obtained for all liposomal aggregates are listed in Table 2. Table 2. Average size, polydispersity index (PDI) and zeta potential ( ζ ) values obtained for all the liposomes prepared. All values are referred to a mass of 6 × 10 −4 g (or 6.51 × 10 −7 mol dm −3 ) of RuC1C19. Errors represent standard deviation. SERIES SAMPLE A Size/nm PDI ζ/mV A0.76 206 ±15 0.669 67.3 ±3.2 1 B 0.43 130 ±10 0.0953 46.7 ±1.6 C0.32 136 ±8 0.0686 38.8 ±1.5 D0.69 147 ±12 0.636 55.2 ±2.2 2 E 0.57 135 ±15 0.262 51.4 ±1.2 F0.52 146 ±6 0.241 43.5 ±3.0 G0.65 128 ±15 0.210 40.2 ±1.2 3 H 0.57 143 ±10 0.289 48.6 ±1.5 I0.35 135 ±14 0.089 52.9 ±0.8 Three different series of liposomes were prepared. Only the mass of one of the species was changed in each series. So, the mass of cholesterol was changed in Series 1, while the masses of PC and RuC1C19 were maintained invariant. The PC mass was changed in the Series 2 and that for RuC1C19 in the Series 3. With respect to Series 1, an increase of the cholesterol mass (and therefore a decrease in α parameter) diminishes both the size and the polydispersity index of the liposomes. Samples B and C shows a good polydispersity index, that is, a large homogeneity in size of the liposomal solution. In fact, a unique population of liposomes with a size about 130 nm was observed (see histograms in Figure 3A). On the contrary, a high PDI value and a bimodal size distribution (see Figure 3A) are observed for the sample A, the sample with Chemosensors 2021,9, 90 8 of 19 the lowest cholesterol mass. Therefore, the presence of cholesterol molecules at the lipid bilayer seems to favor the compaction of the lipid membrane containing both the Ru(II) metallosurfactant and the PC lipid, diminishing the size of the liposomes and forming unimodal populations. Chemosensors 2021, 9, x FOR PEER REVIEW 9 of 19 Figure 3. (A) Size distribution histogram of liposome samples based on dynamic light scattering measurements. (B) TEM images of Ru(II)-based liposomes with different α values (i: sample D magnification 3 k; ii: sample I magnification 3 k; iii: sample B magnification 4 k; iv: sample F magnification 5 k; v: sample H magnification 5 k; vi: sample C magnification 40 k). Red circle shows the micellar structure. (C) Plot of zeta potential values versus α values. No differences were found for the three α studied. In general, the nanoaggregates seem to be less toxic for both the human liver carcinoma cells HepG2 and the non tumoral RPE-1 cells within the range of concentration studied. On the contrary, the cancer cell lines Figure 3. Cont. Chemosensors 2021,9, 90 9 of 19 Chemosensors 2021, 9, x FOR PEER REVIEW 9 of 19 Figure 3. (A) Size distribution histogram of liposome samples based on dynamic light scattering measurements. (B) TEM images of Ru(II)-based liposomes with different α values (i: sample D magnification 3 k; ii: sample I magnification 3 k; iii: sample B magnification 4 k; iv: sample F magnification 5 k; v: sample H magnification 5 k; vi: sample C magnification 40 k). Red circle shows the micellar structure. (C) Plot of zeta potential values versus α values. No differences were found for the three α studied. In general, the nanoaggregates seem to be less toxic for both the human liver carcinoma cells HepG2 and the non tumoral RPE-1 cells within the range of concentration studied. On the contrary, the cancer cell lines Figure 3. ( A ) Size distribution histogram of liposome samples based on dynamic light scattering measurements. ( B ) TEM images of Ru(II)-based liposomes with different α values (i: sample D magnification 3 k; ii: sample I magnification 3 k; iii: sample B magnification 4 k; iv: sample F magnification 5 k; v: sample H magnification 5 k; vi: sample C magnification 40 k). Red circle shows the micellar structure. (C) Plot of zeta potential values versus αvalues. Similar behavior is observed in samples D (Series 2) and G (Series 3). In these cases, although the cholesterol mass used is the double than that in sample A, bimodal size distributions and a high PDI values are also observed. It must be noted that samples A, D, and G have a high α value (>0.6). Therefore, the obtaining of liposome solutions with unimodal populations does not depend on the lipid (or surfactant) concentration but on their molar fraction. The presence of different populations was also observed from TEM images (Figure 3B). This microscopy technique demonstrated the spherical structure of the liposomes prepared. The smallest population observed in samples A, D and G, with sizes about 20 nm, could correspond to the formation of micelles (or pseudomicelles) due to the presence of higher surfactant concentrations in the solution [31]. The sizes and the PDI values of the liposomes depend neither on the metallosurfactant mass nor on the PC mass for αvalues below 0.6. Table 2also contains the zeta potential values obtained for the different samples. Positive values were observed in all cases, as was expected. According to the results, ζ increases when the concentration of RuC1C19 augments and depends on neither the PC mass nor the CHO mass. This is demonstrated in Figure 3C, where a linear relationship between the zeta potential values and αparameter is observed. It is important to have information about the toxic character of any nanosystem being used as a drug carrier. Bearing this in mind, cytotoxicity measurements of RuC1C19/PC/CHO liposomes were carried out in different cancer cell lines (MCF7, LS180, A549, U2OS and HepG2) and the normal cell line RPE-1 (Figure 4). No differences were found for the three α studied. In general, the nanoaggregates seem to be less toxic for both the human liver carcinoma cells HepG2 and the non tumoral RPE-1 cells within the range of concentration studied. On the contrary, the cancer cell lines MCF7 and LS180 show a higher sensibility to the metallo-liposomes for equivalent doses. The A549 cells showed an intermediate situation. The adenocarcinomic human alveolar basal epithelial cells A549 seem to be less sensible than the breast adenocarcinoma MCF7 and the human colon cells LS180, but more sensible than the HepG2 and RPE-1 cell lines. The liposomes also show an acceptable viability into the cancer cell line U2OS, being something less toxic for bimodal (α= 0.69) than for unimodal (α= 0.32) populations. Chemosensors 2021,9, 90 16 of 19 ance of the liposome structure after 3 days. Therefore, a gradual DOX release is expected during these 3 days. In order to know the effectivity of the encapsulated drug, a study about the effect that the encapsulated DOX causes on DNA was carried out. For this, a DOX-loaded metalloliposome solution ( α = 0.32, [liposome] = 3 × 10 −5 g/mL) was mixed with ct-DNA solutions of different concentrations (L/D = 0.07, 0.10, 0.12, 0.15, 0.22, 0.25 and 0.30). Bearing in mind that RuC1C19-based liposomes generate conformational changes in ct-DNA by themselves, as was previously discussed, L/D values lower than 0.3 were chosen in this study in order to diminish the effect of the liposomes on the polynucleotide secondary structure (see Figure 5B) and, thus, ensure the presence of DNA in its B-form. A DOX concentration of 3 × 10 −5 g/mL was used and dialysis assays of the DOX-loaded liposome solutions were carried out for 12 h, previous to the addition of the polynucleotide, to eliminate the presence of non-encapsulated drug in the solution. Results show the release of DOX from the metallo-liposomes to the solution in the presence and absence of polynucleotide. This release is confirmed by the increase in the emission intensity of the DOX antibiotic observed over time (Figure 8A). The addition of ct-DNA to the drug-loaded liposome solution reduces this observed increase in emission intensity (Figure 8B). Knowing that the emission intensity of DOX decreases when the molecule is intercalated between the nitrogen base pairs of the nucleic acid, the experimental data demonstrate that the released DOX molecules from the metallo-liposomes are intercalated between the base pairs of the polynucleotide. This indicates that the released drug does not lose its effectiveness in interacting with DNA. Besides, this reveals the ability of these Ru-based liposomes to be used as drug nanocarriers. Chemosensors 2021, 9, x FOR PEER REVIEW 16 of 19 membrane can release cationic surfactant molecules from the bilayer to the solution, decreasing the charge of the liposomes and generating larger aggregates by fusion among them. Figure 7B,C show the results of biodegradation obtained for two different α values. A gradual degradation of the nanocarriers with time is observed, as well as a total disappearance of the liposome structure after 3 days. Therefore, a gradual DOX release is expected during these 3 days. In order to know the effectivity of the encapsulated drug, a study about the effect that the encapsulated DOX causes on DNA was carried out. For this, a DOX-loaded metallo-liposome solution (α = 0.32, [liposome] = 3 × 10 −5 g/mL) was mixed with ct-DNA solutions of different concentrations (L/D = 0.07, 0.10, 0.12, 0.15, 0.22, 0.25 and 0.30). Bearing in mind that RuC1C19-based liposomes generate conformational changes in ct-DNA by themselves, as was previously discussed, L/D values lower than 0.3 were chosen in this study in order to diminish the effect of the liposomes on the polynucleotide secondary structure (see Figure 5B) and, thus, ensure the presence of DNA in its B-form. A DOX concentration of 3 × 10 −5 g/mL was used and dialysis assays of the DOX-loaded liposome solutions were carried out for 12 h, previous to the addition of the polynucleotide, to eliminate the presence of non-encapsulated drug in the solution. Results show the release of DOX from the metallo-liposomes to the solution in the presence and absence of polynucleotide. This release is confirmed by the increase in the emission intensity of the DOX antibiotic observed over time (Figure 8A). The addition of ct-DNA to the drug-loaded liposome solution reduces this observed increase in emission intensity (Figure 8B). Knowing that the emission intensity of DOX decreases when the molecule is intercalated between the nitrogen base pairs of the nucleic acid, the experimental data demonstrate that the released DOX molecules from the metallo-liposomes are intercalated between the base pairs of the polynucleotide. This indicates that the released drug does not lose its effectiveness in interacting with DNA. Besides, this reveals the ability of these Ru-based liposomes to be used as drug nanocarriers. Figure 8. ( A ) Variation of the emission intensity of DOX in the ternary system liposome/DOX/ct-DNA at α = 0.32 and [DNA] = 0 g/mL at 310.1 ± 0.1 K. ( B ) 3D-plot of DOX emission intensity versus time and ct-DNA concentration at 310.0 ±0.1 K. (C) Sigmoidal dependence of EI values of DOX on the polynucleotide concentration at α= 0.32. Chemosensors 2021,9, 90 17 of 19 The maximum EI values of DOX observed in the release process for each DNA concentration (see Figure 8B) was plotted versus the polynucleotide concentration in Figure 8C. A sigmoidal dependence was observed. This behavior demonstrates that all released DOX molecules are intercalated between the nitrogen base pairs of the nucleic acid at the highest DNA concentrations. In other words, the drug presents a strong affinity for the polynucleotide. The released drug maintains its intercalation capacity. 4. Conclusions Cationic metallo-liposomes containing PC, CHO and RuC1C19 were prepared varying the masses of both the lipids and the surfactant. Unimodal and bimodal populations were obtained depending on the mole ratio used. Cholesterol favors the compaction of the lipid bilayer forming small liposomes and unimodal populations. TEM images showed the spherical structure of these nanoaggregates. A certain specificity of the nanoaggregates by some cell line was seen. In general, the metallo-liposomes were less toxic for the cancer cells HepG2 and U2OS, and for the normal cell line RPE-1. The formation of the lipoplexes liposome/ct-DNA has been studied and its binding constant estimated. The own liposome was used as a biosensor due the luminescence properties of the [Ru(bpy) 3 ] 2+ -based surfactant. Changes in the circular dichroism spectra of the nucleic acid in the presence of the liposomes demonstrated a polynucleotide condensation at L/D values higher than 5. A good internalization of the metallo-liposomes into cancer and normal cell lines was observed. Again, the luminescence properties of these nanoaggregates allowed to carry out these measurements without the addition of another fluorescence agent, which could provoke changes in the biosystem studied. Transfection viability of about 1–6% was obtained in the U2OS cell line, somewhat higher for unimodal populations than for the bimodal ones. A comparison between different populations of metallo-aggregates has not been made previously. These results encourage us to continue working with these nanosystems. Their optimization could lead to interesting results. Author Contributions: Conceptualization and methodology, J.A.L., M.L.-L. and P.L.-C.; software, J.A.L., I.M. and D.G.; validation, F.J.O. and J.A.L.; formal analysis, M.L.M. and P.L.-C.; investigation, all researchers; resources, I.M. and D.G.; writing—original draft preparation, P.L.-C.; writing—review J.A.L., F.J.O., M.L.-L., M.L.M. and P.L.-C.; and editing, P.L.-C.; visualization, J.A.L., F.J.O., M.L.-L., M.L.M., P.L.-C. and P.M.-G.; supervision, F.J.O., M.L.-L., and P.L.-C.; project administration, M.L.M., I.V.R. and P.L.-C.; funding acquisition, M.L.M., I.V.R. and P.L.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ayudas a Consolidación de Grupos de la Junta de Andalucía (2019/FQM-206 and 2019/FQM-274) and the European Union (FEDER Funds). IVR’s lab is recipient of a Ramón y Cajal Contract RYC2015-18670, and supported by grants RTI2018-100692-BI00, PI-0005-2018 and P18-RT-1271. 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