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Niosomes as Drug Nanovectors: Multiscale pH-dependent Structural Response Carlotta Marianecci, Luisa Di Marzio, Elena Del Favero, L. Cantù, P. Brocca, Valeria Maria Rondelli, Federica Rinaldi, Luciana Dini, Antonio Serra, Paolo Decuzzi, Christian Celia, Donatella Paolino, Massimo Fresta, and Maria Carafa Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04111 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 10, 2016

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Niosomes as Drug Nanovectors: Multiscale pH-dependent Structural Response

Carlotta Marianecci1,#, Luisa Di Marzio2,#, Elena Del Favero3, Laura Cantù3, Paola Brocca3, Valeria Rondelli3, Federica Rinaldi4, Luciana Dini5, Antonio Serra6, Paolo Decuzzi7,8,9, Christian Celia2,10, Donatella Paolino11,12, Massimo Fresta13,12, Maria Carafa1,*

1. Department of Drug Chemistry and Technology, University of Rome “Sapienza”, Rome, Italy. 2. Department of Pharmacy, University of Chieti - Pescara “G d’Annunzio”, Chieti - Pescara, Italy. 3. Department of Medical Biotechnologies and Traslational Medicine, University of Milan, LITA, Milan, Italy. 4. Center for Life Nano Science@Sapienza, Fondazione Istituto Italiano di Tecnologia, Rome, Italy. 5. Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. 6. Department of Physics Applied to Materials Science Laboratory (PAMS-Lab), University of Salento, Lecce, Italy. 7. Department of Translational Imaging, Houston Methodist Research Institute, Houston, TX, USA. 8. Department of Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Genoa, Italy. 9. Department of Experimental and Clinical Medicine, University of Catanzaro “Magna Græcia”, Catanzaro, Italy. 10. Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX, USA. 11. Department of Pharmacobiological Sciences, University of Catanzaro “Magna Græcia”, Catanzaro, Italy

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12. IRC FSH-Interregional Research Center for Food Safety & Health, University of Catanzaro “Magna Græcia”, Catanzaro, Italy. 13. Department of Health Sciences, University of Catanzaro “Magna Græcia”, Catanzaro, Italy.

AUTHOR INFORMATION *Corresponding Author Sapienza University of Rome E-mail: [email protected]. Phone: +39 0649913603. Fax: +39 0649913133. Notes The authors declare no conflict of interest. #These authors equally contribute.


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ABSTRACT: The use of nanocarriers, that respond to different stimuli controlling their physicochemical properties and biological responsivness, shows a growing interest in pharmaceutical science. The stimuli are activated by targeting tissues and biological compartments, e.g. pH modification, temperature, redox condition, enzymatic activity, or can be physically applied, e.g. a magnetic field and ultrasound. The pH modification represents the easily way of passive targeting, which is actually used to accumulate nanocarriers in cells and tissues. The aim of this paper was to physicochemical characterize pH-sensitive niosomes using different experimental conditions and demonstrate the effect of surfactant composition on the supramolecular structure of niosomes . In this attempt, niosomes, made from commercial (Tween® 21) and synthetic surfactants (Tween®20-derivatives), were physicochemical characterized by using different techniques, e.g. transmission electron microscopy, raman spectroscopy and small-angle X-ray scattering. The changes of niosome structure at different pHs depend on surfactants, which can affect the supramolecular structure of colloidal nanocarriers and their potential use both in vitro and in vivo. At pH 7.4, the shape and structure of niosomes have been maintained; however, niosomes show some differences in terms of bilayer thicknesses, water penetration, membrane coupling, and cholesterol dispersion. The acid pH (5.5) can increase the bilayer fluidity, and affect the cholesterol depletion. In fact, Tween®21 niosomes form large vesicles with lower curvature radius at acid pH; while Tween®20derivative niosomes increase the intra-chain mobility within a more inter-chain correlated membrane. These results demonstrated that the use of multiple physicochemical procedures provides more information about supramolecular structure of niosomes and improves the opportunity to deeply investigate the effect of stimuli responsiveness on the niosome structure.

KEYWORDS: non-ionic surfactant vesicles, niosomes, pH-sensitivity, Raman, TEM, SA


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1. INTRODUCTION During last decade, nanotechnology had a significant impact in medicine and supported its improvements. In this scenario, scientists developed innovative drug delivery systems showing long physical and chemical stability, easily and reliable productions, useful features and reasonable costs. Nanoparticles, liposomes, solid lipid particles, micelles, surfactant vesicles, quantum dots, were used to deliver payloads, e.g. drugs, proteins, peptides, nucleic acids, with different physicochemical and therapeutic properties.1-4 Nanocarriers, made from organic and biocompatible materials, represent the best solution for the delivery of therapeutic agents. In fact, they can entrap different drugs,5 show a low toxicity and can be modified by conjugating targeting and selective molecules. Nanocarriers can also co-deliver diagnostic and therapeutic agents, thus supporting the theranostic potentiality of nanotherapy.6 Based on Bangham’s vesicles or liposomes, which show a bilayer structure similar to plasmalemma, the electron microscopy analysis,7,8 has been widely used to study the morphology and shape of different colloidal nanocarriers, such as ethosomes, Transferosmes® and niosomes. These nanovectors show a bilayer structure similar to liposomes and can deliver therapeutic agents in different biological compartments and tissues, as well as interact with biological membranes.9-11 In particular, niosomes, which are unilamellar or multilamellar non-ionic surfactant vesicles with a liposome-based structure, can be used as therapeutic and theranostic nanocarriers.11,12 Recently, the increase of theranostic-based therapy in medicine needed the development of smart nanovectors activated by environmental factors and physical stimuli. These smart nanovectors should increase the efficacy of therapy and provide innovative nanoplatforms to obtain better performances compared to conventional drug delivery systems and/or imaging agents. In particular, the theranostic strategy can take some advantages from specific symptoms

of diseases, get a selective diagnosis and targeting.13,14 For this reason, pH

modification could represent a promising parameter to be taken into consideration for theranostic 4 ACS Paragon Plus Environment

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therapy. In fact, the inflamed tissues show different pH compared to healthy ones. Furthermore, several big tumors show a significant decrease of pH from physiological (pH≈ 7.4) to acidic (pH≈ 4-6) conditions.15 The endosomal and lysosomal compartments of cells show also an acidic pH (pH≈ 5-6), which can promote the degradation of xenobiotics and molecules. The decrease of pH can trigger the local release of chemotherapeutics or oligonucleotides from pH-sensitive nanovectors.16-21 Surfactants and pH responsiveness could predict and control in vivo fate of niosomes, and modulate their metabolism, accumulation and distribution. The pH-responsiveness of smart nanovectors depends on bilayer fluidity and can be deeply investigated using both conventional techniques, such as fluorescence spectroscopy,22,

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and non-conventional procedures, such as dynamic light scattering

analysis, transmission electron microscopy and small-angle X-ray scattering.24-26 In this work, we applied a multidisciplinary approach to study the supramolecular structure and morphology of pH-sensitive non-ionic surfactant vesicles, or niosomes (NSVs), made from commercial polysorbates, e.g. Tween®21, and/or Tween®20 synthetic derivatives, which have been synthesized by modifying the head group of surfactant with different glycine derivatives (Figure 1). 18 It was previously demonstrated that pH-sensitive nanovesicles can be synthesized using pH-reactive macromolecules, e.g. cholesterol hemisuccinate or oleic acid.27,28 Furthermore, our research group recently demonstrated in vitro pH sensitivity of NSVs made from Tween®21 and Tween®20-derivates, using a fluorescent amphiphilic probe (Nile Red).29 This effect may depend on the potential vesicle-tomicelle transition of NSVs by decreasing pH from 7.4 to 5.5. The decrease of pH from neutral to acid value modifies surfactant bilayers from stable and ordered structure to fusogenic core-shell convex or rod-shaped structures30,31. This transition of niosome bilayer may be also affected from release of payloads, which shows a time dependent effect. The physicochemical characterization of nanovectors plays also a pivot role for their therapeutic applications and needs to be deeply investigated to translate in vivo nanoparticles and tailor their 5 ACS Paragon Plus Environment

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targeting after systemic injection. In fact, physicochemical parameters, e.g. size, size distribution, charge, shape and morphology, depend on the preparation procedures, material composition and surface properties as previously demonstrated.32-34 The aim of this work was to physicochemical characterize the bilayer structure of pH-sensitive NSVs affecting from pH modifications and correlate these changes with nanocarrier properties. In this attempt, the supramolecular structure of niosomes has been deeply investigated using transmission electron microscopy (TEM), raman spectroscopy and small-angle X-ray scattering (SAXS), respectively. These procedures allowed to provide several information about the supramolecular structure of NSVs.35,36

2. MATERIALS AND METHODS Tween®20 (Tw20), Tween®21 (Tw21), Sephadex G-75, Hepes salt {N-(2-idroxyethyl) piperazine-N(2-ethanesulfonicacid)}, calcein, and sodium acetate anhydrous were purchased from Sigma-Aldrich (Sigma-Aldrich SRL, Milan, Italy). Cholesterol (CHOL), was obtained from Acros Organics (Acros Organics BVBA, Geel, Belgium). All other products and reagents were of analytical grade and used without further purification. 2.1. Synthesis of Tween®20 derivatives Tw20 derivatives were synthesized as previously reported.18 Briefly, terminal hydroxyl groups were activated using glycine (Gly) or N-methyl-glycine (Mgly) or N,N-dimethyl-glycine (Dmgly), and the Eschweiler-Clarke reaction, applied to synthesize methyl derivatives of primary or secondary amines, was carried out.37 2.2. Niosome preparation NSVs, made from commercial, and/or synthetic surfactants, and CHOL, at different molar ratio (Table 1), were synthesized using the thin layer evaporation (TLE) method as previously reported.38 All samples show a surfactant concentration below the critical micellar concentration (CMC).18Niosomes 6 ACS Paragon Plus Environment

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were then purified using size exclusion chromatography on Sephadex G75 glass columns as previously reported.38 2.3. Raman spectroscopy Raman spectroscopy analysis was performed using a micro-Raman spectrometer (INVIA, Renishaw, Gloucestershire, United Kingdom) with a 514.5 nm air-cooled Ar ion laser source and an 1800 lines/mm grating polychromator with RenCam CCD detection, providing a resolution of 1 cm-1. The laser source was focused on niosome suspension through a long working distance, 100× long working objective to a spot diameter of approximately 1 µm. and the Raman signal from the vesicular system in fluid was collected in backscattering geometry. The samples were analyzed using suitable hand-made glass holes. The acquisition time for Raman spectra was carried out every 10 min, depending on the strength of the Raman signals, until a suitable signal-to-noise ratio was achieved. Niosomes, which show high surface area per unit volume, generate strong Raman signals. The potential laser-heating effects, occurred during the Raman spectra collection, were deleted by limiting the argon laser power density to testing levels. This set up prevents the effect of continuous laser irradiation on the spectra during the analysis. Data was acquired and analyzed using the Renishaw Wire 2.0 software (INVIA, Renishaw, Gloucestershire, United Kingdom). 2.4. Transmission electron microscopy (TEM) analysis of niosomes NSVs incubated at pH 7.4 and 5.5, respectively, were suitable diluted before the TEM analysis as herein reported: a drop of colloidal suspensions was loaded to a carbon coated copper grid, and after 20 s of incubation, the excess of NSVs was removed at the boarder of the grid using filter paper. The resultant samples were air-dried at room temperature for 10 min. Finally, NSVs were negatively stained with 0.5% (v/v) uranyl acetate for 20 s, and the excess of staining solution was removed at the border of the grid using filter paper. TEM images and electron diffraction patterns were carried out at 100 KV, 7 ACS Paragon Plus Environment

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which is the suitable acceleration voltage to obtain a good resolution and minimal radiation damage of nanomaterials. 2.5. Synchrotron Small X-ray Scattering (SAXS) SAXS measurements were performed at the ID02 high-brilliance beam line (ESRF, Grenoble, France), with a beam cross section of 0.3 mm × 0.8 mm and wavelength 0.1 nm, in the region of momentum transfer 0.017 nm-1 ≤ q ≤ 5 nm-1 (q = (4π/λ)sin(θ/2), where θ is the scattering angle). NSVs were loaded into plastic capillaries (KI-beam, ENKI, Concesio, Italy) mounted horizontally onto a six-places sample holder, which allow nearly contemporary measurements for samples and reference cells in the same experimental conditions. All measurements were performed at 25 ± 1 °C. The exposure time of each measurement was 0.1 s, in order to minimize radiation damage. Several frames were collected on each sample, with 1 s sleeping time, carefully compared and mediated to superimposable within experimental error. The resultant SAXS profiles report the scattered radiation intensity as a function of momentum transfer, q. Several spectra for empty holder and solvent were collected, carefully compared and subtracted to each samples. To investigate a wide q region, SAXS spectra relative to different q ranges were compared and connected. Analysis was carried out to obtain information on the niosome structure on a local scale, beyond the particle size, where NSVs can be treated as a bilayer structures. The scattered intensity, which decays for a statistically isotropic dispersion of bilayers, is proportional to I(q) ÷ [t1ρ∆12 sin(qt1)/qt1 + t2ρ∆23 sin(qt2)/qt2]2, where t1 and t2 are the hydrophobic and total thicknesses of each layer39; ρ∆ij represents the contrast differences between media i and j; 1 and 2 represent the hydrophobic and hydrophilic moieties, respectively, and 3 represents water layer around the samples.


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3. RESULTS AND DISCUSSION NSVs were physicochemical characterized using different procedures. Changes of their supramolecular structure and morphology were carried out at different pHs. The pH-responsiveness of NSVs provides suitable information to predict their behavior both in vitro and in vivo. The decrease of pH can modify the physicochemical properties of NSVs as previously reported

17,18,20

; however, further

physicochemical characterizations would be carried out to investigate the effect of pH changes on the supramolecular structure and morphology of NSVs. In this attempt, NSVs incubated at different pHs, e.g. 7.4 and 5.5, were analyzed using TEM, Raman spectroscopy and SAXS techniques, and the resultant data has been compared to DLS analysis and fluorescence anisotropy to select nanovectors showing the best physicochemical properties for a potential in vivo application (Figure 2). pH-sensitive NSVs were made from glycine derivatives of Tw20, i.e. Tw20-Gly (with glycine), Tw20Mgly (with N-methylglycine) and TW20-Dmgly (with N,N-dimethyl-glycine), or Tw21 and cholesterol (Table 1, Figure S1a and S1b in Supporting Information).


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3.1. Transmission Electron Microscopy (TEM) analysis The TEM analysis allows to analyze and discriminate single particle, closed to spherical structure, in NSVs at whole-particle length scale. The experimental set up of TEM analysis and the limit of detection for nanovectors can restrict the field of analysis. The NVSs are closed to a selective portion of microscopy field and the analysis does not provide a wide overview of colloidal samples. Although its limits, TEM is widely used to physicochemical characterize nanoparticles and provide some information about the morphology of NVS and the effect of surfactants on their supramolecular structure. Results demonstrated that pH changes can affect the morphology of NSV (Figure 3). Changes of niosome structure do not only depend on the pH modifications from 7.4 (top row) to 5.5 (bottom row), but also on the composition of NSVs (Figure 3a-h). In fact, they show a spherical shape and closed bilayer structure in Mgly-NSV2 and Dmgly-NSV3, respectively, at pH 7.4 and an average 10 ACS Paragon Plus Environment

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size around 200 nm as previously demonstrated;20 while Gly-NSV1 shows a flip-flop of surfactants in the niosome bilayer at the same pH value (Figure 3a). This effect may depend on the rearrangement of NSVs, which form a double bilayer, as shown from dark and intense circles in niosome membrane (Figure 3a). Tw21-NSV4 shows a multilamellar structure (Figure 3g). Conversely, the average sizes of NSVs increase by decreasing pH of reaction medium from 7.4 to 5.5 (bottom row, Table 1). In particular, pH 5.5 modifies the bilayer structure of Tw21-NSV4 and forms pore channel through their membrane (Figure 3h). The magnification of TEM images shows the electron diffraction patterns generated during analysis of samples, which were obtained from the electron beam microscopy crossed through bilayer, and affected from its modifications. A maximum diffraction of electron beam microscopy (arcs or rings) demonstrated that an ordered structure occurs for few Å value in local length scale. Furthermore, it is possible to demonstrate that despite of the lack of extended directional order, as in multilayer depositions, arcs or spots can be carried out for TEM images, and they are more evident for samples incubated at low pH (5.5) compared to samples incubated at neutral pH (7.4). This effect can depend on the growing of cholesterol crystallites inside the membrane bilayer at acid pHs.40,41

3.2. Raman spectroscopy The Raman spectroscopy can be used to investigate specific interactions of surfactants in the niosome structure, by investigating the changes of vibrational modes of molecular groups within the bilayer. In fact, the functional groups of surfactants can respond differently to pH modifications and affect the bilayer structure of NSVs.42 However few Raman data are actually available about NSVs made from commercial and/or synthetic polysorbates and cholesterol. Conversely, the effect of cholesterol and other steroidal macromolecules on phospholipid membranes has been extensively investigated and demonstrated

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that the Raman spectra of binary vesicles,

containing cholesterol in the bilayers, show a prevalence of vibrational modes associated with the acyl 11 ACS Paragon Plus Environment

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chains of phospholipids. In fact, the cholesterol can modify vesicle bilayer through segregation or modification of phospholipid transition temperature

44,45

. The full scan of Raman spectra of NSVs at

different pHs has been reported in Figure S2 (in Supporting Information). The decrease of pH (5.5) modifies the bilayer of NSVs;46 in particular, the Raman spectroscopy analysis allows to disclose changes of bilayer fluidity in ordering of hydrophobic derivatives. Results demonstrated that the band of Raman spectra corresponding to the C-H stretching of hydrophobic chains (~ 2900 cm-1) is enlarged for all NSVs both at pH 7.4 and 5.5, respectively (Figure 4). This region of the spectra consists of a large serious of overlapping peaks, which shows both C-H stretching vibrations of the backbone structure, and Fermi resonance bands of the methyl and methylene groups. The changes of Raman spectra depend on the intra- and inter- molecular interactions between surfactants and cholesterol in the bilayer (Figure 4). In fact, the relative intensities of each peak change significantly upon variations of hydration, head group modifications, molecular packing and conformational order of bilayer. Furthermore, results demonstrated that changes of Raman spectra at these specific bands show an high sensitivity of NSVs to pH modifications. The resultant spectrum showed that the peaks of C-H stretching bands at 2850, 2890 and 2930 cm-1, respectively, and their relative intensities allowed to describe the packing of acylic chains and their inter-chains interactions in the surfactant bilayer. An increase of inter-chain disorder between surfactants of niosome bilayer can depend on the decrease of relative intensity for peak at 2890 cm-1 compared to peak at 2850 cm-1, and the decrease of both peaks compared to peak at 2930 cm-1. These peaks change their relative intensity in niosome bilayer due to the presence of cholesterol. In fact, cholesterol decreases peaks at 2850, 2890 and 2930 cm-1, respectively, as a consequence of cholesterol content leakage and cholesterolrich/cholesterol-poor phase separation.43 A significant increase of intensity was observed at pH 5.5 for peak at 2930 cm-1 compared to the full scan spectra (Figure 4). This increase is marked for Tw21NSV4 series, where the peak at 2930 cm-1disappeared at pH 7.4, and appeared at pH 5.5. This is a 12 ACS Paragon Plus Environment

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physicochemical properties of different NSVs made from Tw21. Conversely, the fitting of spectra showed that the relative intensity ratio I2890/I2850 is larger at pH 5.5 than pH 7.4 (Table S1 in Supporting information). These results are opposite to data achieved from Gly-NSV1 and Dmgly-NSV3. MglyNSV2 shows a lower relative intensity ratio I2890/I2850 than Tw21-NSV4. The C-C stretching skeletal modes of different NSVs, corresponding to the Raman spectra at 1100 cm-1 and showing a low sensitivity to pH modifications, was also performed (Figure 5). The three bands at 1050 cm-1, 1125 cm-1 and 1090 cm-1, corresponding to the skeletal stretching modes for trans and gauche conformations of acylic chains, were selected to discuss the intra-chain arrangement of surfactants in the niosome bilayer. The I1090 increased for surfactants closed to transition temperature of acylic chains, which further shows an increase of I1125/I1050 ratio during the analysis. NSVs made from Gly-NSV1 and Dmgly-NSV3 show a significant increase of I1125/I1050 ratio at pH 5.5; while no changes of I1125/I1050 ratio occurred at acid pH for Tw21-NSV4 (Table S1 in Supporting information). The resultant data seems to demonstrate that niosome bilayer of Gly-NSV1 and Dmgly-NSV3 is modified at pH 5.5, probably due to the segregation or leakage of cholesterol, which can increase the fluidity of bilayer made from high hydrophobic surfactants. Conversely, the Tw21-NSV4 is the less sensitive to pH changes due to the presence of Tw21, which protects NSVs and prevents the modifications of bilayer structure during the analysis18. 3.3. Small Angle X-ray Scattering SAXS measurements were performed to characterize the overall structure of NSVs. Multiscale information can be carried out by analyzing different regions of SAXS spectra in the scale from few to hundred nanometers (Figure 6). The overall structure of NSVs, related to particle size and shape, were characterized at low for q region (q < 0.08 nm-1); while the internal structure of NSVs can be characterized at high q region (q > 0.08 nm-1).


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3.4. Niosome properties at pH 7.4 Figure 6 shows the vertical shift of SAXS intensity spectra for GlyNSV1, Mgly-NSV2, Dmgly-NSV3, and Tw21-NSV4, respectively, at pH 7.4. The diffraction peak at 1.84 nm-1,which corresponds to a characteristic distance of 3.41 nm, demonstrates that the presence of cholesterol crystallites after the interaction between surfactants and cholesterol.46,47 Results also showed different peaks in the Wide-Angle region of the spectrum corresponding to crystallites originated from anhydrous cholesterol (Table S2 in Supporting information). The amount of cholesterol crystallites are different in NSVs, and decreases in the following order: Mgly-NSV2 > Gly-NSV1 > Dmgly-NSV3. Crystallites could be either excluded from bilayer, in the border of membrane, and either included within bilayer and confined inside its internal structure.46,47 3.5. Bilayer structure at pH 7.4. On the local scale NSVs display a bilayer structure, with an hydrophobic core, surrounded by two hydrophilic region (Figure 6). Figure 7 reports the reconstructed electron density profiles of surfactant bilayer (for fit details see Method section). The electron density profile of Gly-NSV1 shows that the synthetic surfactant aggregates, thus making a sharp lamellar structure. Gly-NSV1 showed a single small diffraction peak at q = 0.966 nm-1, which corresponds to d = 6.5 nm, and suggests the presence of bilayer pairs. This data agreed TEM analysis for Gly-NSV1 (Figure 3a). Conversely, Mgly-NSV2 showed a single lamellae with a rough profile. In fact, the boundaries between hydrophilic and hydrophobic chains of surfactants were blurred, thus allowing a light water penetration in the bilayer structure. Furthermore, a significant fraction of cholesterol in Mgly-NSV2 seems to be segregated in crystallites as demonstrated from the height of the peak at 1.84 nm-1. This fraction of cholesterol does not take part of the mean structure of bilayer, which is made from only surfactants with short hydrophobic chains. This composition of surfactant bilayer provides a low order degree in the hydrophobic region of NSVs. Conversely, Dmgly-NSV3 shows higher thickness layer than other NSVs where the hydrophilic counterpart are highly packed, and the lipid chains of lauric 14 ACS Paragon Plus Environment

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acid are stretched in the hydrophobic core and packed to cholesterol. This structure seems to suggest that cholesterol is finely dispersed in the surfactant bilayer of NSVs (Figure 7). Conversely, Tw21NSV4 shows unilamellar structure and thin bilayer (Figure 7). 3.6. Large-scale structure at pH 7.4. The lamellar structure of different samples was supported by the typical slope I(q) ÷ q-2 for more or less extended q-range (Figure 6). Downward deviation at low-q corresponds to the lamellae closed up to 100 nm of particles. The average sizes of NSVs (Table 1) are measured and investigated using laser light scattering technique as previously reported.

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Dmgly-

NSV3 shows an intensity SAXS spectrum of colloidal vesicles narrow size distributed, with a characteristic particle form factor (Figure 6);while Mgly-NSV2 shows a SAXS spectrum of polidispersed colloidal vesicles with a flat-lamella geometry up to 220 nm. 3.7. Modifications of niosome properties at pH 5.5. The SAXS analysis was further performed by decreasing the pH from 7.4 to 5.5. The analysis was carried out using different NSVs and the resultant spectra was compared to those, which have been carried out at pH 7.4 (Figure 8) Results demonstrated that there are some differences in the low-q region of the spectra by decreasing pH from 7.4. to 5.5 (Figure 8). Differences depend on the surfactants used to make NSVs. In particular, Gly-NSV1 and Dmgly-NSV3 showed deepness and different position of the minimum value for q-region compared to Mgly-NSV2 and Tw21-NSV4. This q value is specific for the bilayer structure of NSVs. 3.8. Bilayer structure at pH 5.5 Figure 8 showed the effect of pH changes (bottom panel) in the surfactant bilayer of Gly-NSV1 and Dmgly-NSV3. At pH 5.5 the hydrophilic layer of NSVs shows a thin thickness due to the decrease of electrostatic repulsion occurred between polyoxyethylene chains of surfactants, which lead to a different packing of their head groups in the bilayer structure. This modification of the hydrophilic region in NSVs decreases the density of hydrophobic packing chains and increases the bilayer fluidity at pH 5.5. The SAXS analysis shows that changes of bilayer thickness and fluidity in Gly-NSV1 decrease of 0.4 nm the double-bilayer inter-lamellar distance and shift the 15 ACS Paragon Plus Environment

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multilayer diffraction peak to q = 1.03 nm-1, which corresponds to d = 6.1 nm. The modification of internal bilayer structure and inter-bilayer distance at pH 5.5, demonstrated that Gly-NSV1has a low number of bilayer but maintain its specific double bilayer structure . Conversely, Dmgly-NSV3 has a floppier bilayer and is polydispersed at acidic pH (5.5) (Figure 8), according to the lack of particle form-factor details in the very low q region. Mgly-NSV2 is less affected by acid pH than other formulations. At pH 5.5, Mgly-NSV2 did not modify their bilayer structure (Figure 8). The boundaries between hydrophilic and hydrophobic region of surfactants still remain blurred and the hydrophobic chains are packed in a low density region. Differences occurred for the absolute intensity of Mgly-NSV2 spectra, related to particle number and mass, and in the steeper slope at low-q values. Results showed the presence of large aggregates for Mgly-NSV2 (Figure 8). The resultant data may suggest that Mgly-NSV2 partially lacks the bilayer structure at pH 5.5 (Figure 8). Tw21-NSV4 is not sensitive to pH modifications (Figure 8) on the local scale of bilayer structure (Figure 8). In fact, the thickness of bilayer is not changed at pH 5.5. Conversely, the hydrodynamic radius increased up to 88 nm, as shown from the left-shift of the first minimum of the particle form factor (Figure 8). These results may suggest that Tw21-NSV4 aggregates by changing pH environment. The closed large-scale structure is modified and destabilized, with any significant local changes for bilayer structure, shell thicknesses and roughness. From the reported data it can be assumed that at pH 7.4, all the systems here examined have a niosome structure, although with different features, like bilayer thicknesses, water penetration, membrane coupling, and cholesterol solubilization. All of the systems display structural pH dependence, on the local and overall niosome scale, paralleling the already assessed size- and charge- pH sensitivity 18-20, 48

. Increased bilayer fluidity and likely cholesterol depletion on acidification are shared features. All of

the experiments here presented concur to this conclusion. We recall the prominent role of cholesterol 16 ACS Paragon Plus Environment

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(packing/hydrophobicity) in turning micelle-forming Tween-based surfactants into bilayer forming mixtures.

38, 49

Cholesterol exclusion or demixing is then expected to play a role in niosome structure

and stability. When mixed with phospholipids in membranes, in some range of relative concentration, cholesterol is known to give rise to a remarkable phase, known as the lo phase,

50

where crystallinity

and local order are decoupled. Intra-chain and inter-chain mobility loose the usual parallel behaviour due to cholesterol intercalation. Changes in the quality and quantity of cholesterol mixing on pH lowering, then, may give rise to apparently contrasting behaviours, as inferred by Raman spectroscopy. The same general features result in specific behaviours in the different niosome systems. Tw21-NSV4 sample seems to be less pH-sensitive as far as the local scale is concerned. All results concur to this conclusion. Niosomes are formed by a well-defined bilayer, finely dispersing a large amount of cholesterol into the hydrophobic moiety and well packing the small polar head of Tween21 (4 PEG units) to give rise to a local arrangement, similar to the lo phase, very resistant to pH change. Rather, at acidic pH, SAXS and TEM analysis agree in suggesting that Tw21-NSV4 niosomes fuse in larger vesicles with lower curvature, in accordance with the increase in vesicle size recorded by DLS analyses.18 In samples based on Tween20 derivatives, the pH decrease seems to lead to an increase in intra-chain mobility within a more inter-chain correlated membrane. Gly-NSV1 and Dmgly-NSV3 display the stronger modification on the local scale, on pH lowering. We recall that the drop in pH from 7.4 to 5.5 promoted a corresponding decrease in sample ζ-potential, indicating that the amino (or methylamino) groups are protonated at acidic pH (5.5) and the electrostatic repulsion on the outer surface are minimized. Thus it can be assumed that reduced head group repulsion allows for better packing in a niosome low curvature bilayer, also with a reduced amount of “flattening” cholesterol. The thick Dmgly-NSV3 niosome bilayer shrinks, and the interbilayer distance of the Gly-NSV1 bilayer-pairs reduces, for both membrane thinning and reduced interbilayer repulsion.


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The Mgly-NSV2 system displays a still different behaviour. A large amount of cholesterol crystallites are present, embedded or in peripheral contact with the aggregate. The bilayer structure, already blurred at higher pH, seems to be destabilized on pH lowering, losing the vesicular structure, although the molecular response, as seen by Raman spectroscopy, is similar (but less pronounced) to that of GlyNSV1 and Dmgly-NSV3.

4. CONCLUSION The pH modification can affect the supramolecular structure and morphology of niosomes. At neutral and acid pH, niosomes show different physicochemical properties. This effect depends on surfactants and cholesterol ratio, which is self-assembled to make niosomes, accordind to the evidence that cholesterol exclusion or demixing plays a role in niosome structure and stability. Niosomes containing pH-sensitive components, such as Gly-NSV1, Mgly-NSV2 and Dmgly-NSV3, that protonate at lowered pH, show a destabilization of the carrier bilayer; this approach could be useful in tumor targeting, overcoming problems related to drug efflux and other mechanisms of drug resistance51. On the other hand, pH-sensitive nanocarriers formed by a well-defined bilayer resistant to pH change at the local scale, as Tw21-NVS4 samples, but able to release their content at acidic pH after fusion in larger vesicles could be applied as an alternative procedure for inflamed site detection by means of scintigraphic images and in this case, it could be useful to employ vesicles,.. The combination of multiple techniques, allowing to deeply investigate the physicochemical features of nanocarriers and to predict the potential arrangement of bilayer structure responding to specific external stimuli, can be an essential tool to understand the potential interaction between nanocarriers, biological membranes and in vivo compartments.


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ACKNOWLEDGMENT This research was supported by a grant from the Inter-regional Research Center for Food Safety and Health (PONa3_00359), and Italian Institute of Technology (IIT) Center for Life Nano Science@Sapienza. Authors are grateful to D. Manno, Ph.D. (Department of Physics Applied to Materials Science Laboratory (PAMS-Lab), University of Salento, Lecce, Italy) for the technical acquisition of TEM images.

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Figure captions Figure 1. Schematic representation of Tween®20-derivatives and Tween®21. Figure 2. The cartoon describes the bilayer structure of niosomes. The multiple approach represents a useful tool, which provides a suitable physicochemical description of supramolecular structure of niosomes at different length scales. Figure 3. Transmission Electron Microscopy (TEM) images of niosomes at pH 7.4 (top row) and 5.5 (bottom row). The inserts represent the corresponding Electron Diffraction patterns, which can be carried out during the analysis. Arrows indicate the presence of a bilayer. Figure 4. Raman spectra of niosomes in the C-H stretching vibration region (~2900 cm-1) at pH 7.4 (top lines) and pH 5.5 (bottom lines). Figure 5. Raman spectra of niosomes in the region corresponding to the C-C stretching skeletal modes (~1100 cm-1) at pH 7.4 (top lines) and pH 5.5 (bottom lines). Figure 6. Small angle X ray scattering (SAXS) intensity spectra of niosomes at pH 7.4 in the range of q 0.02 nm-1 ≤ q ≤ 4 nm-1. The spectra are vertically shifted for improving the quality of analysis. From bottom to top: Gly (red), Mgly (violet), Dmgly (green), Tween21 (blue). Figure 7. The local bilayer structure. Electron density profiles, obtained from the fit of bilayers, show the physicochemical properties of niosomes (left side). Gly (red), Mgly (violet), Dmgly (green), Tween21 (blue). The panel (right side) reports the cartoon corresponding to bilayer of niosomes. Figure 8. The effect of pH on niosome bilayer. Top panel: small angle X ray scattering (SAXS) intensity spectra of niosomes in the range of q 0.02 nm-1 ≤ q ≤ 4 nm-1 at different pH values. The spectra are vertically shifted for improving the quality of analysis. Niosomes at pH 7.4 are reported by using the following colored curves: from bottom to top, Gly (red), Mgly (violet), Dmgly (green), Tween21 (blue). Niosomes at pH 5.5 are showed by using the black curves. Bottom panel: effect of pH on the local bilayer structure. The electron density profiles, obtained from the fit of bilayers, show 27 ACS Paragon Plus Environment

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physicochemical properties of: Gly (pH 7.4 red – pH 5.5 black) and Dmgly (pH 7.4 green – pH 5.5 black).


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TOC graphic


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Tween20 polar head

Tween 21 polar head

Tween 20 derivatization

Lauric acid apolar tail Gly-NSV1 Mgly-NSV2 Dmgly-NSV3

Tw21-NSV4

Figure 1 ACS Paragon Plus Environment

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