A study of synthesis, characterization and metalloplex formation ability of cetylpyridinium chloride based metallosomes

Journal Pre-proof A study of synthesis, characterization and metalloplex formation ability of cetylpyridinium chloride based metallosomes

Gurpreet Kaur, Baljinder Kaur, Preeti Garg, Ganga Ram Chaudhary, Santosh L. Gawali, P.A. Hassan PII:

S0167-7322(19)35868-4

DOI:

https://doi.org/10.1016/j.molliq.2019.112326

Reference:

MOLLIQ 112326

To appear in:

Journal of Molecular Liquids

Received date:

23 October 2019

Revised date:

10 December 2019

Accepted date:

14 December 2019

Please cite this article as: G. Kaur, B. Kaur, P. Garg, et al., A study of synthesis, characterization and metalloplex formation ability of cetylpyridinium chloride based metallosomes, Journal of Molecular Liquids(2019), https://doi.org/10.1016/ j.molliq.2019.112326

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© 2019 Published by Elsevier.

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A study of synthesis, characterization and metalloplex formation ability of cetylpyridinium chloride based metallosomes Gurpreet Kaura*, Baljinder Kaura, Pr++eeti Garga, Ganga Ram Chaudharya, Santosh L Gawalib,c, PA Hassanb,c

Department of Chemistry and Centre of Advanced Studies in Chemistry, Panjab University,

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Chandigarh 160 014, India.

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Corresponding author : Tel: +91-1722534431; Fax: +91-1722545074, Email:

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[email protected] (Gurpreet Kaur) Homi Bhabha National Institute, Training School Complex, Anushakti Nagar,

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Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400 085, India

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c

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Mumbai-400 094, India

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Abstract Controlled stoichiometry in organic–inorganic hybrid surfactants (metallosurfactants) leads to the formation of liposomal structures (metallosomes). This versatile approach provides a new strategy for preparing metallosomes with transition metal salts from single tail cationic surfactant as a precursor. Four different types of metallosomes having same organic counterpart but different transition metals (Fe, Co, Ni and Cu) were synthesized from

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metallosurfactants. The spherical morphology of synthesized metallosomes was confirmed with transmission electron microscopy (TEM), atomic force microscopy (AFM) and small

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angle X-ray scattering (SAXS) studies. The multivesicular nature of metallosomes was

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supported by TEM results. From DLS studies, hydrodynamic size and polydispersity index

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was found to vary with counter metal-ion. Presence of bilayer within metallosomal structure

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was supported with XRD. More detailed information on lamellarity of the metallosomal structure was obtained with SAXS experiments. As metallosomal structures are able to mimic

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biological membranes; hence, their binding behavior towards calf thymus DNA (CT-DNA) was investigated. Appreciable binding efficiency of metallosomes towards DNA was

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affirmed with fluorescence spectroscopy and ζ-potential studies. Conformational changes were observed from gel retardation assay, providing vivid proof of DNA-metallosomes interactions.

Keywords Metallosurfactants; metallosomes; metal-embedded liposomes; metalloplex; DNA Binding

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1. Introduction A surfactant molecule self-assembles to engender a range of structures e.g. micellar/reverse micellar structures, lamellae, ellipsoids, disks, cylinders and vesicles/reverse vesicles, which vary in nature and size [1,2]. Past two decades have witnessed the utilization of liposomes as synthetic non-viral vectors in gene delivery and drug delivery [3]. Presence of positive charge enables cationic lipids to interact strongly with anionic surfaces such as bilayer membranes of

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biological cells. As a result, they are able to modify the properties of the surface at which they are adsorbed [4]. Researchers have used various combinations of cationic lipids and co-

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lipids, and evaluated their efficiency as transfection reagents. By optimizing the lipid

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composition, liposomal size, membrane fluidity, surface charge, and steric stabilization, it is

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possible to extend the therapeutic index of such carriers. So, from both biological and clinical

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applications view point, the understanding of the mechanism of interaction of DNA and the cationic surfactant is of great interest [5].

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Interaction of metal-complexes with DNA have also been studied for achieving a number of goals like developing probes for nucleic acid structures, chemotherapy agents, design of new

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drugs, and also to determine the mechanism of metal ion toxicity [6]. It has also been proposed that divalent positive ions are required for the replication, transcription, and translation of the genetic code. Metal-based complexes have always been an interesting field of research due to their interesting physical and chemical properties [7]. Various properties associated with them such as ligand exchange rate, redox properties, coordination affinities, solubility, oxidation sates, and biodistribution can be easily modified to increase the therapeutic effect. Along with this side effects associated with these structures can also be controlled [8]. In biological systems, interactions between metal-ions and lipid membrane play a significant role in the various biological processes. Conformational changes are observed in lipid 3

Journal Pre-proof bilayers when different divalent cations such as Ca+2, Fe+2, Mg+2 are adsorbed on their surface. Ion-dipole interactions between lipids molecules and metal ions cause conformational modifications in headgroup as well as tail region of membrane bilayer [9,10]. The presence of metal-ions near lipid membrane can modulate its surface potential, so it is very crucial to understand the complexation of lipid bilayer with proteins and DNA in presence of metal-ions. These types of studies have attracted the attention of various researchers due to their significant role in gene delivery [11,12]. Disadvantage associated

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with these interaction studies is that metal-ions have to be introduced separately for

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individual experiment [9].

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There is a vast literature on the effect of metal ion in surfactant aggregation but that deals

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with anionic surfactants or anionic lipids and positively charged divalent transition and alkali metal ion [13–17]. The anionic surfactant or lipid easily interacts with positively charged

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metal ions or electrolytes due to electrostatic interactions. Also, anionic amphiphiles can form

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vesicular aggregates known as catanionic vesicles in the presence of cationic surfactant at a specific molar ratio [18–20]. However, this work takes into account the water soluble cationic

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metallosurfactants for the preparation of liposomal like bilayer assembly known as metallosomes by virtue of controlling the stoichiometry of metal-salt and single chain surfactant. The cationic metallosurfactants were preferred because of following reasons (i) Metal ion added in anionic surfactant system i.e. micelles and liposomes usually precipitate out [21] (ii) Double chained surfactants used for the preparation of liposomes have very low solubility. (iii) The negatively charged lipids in addition with metal ion form liposomes that show inferior characteristics for in vivo applications such as slower cellular uptake, diminished lysosomal evasion and nuclear localisation etc. [22,23]. Metallosomes are considered superior as they possess metal-ions embedded in their molecular structure. As a result, the experiments can be performed more conveniently with a metallosomal system,

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Journal Pre-proof where metal-ions need not to be introduced separately and interactional studies of metal-ions as well as metallosomal bilayer with DNA can be carried out concurrently. Various examples are reported in literature regarding such vesicles, as one published by Marín-García et al. They synthesized photosensitive CO releasing molecules from Mo based metallosurfactants [24]. In another report, Cu-based metallovesicles were synthesized by Zha et al. and were employed as drug delivery vehicle for doxorubicin-hydrochloride (DOX·HCL) [25].

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Considering all the possibilities, we herein, report the fabrication of metal embedded

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liposomes (known as metallosomes) from a single tailed cationic surfactant cetylpyridinium

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chloride (CPC) based metal-complexes (metallosurfactant). This moiety has both positive

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charge and metal ion which serves as an advantage. Different transition metal salts (Fe, Co, Ni and Cu) were used in 1:2 stoichiometries with CPC to prepare double tailed organic-

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inorganic hybrids as per the method described in our previous paper [26–29]. These

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metallosurfactants have been characterised comprehensively and have been reported in our previously published papers [26,28].

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These as-synthesized double tailed metallosurfactants were utilised in synthesis of metallosomes with and without incorporation of cholesterol using film hydration method. The synthesized metallosomes were characterised using various analytical techniques such as TEM, AFM, XRD, DLS and SAXS. Further, to examine the binding and possible synergetic effects of metallosomes with CT-DNA would be quite interesting because it will take into account the dual functionality of the prepared cationic metallosomes. Techniques such as fluorescence spectroscopy, ζ-potential and gel retardation assay were employed for the investigation. In addition to this, TEM spectroscopy was also employed to study the structural effect on metallosomal membrane in vicinity of calf-thymus DNA.

2. Experimental 5

Journal Pre-proof Scheme 1 Methodology involving the synthesis of metallosomes through thin film hydration

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method.

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2.1 Materials

Cetyl pyridinium chloride (CPC) (Sigma, 99%), Cholesterol (Sigma, 99%), Chloroform

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(Fischer scientific, 99.5%), CT-DNA (Sigma), ethidium bromide (Etbr) (95%, Sigma-Alrich),

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agarose (Sigma-Alrich), ethanol (99.5%, Changshu Hongsheng Fine Chemicals), methanol (99%, Emplura), FeCl2 (sigma, 98%), CoCl2 (Himedia, 98%), NiCl2 (CDH chemicals, 98%), CuCl2 (Himedia, 98%), Bromophenol blue dye (Sigma, 97%), Trizma base (Sigma, 99%) EDTA (CDH, 95%). All aqueous solutions were prepared in triple distilled water. 2.2 Synthesis Procedure of Metallosomes Firstly, four different metallosurfactants named bishexadecylpyridiniumiron tetrachloride (Fe:CPC

II),

bishexadecylpyridiniumcobalt

tetrachloride

(Co:CPC

II),

bishexadecylpyridiniumnickel tetrachloride (Ni:CPC II) and bishexadecylpyridiniumcopper tetrachloride (Cu:CPC II) were synthesized as per our previous reports [26,28]. For synthesis of metallosomes, these double tailed metallosurfactant and cholesterol were mixed in equal molar ratio in chloroform. Mixture was stirred at 55 ºC for three hours. Solvent was slowly 6

Journal Pre-proof evaporated through rotatory evaporator at low vacuum to get thin film deposited on inner walls of flask. Obtained thin film was dried under vacuum for 24 hours and after that hydrated with approximate volume of phosphate buffer (pH 7) making final concentration of 2 mM of double chained metallosurfactant. Hydrated solution was then sonicated for 3 hours using bath sonicator and filtered (10 times) through 0.2 µm pore size syringe filter.

3. Characterisation

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3.1 X-ray Diffraction (XRD) XRD studies were performed with Panalytical D/Max 2500 X-ray diffractometer. The liquid

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samples were dried on a glass slide prior to irradiation with Cu-Kα radiation at 40 kV. Angle

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was established from diffraction spectra.

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2θ was varied from 5-40° with scanning speed of 8°/min. Lamellarity of metallosomal bilayer

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3.2 Transmission Electron Microscopy (TEM)

Microscopic analysis of prepared metallosomes was carried out to depict the internal

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structure by Hitachi H-7500 instrument. Freshly prepared samples were gently placed over 200-mesh formvar copper grid uniformly coated with carbon film and later allowed to be

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adsorbed. After 2 min of deposition, the grid was tapped with a filter paper to remove the surplus fluid and the grid surface was dried at room condition before imaging with a transmission electron microscopy operating at an accelerating voltage of 100 kV. 3.3 Atomic Force Microscopy (AFM) AFM studies were performed using Bruker Nanoscope “V-multimode 8” with an operating frequency of 312.1309 kHz and tapping mode was used for image scanning. Aqueous solution of metallosomes was gently poured on ultrapure silicon wafer 1cm × 1cm and was dried overnight in vacuum desiccator to obtain thin homogeneous coating of metallosomes. The analysis further confirmed the formation, size and shape of metallosomes. 3.4 Dynamic light scattering (DLS) 7

Journal Pre-proof Size and stability studies of synthesized metallosomes were performed using dynamic light scattering experiments. These studies were performed at constant temperature of 25 °C using Malvern NANO-S90 Zetasizer having a He–Ne laser with a wavelength of 633 nm. 3.5 Small Angle X-ray Scattering (SAXS) SAXS experiments were performed using Anton Paar SAXSpace instrument which employs line collimated sealed tube X-ray source (Cu- Kα) operated at 40kV, 50mA. The scattering intensities were monitored in transmission geometry using a 2D CCD detector (pixel size 24

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micron) to span a Q (momentum transfer) range of 0.01 Å−1 to 0.65 Å−1. The data were

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processed using standard protocols.

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3.6 Binding Studies with CT-DNA

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3.6.1 Fluorescence studies

Fluorescence spectra were recorded on a Hitachi F-7000 Fluorescence Spectrophotometer

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with slit width of 10 nm for excitation and emission beams. Samples were illuminated at 490

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nm and emission was analysed between 550-650 nm wavelengths. The fluorescence emission spectra of intercalatively bound Ethidium Bromide-CT-DNA (EB-DNA) complex were

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examined in presence of metallosomes. Increasing amount of metallosomes (2 mM) was added directly to the 1 cm cell, containing EB-DNA complex (keeping the [DNA] / [EB] = 1). The solution in the cuvette was thoroughly mixed before each scan. All measurements were performed at 25C.

3.6.2 ζ- Potential measurements Zetasizer Nano ZS from Malvern Instrument Ltd was used to estimate the surface charge of CT-DNA in presence and absence of metallosomes. Complexation between metallosomes and DNA lead to the formation of metalloplex. Metallosomes and DNA were mixed in a particular mass ratio (M/D). DNA was solubilised overnight prior to metalloplex formation. Metalloplexes were incubated for two hours prior to every measurement.

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Journal Pre-proof 3.6.3 Gel retardation assay To evaluate binding ability of metallosomes with DNA, gel retardation assay was performed. Prepared metalloplexes were loaded on 1% agarose gel and was run at 90V for 2 hours. Ethidium bromide was used to visualise the bands. To compare the effect of counter metalion, this assay was performed for same M/D ratio in each case i.e M/D= 0.01, 0.05, 0.1,0.3, 0.5, 1, 2.

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4. Results and discussion Normally, the single chain surfactants on aggregation result spherical micelles due to its

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critical packing parameter [30]. Here, in this work we are altering the packing parameter of

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single chain surfactant by the virtue of a metallic counter ion which in selected stoichiometry

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of 1:2 (metallic counter ion MCl4: hydrophobic tail cetyl pyridinium), leads to the formation

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of a double tailed metallosurfactant. Thus, we are able to control the packing of single chained surfactant monomers into bilayer arrangement, which upon hydration yields

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multivesicular liposome like structures known as metallosomes. Bilayer vesicular structures i.e. metallosomes in this work have been prepared by water soluble double-chained cationic

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metallosurfactants (prepared from single chain surfactant CPC), which is usually the property of lipids or hydrophobic surfactants. There is a very rare mention in literature where such structures have been obtained using cationic single chain surfactants as precursors. First of all, the molecular arrangement of synthesized metallosomes was investigated using XRD analysis. Compared to XRD spectra of powdered metallosurfactants [26] (spectrum for Fe, Co and Ni based metallosurfactants are given in Fig. ES1), more ordered structures have been obtained for their respective metallosomes. High intensity of individual peaks was obtained for each system, indicating the presence of high order of crystallinity in the structure of metallosomes. Moreover, sharp diffracted peaks were found to be repeating at an interval of 2θ; implying the presence of lamellar layered phases [31] (Fig. 1 (a)-(d)). For Fe, Co and

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Journal Pre-proof Cu based metallosomes diffracted peaks were found to present at interval of 2θ value of 3.1. This trend was not followed by Ni based metallosomes in which additional peaks were observed indicating the complexity in their molecular arrangement. Some small additional peaks were also observed in case of Cu decorated metallosomes, referring to presence of additional disorder in the molecular arrangements. The interplaner spacing (d) was calculated for each metallosomal system using Bragg’s equation (ES1) corresponding to the first peak in diffraction spectra (Table 1). For Fe-metallosomes ‘d’ value came out to be 1.41 nm

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corresponding to 2θ value of 6.24; with Co-metallosomes this value was 1.40 nm with 2θ of

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6.27; for Ni-metallosomes value obtained was 1.29 nm corresponding to 6.80 to 2θ value

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6.80 and with Cu-metallosomes this was 1.41 nm for 2θ of 6.23. The minimum bilayer

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thickness was witnessed with Ni-metallosomes whereas other three had same d-value.

Fig. 1 (a)-(d). XRD patterns obtained with different metallosomal suspensions.

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Journal Pre-proof The metallosomes formed are of multi-vesicular nature as observed from TEM images given in Fig. 2 (a)-(d). These are fabricated from the hydration of lamellar structures formed from the bilayer organization due to cylindrical shape taken by monomers [30,32]. In the present case, the chosen stoichiometry of ligand and metal counter-ion alter the packing parameter and lead to cylindrical packing. In this work, metal is not entrapped as usually is the case of conventional lipid-based liposomal assembly but rather it is a part of surfactant and does not show any kind of precipitation nor does it require any chelating agent. These as synthesized

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metallosomes, as examined from TEM analyses, exhibited size range of 50-150 nm.

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However, with thin film hydration method, we usually get polydisperse vesicles. Therefore,

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few metallosomes in the range of 250 nm were also observed. AFM studies also support these

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outcomes and confirmed the spherical morphology of the metallosomes for all four transition

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metals (Fig. 2 (e)-(h)).

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(a)

100 nm

100 nm

(c)

(d)

100 nm

20 nm

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(e)

(f)

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(h)

Fig. 2 TEM images of (a) Fe-metallosomes (b) Co-metallosomes (c) Ni-metallosomes (d)

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Cu-metallosomes; AFM images of (e) Fe-metallosomes (f) Co-metallosomes (g) Ni-

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metallosomes (h) Cu-metallosomes;

(b)

(a)

(d)

(c)

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Journal Pre-proof Fig. 3 Dynamic light scattering (DLS) graphs of size distribution (Intensity (%)) for different metallosomes (a) Fe-metallosomes (b) Co-metallosomes (c) Ni-metallosomes and (d) Cumetallosomes. The synthesized metallosomes were further studied using DLS, which is a convenient technique to study the stability and size of metallosomal suspension in aqueous phase. The size distribution graphs for all the four metallosomes are given in Fig. 3. As can be observed from the graphs, bimodal distributions were clearly observed with each transition metal. This

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indicates that sizes of the particles are deviating from a range of 50 nm to 200 nm. From Fig.

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3, in case of Fe and Co containing metallosomes, majority of particles were present in the

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size range of above 100 nm; but for Ni and Cu containing metallosomes maximum no. of

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particles were in the range of less than 100 nm. As Z-averaged diameter cannot be calculated in these kinds of systems, we have reported peak maxima in each case in Table 1. Along with

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this, polydispersity index (PDI) obtained from DLS experiments are also given in Table 1.

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High values of PDI affirm the size variation of metallosomes as obtained from TEM images. As crowded colloidal solution can lead to multiple scattering phenomena due to interactions

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between particles; the metallosomes were diluted three times prior to DLS measurements. Table 1 Z-averaged diameter (DZ), PDI values and Inter-planer spacing (d) for different metallosomes Metallosome

Peak maxima (nm)

Polydispersity Index (PDI)

Inter-planer spacing (d) (nm)

Fe-metallosome Co- metallosome Ni- metallosome Cu- metallosome

40, 200 30, 125 50, 160 42, 170

0.54 0.44 0.40 0.46

1.41 1.40 1.29 1.41

Along with this, we also tried the preparation of metallosomes with higher stoichiometries of metal-chlorides to CPC i.e. 1:3 and 1:4. For that matter we chose CuCl2. TEM images in Fig. 4 (a) shows clear image of multivesicular vesicle at 1:3 stoichometry and Fig. 4 (b) at 1:4

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Journal Pre-proof stoichometry. These results clearly indicate that these metallosurfactants are able to form metallosomes with higher stoichiometries as well. Further, the experiments for formation of metal embedded liposomes i.e. metallosomes was also carried out without cholesterol. In this situation too metallosurfactants are able to form spherical metallosomes as confirmed by the TEM image (Fig. 4 (c)). It was very interesting to observe that the given methodology works in different conditions such as variation in stoichiometry and we believe that this process

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would find applications in diverse areas in future.

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(a)

20 nm

(c)

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20 nm

100 nm

Fig. 4 TEM images of multivesicular Cu-metallosomes formed from (a) 1:3 stoichiometry (b) 1:4 stoichiometry (c) 1:2 stoichiometry without cholesterol. The internal structural within the mutivesicular struture, their lamellarity and their thickness has been further estimated by SAXS analysis. Fig. 5 displays the normalized X-ray scattering intensity I(q) of metallosomes prepared from different metallosurfactants. The scattering intensity includes two factors which shows the shape of metallosomes and express as in equation given below. 14

Journal Pre-proof I(q) α NP(q)S(q)

(I)

P(q) is the intraparticle structure factor and depends on the shape and size of the particles. S(q) is the interparticle structure factor and is decided by the spatial distribution of the particles. The metallosomes Co:CPC shows the characteristic peaks due to multilamellar structures, as indicated by the relative position of peaks 1:2:3 (dotted lines) with a lamellar spacing of 10.12 nm. Cu:CPC metallosomes also display characteristic lamellar patterns,

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though the intensities of the peaks are low. For Cu:CPC the lamellar spacing is found to be 10.77 nm. However, Ni:CPC shows weak scattering peaks corresponding to a lamellar

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spacing of 10.18 nm with an additional peak at 1.0 nm-1. This additional peak arises from

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slight inhomogeneity in the bilayer spacing or additional structures that could be present in

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the system. The different packing behaviours depend upon the ionic radius and coordination

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geometry of metal ion binding to ligand during the film formation [33]. In case of Fe:CPC metallosomes, a broad scattering pattern was obtained which indicated the formation of

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uni/oligo lamellar structures with a few bilayers. Since well-defined characteristic peaks of lamellar structures could not be observed, no attempts were made to estimate bilayer spacing

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in these metallosomes. The interaction between metal ions and ligand in the membrane induce the multiple fusions in multivesicular metallosomes, resulting in the formation of multilamellar metallosomes [34].

Co:CPC

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Co-metallosomes

-1

I (q) [cm ]

-1

I (q) [cm ]

1

0.1

Cu:CPC

Cu-metallosomes

1 0.1

0.01 0.01

1E-3

1E-3

0.1

1 -1 q (nm )

0.1

1 -1

q (nm )

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Ni:CPC Ni-metallosomes

Fe:CPC

Fe-metallosomes I (q) [cm ]

1

-1

-1

I (q) [cm ]

1

0.1

0.1

0.01

0.01

1E-3

1E-3

1 -1 q (nm )

0.1

1 q (nm ) -1

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0.1

Fig. 5 SAXS intensity patterns of Co, Cu, Ni and Fe metallosomes.

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After the authentication of formation of metallosomes, their binding abilities with CT-DNA

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were explored. Fluorescence spectroscopy is probably the most commonly used technique to

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study interactions of DNA [35]. The as-synthesized metallosomes show no fluorescence at

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room temperature in solution nor in the presence of CT-DNA and their binding cannot be directly predicted through emission spectra, therefore, competitive ethidium bromide (EB)

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binding studies have been undertaken in order to examine the mode of binding of our formulations with CT-DNA. The fluorescence spectra were collected in phosphate buffer (pH

binding to DNA.

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7.2) which is approximate physiologically relevant condition) to minimize the electrostatic

EB is preferred because it is one of the most sensitive fluorescent probes, is easily available, and more economical substituent for such studies. This dye exhibit minute fluorescence in aqueous media, but shows increased intensity when DNA molecules are present in its vicinity. EB is an intercalating agent and intercalate between the base pairs of DNA molecules. Thus, if a molecule is able to replace EB from its DNA intercalated complex, that molecule is able to interact with DNA molecule and hence possess significant DNA binding ability. These results are reflected by decrease in fluorescence intensity of DNA-EB complex by addition of test molecules [36,37]. With metallosomes consisting different transition in 16

Journal Pre-proof their counter-ion, this trend was followed i.e. a decrease in intensity of DNA-EB was observed with addition of increasing concentration of different metallosomal suspensions concluding replacement of EB with metallosomes (Fig. 6 (i)). This gives inference of intercalating nature of metallosomes. Also, it has been reported in the literature that molecules that are intercalating through DNA base pairs can also be involved in another type of binding mode such as groove binding. When unfused rings are present in a system which is attached through flexible bonds to the

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neighbour moieties, are efficient groove binders. The presence of such rings in our

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metallosomes raises the possibility that our metallosomes may also be interacting with DNA

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through groove binding mode [35,38]. The corresponding Stern-Volmer plots are given in

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Fig. ES2. As can be observed from binding constant values from Table ES1 (using equation

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(i)

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metallosomes.

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ES2), Cu-metallosomes interact most efficiently with CT-DNA than the Fe, Co and Ni based

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(a)

(b)

100 nm

100 nm

(d)

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100 nm

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(c)

100 nm

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Fig. 6 (i) Fluorescence spectra of variation in concentration of different metallosomes with

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constant CT-DNA concentarion (10µM), displaying prominent interaction ability, (ii) DNAmetallosome complex ([DNA]/[metallosome]=1) of (a) Cu-metallosomes (b) Ni-

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metallosomes (c) Co- metallosomes (d) Fe-metallosomes. The binding of CT-DNA with all the four metallosomes was also assessed using TEM as shown in Fig. 6 (ii). From the TEM images, it was confirmed that metallosomes retain their spherical morphology even in the vicinity of DNA. With metalloplex (DNA-metallosomes complex) a transparent layering in the surrounding of metallosomes was observed. Experiments were repeated to confirm this observation and equivalent results were obtained in the repeated experiments. To understand variation in surface charge (ζ-potential) of CT-DNA in vicinity of metallosomes, zeta potential studies were carried out. In absence of metallosomes, zeta potential of CT-DNA was found to be -26.1 mV. When metallosomes were added to DNA,

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Journal Pre-proof negative potential of DNA decreases due to positive potential present on metallosomes. This shows that there are interactions between metallosomes and DNA and they are present in a close proximity. Other than this, zeta potential is way

to

determine

the

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effective

Fig. 7 Zeta potential of metallosomes with CT-DNA at different M/D ratio.

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isoneutrality ratio for a given liposome and

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DNA system [39]. Isoneutrality is a stage at which negative charge present on DNA is

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neutralised by positive charge present on liposomes. Due to charge reversal, the properties of lipoplexes changes at this ratio [40]. It was very interesting to note that for every

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metallosomal system, different isoneutrality ratio was observed although the liposomal

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systems differ in terms of metal-ions present in counter-ions. This illustrate the fact that how change in metal-ion (as part of counter-ion) is affecting the interaction of metallosomes with

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DNA. Maximum isoneutrality ratio (5.1) was observed in case of metallosomes fabricated with Fe (as its counter-ion) and minimum isoneutrality ratio (1.1) was observed with Co fabricated metallosomes (Fig. 7).

Gel retardation assay was employed in order to identify the ability of different metallosomes to promote DNA condensation and inhibition of DNA migration. For this assay metallosomes and CT-DNA were incubated in definite mass ratios (M/D=0.01, 0.05, 0.1, 0.3, 0.5, 1 and 2). CT-DNA possesses a highly polymeric structure which is illustrated from its single band in electrophoresis [41]. Changes observed in intensity and mobility of a DNA band are indication of modification in its molecular structure. These changes can take place on conformational or structural level. Decrease in intensity of DNA bands indicates the

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Journal Pre-proof fragmentation of DNA into smaller units while increase in intensity is visualized as decrease in electrophoretic mobility due to complexation of DNA with external agents [42]. As can be observed from the images (Fig. 8), with increasing M/D ratio the slight change in intensity and mobility of bands can be observed with respect to pure DNA band in lane 1 of Fig. 8. As no decrease in the intensity of bands was observed, so this rule out the possibility of fragmentation of DNA strands in presence of metallosomes. In presence of Femetallosomes compared to pure DNA band in lane 1, the mobility of DNA bands was found

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to be increased (lane 7, 8) with higher M/D ratio. In addition to this slight increase in

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intensity of bands was also observed. These slight changes in mobility and intensity of DNA

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bands point in the direction that there is effective complexation between DNA and

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metallosomes. Similar changes were observed with Co, Ni and Cu based metallosomes; thus

1

2

3

4

5

6

7

1

8

(ii)

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(i)

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indicating effective interactions between CT-DNA and these metallosomes.

(iii)

(iv)

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2

3

4

5

6

7

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Journal Pre-proof Fig. 8 Gel electrophoresis of CT-DNA in with metallosomes synthesised from different metal-surfactants. With (i) Fe:CPC metallosomes, (ii) Co:CPC metallosomes (iii) Ni:CPC metallosomes (iv) Cu:CPC metallosomes with different mass ratio of metallosomes to DNA (M/D ratio) (lane 2-8). Lane 1: DNA, lane 2-8: 0.01, 0.05, 0.1, 0.3, 0.5, 1, 2.

5. Conclusion In this report, four metallosomes embedded with four different transition metals were

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synthesized using a single tail surfactant (CPC) based metallosurfactant by thin film hydration method. For the confirmation of formation of these metallosomes, measurements

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have been carried out using TEM, AFM, XRD, DLS and SAXS techniques. The spherical

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and multivesicular nature of metallosomes has been authenticated by TEM studies. AFM

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results support the spherical morphology of metallosomes. The size and stability studies were

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further affirmed using DLS measurements. The lamellar structure of metallosomes has been confirmed by the XRD spectra and the scattering curves of SAXS. Interestingly, it was

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observed that Fe-metallosomes shows a unilamellar structure as broad scattering peak was observed in this case; while small scattering peaks were observed with Co, Ni and Cu based

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metallosomes displaying their multilamellar nature. Further, the binding ability and interactions of these metallosomes with CT-DNA were explored through different analytical techniques such as TEM, ζ-potential, fluorescence spectroscopy and gel retardation assay. Fluorescence studies displayed efficient intercalating nature of metallosomes with DNA via replacing the Etbr from intercalated Etbr-DNA complex owing to intercalation and groove binding interactions. From gel retardation assay, conformational and structural changes in DNA in the presence of metallosomes were observed as changes in mobility and intensity of bands were observed. With ζ-potential results, it was perceived that incorporation of different metal ion in metallosurfactants affect the isoneutrality ratio for metallosomes and DNA system. Thus, metallosomes opens a new era of wide possibilities due to additional properties 21

Journal Pre-proof associated with them. Their metal-embedded structures are found to be stable and hence, can be employed to study various biological parameters. Significantly, the study of these versatile systems will be highly envisaged them as potential drug delivery system in the biological field and their interaction with biomolecules will illuminate the path to fabricate a potent in vivo diagnosis system in near future.

Conflict of interest

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There is none to declare.

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Acknowledgement

Gurpreet Kaur is thankful to DST for Inspire Faculty award (IFA-12-CH-41) and

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Technology Information Forecasting & Assessment Council (TIFAC), DST, for filing Indian

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Patent Application No. 4055/DEL/2015. Authors are also thankful to DST for PURSE grant

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ӀӀ. Baljinder Kaur is thankful to CSIR for senior research fellowship. Preeti Garg is thankful to UGC for senior research fellowship. Ganga Ram Chaudhary is thankful UGC, India for the

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support under INDO-US 21st century knowledge Initiative project [F. No. 194-2/2016(IC)].

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CRediT author statement 1) Conceptualization: Gurpreet Kaur 2) Methodology: Gurpreet Kaur, PA Hassan 3) Software: Baljinder Kaur, Preeti Garg, Santosh L. Gawali, 4) Validation: Gurpreet Kaur, Baljinder Kaur, Preeti Garg, Santosh L. Gawali, PA Hassan Ganga Ram Chaudhary 5) Formal analysis: Baljinder Kaur, Preeti Garg, Santosh L. Gawali, PA Hassan 6) Investigation: Baljinder Kaur, Preeti Garg, Santosh L. Gawali,

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7) Resources: Gurpreet Kaur, Ganga Ram Chaudhary, PA Hassan

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8) Data Curation: Baljinder Kaur, Preeti Garg, Santosh L. Gawali 9) Writing - Original Draft: Baljinder Kaur, Gurpreet Kaur, PA Hassan

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10) Writing - Review & Editing: Baljinder Kaur, Gurpreet Kaur, PA Hassan 11) Visualization: Gurpreet Kaur, PA Hassan, Ganga Ram Chaudhary

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12) Supervision: Gurpreet Kaur, Ganga Ram Chaudhary

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13) Project administration: Gurpreet Kaur, Ganga Ram Chaudhary

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14) Funding acquisition: Gurpreet Kaur,Ganga Ram Chaudhary

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Declaration of conflict of interest:

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The authors hereby declare that they have none conflict of interest.

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Graphical Abstract:

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Highlights

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Liposome like structures were obtained from single chain metallosurfactants Fabrication of ‘metallosomes’ from water-soluble metallosurfactants TEM, AFM, XRD, DLS and SAXS were carried out to understand membrane parameters Appreciable binding efficiency of these metallosomes was observed towards CTDNA Different metal-ions affects the binding capability with CT-DNA

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  

32