Structural Characterization of Self-Assembling Hybrid Nanoparticles

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Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors Sandra Ristori, Isabelle Grillo, Sara Lusa, Jana Thamm, Gina Valentino, Virginia Campani, Michele Caraglia, Frank Steiniger, Paola Luciani, and Giuseppe De Rosa Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01085 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Molecular Pharmaceutics

Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors

Sandra Ristoria, Isabelle Grillob, Sara Lusac, Jana Thammd, Gina Valentinod, Virginia Campanie, Michele Caragliac, Frank Steinigerf, Paola Lucianid, Giuseppe De Rosae.

a) Department of Chemistry & CSGI, via della Lastruccia 3, 50019, Sesto Fiorentino, Italy b) Institut Laue Langevin, Large Scale Structures group, 71 rue des Martyrs, CS 20156, 38042 Grenoble Cedex 9 c) Department of Biochemistry, Biophysics and General Pathology, University of Campania “Luigi Vanvitelli”, Via S.M. Costantinopoli, 16, 80138, Naples, Italy d) Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich Schiller University, Lessingstrasse 8, 07743 Jena, Germany e) Department of Pharmacy, University of Naples Federico II, Via Domenico Montesano 49, 80131 Naples, Italy. f) Electron Microscopy Center, University Hospital Jena, Friedrich Schiller University, Ziegelmühlenweg 1, 07743 Jena, Germany

Correspondence to: Prof. Giuseppe De Rosa Department of Pharmacy University of Naples Federico II Via Domenico Montesano 49 80131 Naples Italy Tel. +39 (0)81 678 666 E-mail: [email protected]

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ABSTRACT Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, i.e. Small Angle Neutron Scattering (SANS) and Xray Scattering (SAXS), with cryo-Electron Transmission Microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments, suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structure consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insights, also combined with size distribution and zeta potential of the NPs, provides exaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development.

Keywords: Self-assembling nanoparticles, hybrid nanoparticles, small angle neutron scattering, small angle X-ray scattering, cryo-electron transmission microscopy, bisphosphonate, zoledronic acid.


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1. INTRODUCTION Growing efforts in the field of nanomedicine have resulted in a huge number of formulations based on nanoparticles to meet many and varied therapeutic needs. Nanoparticles (NPs) can interact with cells and tissues through pathways that allow for novel diagnostics and drug delivery routes. Cancer therapy certainly represents the largest area of application for nanomedicine, due to the possibility to achieve selective targeting and accumulation of drugs in tumors via the enhanced permeability and retention (EPR) effect.1 In addition, long circulation in the blood stream can be obtained by making stealth nanocarriers, e.g. by covering their surface with polymers such as poly(ethylene glycol) (PEG) or polysaccharides.

2,3

Liposomes were the

first nanovector entered in the clinical practice, thanks to their biocompatible nature of the lipids, versatility of preparation and possibility to prepare them on a large scale.4 Other biomaterials, such as biodegradable polymers and inorganic nanoparticles (made by metals, metal oxides or salts) have also been proposed for application in numerous areas of medicine. Hybrid nanoparticles and nanocomposite materials offer the possibility to perform multifunctional tasks and their potential applications in drug delivery are certainly worthy of further investigation.5 However, only rarely these formulations overcome the preclinical stage of study. The poor knowledge about the architecture of the nanoassemblies based on novel/hybrid materials is one of the reasons hampering further clinical and industrial development of these technologies. Thus, the control of the nanoparticle (NP) architecture, with precise knowledge of the chemical group distribution, is an issue of the utmost importance for producing materials with desired properties to obtain well-tuned formulations and justify their progress to clinical trials. Such fine control can be only achieved by careful design and in-depth structural analysis of the engineered formulations. Generally physico-chemical characterization is limited to the study of the mean size, size distribution and zeta potential of the plain and loaded nanovectors. The overall and detailed structural properties of nanocomposites can be thoroughly investigated by small angle scattering techniques and high resolution microscopy. Small angle scattering techniques either of X-rays (SAXS) or neutrons (SANS) are well-established methods in the study of self-assembly structures over a wide range of scale lengths, ranging from 2-5 nm to 500 nm. 6,7,8,9,10,11


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The combined use of SAXS and SANS, each relying on a different contrast mechanism, is suited for investigating composite systems. Moreover, in this study the high resolution structural investigation was fostered by cryo-transmission electron microscopy (cryoTEM), a wellestablished method to visualize nanoparticles of both soft- and hard-matter nature11. Zoledronic acid (ZOL) is a powerful bisphosphonate (BP), currently used in the clinical practice as anti-resorption agent in bone related diseases.13 ZOL inhibits isoprenylation processes including farnesylation and geranylgeranylation,

suppresses prenylation of small GTPases,

including Ras proteins, that regulate the proliferation, invasive properties and pro-angiogenic activity of human tumour cells.14 Despite the significant antiproliferative activity of ZOL on different cell lines, its in vivo anti-tumor activity is negligible, probably due to the rapid clearance of ZOL from the circulation and preferential accumulation into the bone.15 It has been shown that the use of nanovectors, such as PEGylated liposomes, can “convert” ZOL in a powerful anticancer agent.16,17 Starting from these findings, novel hybrid self-assembling nanoparticles (hsaNPs), based on calcium phosphate (CaP) and lipids, have been developed and patented to deliver ZOL in different tumor models18,19,20,21,22. Indeed, hsaNPS have shown superior delivery efficiency compared to PEGylated liposomes. The inclusion of a targeting agent such as human transferrin in the formulation allowed to use hsaNPs to deliver ZOL into brain tumor with stabilization of the tumor mass or, in a significant number of animals, complete tumor regression.20,23 Owing to these promising pre-clinical findings, the European Medicine Agency and the Food and Drug Administration have recently granted hsaNPs encapsulating ZOL with the orphan drug designation for the treatment of high grade glioma with trade name Edroma (www.ema.europa.eu reference number EU/3/16/1735; and www.fda.gov date designated: 11/29/2016). Further clinical and industrial development of Edroma could be supported by selfassembling approach because this formulation can be prepared immediately before use, thus overcoming the drawbacks of its commercial development, i.e. long-term stability of NPs and scale-up process.23 However, despite the valuable biological data, structural details on these hybrid NPs are still lacking. To fill the gap, the aim of this study was to provide detailed information of the structural properties of hsaNPs encapsulating ZOL by interpreting data from different scattering techniques, specifically DLS, SANS and SAXS, with the support of morphological evidences provided by the cryoTEM analysis.


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2. MATERIALS AND METHODS 2.1 Materials 1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) and 1,2-diacyl-sn-glycero-3phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000) were purchased by Lipoid GmbH (Cam, Switzerland). Human transferrin (Tf) and all the other chemicals, including high purity (99.9%) deuterium oxide for the preparation of samples to be studied by SANS, were obtained by Sigma-Aldrich (Saint Louis, MO, USA). ZOL was kindly gifted by Lisapharma s.p.a. (Erba, Como, Italy). 2.2 Liposome preparation In a first step, PEGylated cationic liposomes (DOTAP/chol/DSPE-PEG2000 1:1:0.5 weight ratio) were prepared by hydration of a thin lipid film, followed by extrusion. The lipid mixture was dissolved in 1 mL of a mixture chloroform/methanol (2:1 v/v). The organic solution was removed by N2, and the film was further dried under vacuum. Then, the lipid film was hydrated with 1 mL of sterile water and the resulting dispersion was extruded using a thermobarrel extruder system, through polycarbonate membranes with progressively lower porosity from 0.4 to 0.1 µm. The liposomes were stored at 4 °C before use. 2.3 Preparation of self-assembling NPs Different self-assembling NPs (SANPs) formulations, namely blank (PLCaP NPs) and encapsulating ZOL (PLCaPZ NPs) SANPs, and SANPs decorated with Tf, blank (Tf-PLCaP NPs) and encapsulating ZOL (Tf-PLCaPZ NPs), were prepared. 2.3.1 Preparation of PLCaP and PLCaPZ NPs. CaP NPs/ZOL complexes (CaPZ) were prepared as previously described by G. Salzano et al.23 Briefly, an aqueous solution of calcium chloride (18 mM) was added, dropwise and under magnetic stirring, to an aqueous solution on dibasic hydrogen phosphate (10.8 mM). The pH of both solutions was adjusted beforehand to 9.5 with NaOH 0.1 M. The resulting dispersion (CaP) was filtered through a 0.22 µm filter and stored at 4 °C before use. The dispersion was then mixed with an aqueous solution of ZOL (0.132 mM in phosphate buffer at pH 9.5), resulting in


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formation of a colloidal dispersion, here named CaPZ. Finally, PLCaPZ NPs were obtained by mixing PEGylated cationic liposomes with CaPZ, at a volume ratio of 1:1 at room temperature for 15 min. Plain SANPs (PLCaP NPs) were prepared similarly, just replacing the ZOL solution with an equal volume of buffer. 2.3.2 Preparation of Tf-PLCaP and Tf-PLCaPZ NPs. Self-assembling NPs decorated with human Tf were prepared as previously reported.23 Briefly, CaPZ were obtained as reported above. PEGylated cationic liposomes were mixed with human Tf 10 mg/mL HBS (NaCl 140 mM, HEPES 25 mM, EDTA 0.1 mM, pH 8) at a volume ratio of 1:1, at room temperature for 15 min, obtaining a colloidal dispersion here named Tf-PEGylated cationic liposomes. Tf-PLCaPZ NPs were prepared by mixing Tf-PEGylated cationic liposomes complex with CaPZ, at a volume ratio of 1:0.5, at room temperature for 15 min. Plain Tfdecorated SANPs (Tf-PLCaP NPs), were prepared similarly but replacing the ZOL solution with an equal volume of buffer. 2.4 Nanoparticle characterization by Dynamic Light Scattering (DLS) and Zeta potential The mean hydrodynamic diameter of NPs was determined at 20 °C by photon correlation spectroscopy (PCS) (N5, Beckman Coulter, Miami, USA). For these measurements, each sample was diluted in deionizer and 0.22 µm filtered (polycarbonate filters, MF-Millipore, Microglass Heim, Italy) water, and analysed with detector at 90° angle. Polydispersity index (PI) was used as measure of the particle size distribution. For each batch, mean diameter and size distribution were the average of three measures, while for each formulation, the mean diameter and PI were calculated as the values averaged over three different batches. The zeta-potential (ζ) of the NPs surface was measured in water by means of a Zetasizer Nano Z (Malvern, UK). For each batch, data of ζ were collected as the average of 20 measurements, while for each formulation the ζ was calculated as the average of ζ over three different batches. 2.5 SANS and SAXS measurement SANS measurements were performed on the D11 small angle instrument at the Institut Laue Langevin, (ILL, Grenoble, France, experiment number 9-13-505).22 The wavelength was fixed at 8 Å and three sample-to-detector distances were used to cover a q-range from 1.5 10-3 to 0.32 Å-1, with q, the wave vector, defined as q=4π/λsin(2θ) where θ is the scattering angle. The sample were placed in 2 mm thick quartz cells and thermostated at 25 °C with a water circulating


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bath. The data were radially averaged, subtracted from empty cell and electronic background and normalized to the absolute scale using the scattering crossed section of water using LAMP software (http://www.ill.eu/data_treat/lamp/the-lamp-book/). A few complementary measurements were carried during test time on the D33 instrument. SAXS experiments were performed at the ID02 beamline of the European Synchrotron Radiation facility (ESRF), Grenoble, France. The wavelength of the incident photons was 0.1 Å and the sample-detector distance was 1m, allowing for scattering vector q in the range 8×10-3–0.48 Å. Samples were placed in quartz capillaries of 1.5 mm diameter. Temperature was 24 °C. Raw SAXS patterns were recorded using a high sensitivity two-dimensional CCD detector (FReLoN), corrected for background and azimuthally averaged to obtain 1D SAXS profiles. Standard background subtraction due to water and capillary contribution were also applied with in housebuilt software (http://www.sztucki.de/SAXSutilities/).

2.6 Fitting of SANS and SAXS Intensity profiles SANS and SAXS characterization aims at determining the size, shape and interaction of scattering particles with typical size ranging from the nm to the tenth of micrometer. In particular, X-rays interact with the electronic cloud of the atoms while neutron-beams interact with the nuclei. This difference is reflected in the contrast mechanism for the two techniques and makes them highly complementary to get the full description of multi-component systems.23 The scattering intensity per unit volume of spherically symmetric particles can be written as: ‫ܫ‬ሺ‫ݍ‬ሻ = Φௌ ܸ௣ ∆ߩ‫ ܨ‬ଶ ሺ‫ݍ‬ሻܵሺ‫ݍ‬ሻ Where Φs is the volume fraction of particles and Vp their unit volume. P(q)= F2(q) is the form factor and S(q) the structure factor equal to 1 in the absence of interactions between particles. Unilamellar liposomes can be described as hollow spheres. By SANS, since the highly hydrated polar heads of the lipid bilayer are almost not visible and a core-shell model represents well the scattering data. By SAXS, on the contrary, the scattering length density (SLD) of the head groups and of hydrophobic tails is different and it is also clearly distinct from the SLD of the solvent. A more complex model with a core plus three shells (core-3shells) is then required to complete the


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data analysis. Similarly, the double bilayer liposomes was fitted with a core-3shells for neutron data and a core 7-shell for X-rays, to take into account the different contrast. Concerning transferrin, an in depth study of this protein structure was out of the aims of the present paper. As a first approach we therefore used a three-axis ellipsoid model.25 The detailed description of the models used in the following can be found in the Supporting Information. All the data analysis was performed using the SasView program (SasView, http://www.sasview.org/).

2.7 TEM measurements For cryo-electron microscopy (Cryo-TEM) analysis, samples were applied to holey carbon coated copper grids (Quantifoil, Germany), blotted and vitrified in liquid ethan at -180°C. The frozen specimen were transferred immediately with a Gatan 626 cryo-holder (Gatan Inc., USA) to the CM120 transmission cryo-electron microscope (Philips, Netherlands) operated at 120 kV accelerating voltage (LaB6 cathode source) and about –178 °C. Images were acquired using a 2k CMOS camera TemCam-F216 (TVIPS-GmbH, Germany). In order to minimize the noise, four images were recorded and averaged to one image.


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3. RESULTS AND DISCUSSION 3.1 Liposomes Cationic PEGylated liposomes with a mean diameter of about 160 nm and a narrow size distribution (IP < 0.2) were used to prepare the hsaNPs15 studied in this work. The small angle scattering profiles of DOTAP/Chol/DSPE-PEG2000 pure liposomes are shown in Figure 1a and b (SANS and SAXS, respectively), together with the corresponding fittings and SLD profiles. The two techniques, though probing the same objects, exhibits very different features, and this issues from the subtle balance of scattering length densities between hydrophobic surfactant tails, hydrophilic heads, polymer shell and solvent. As mentioned above in the experimental section, neutrons are sensitive to the nuclei of atoms. In this case, due to the high hydration of the surfactant head group and PEG chains, there is almost no contrast to the solvent and only the hydrophobic layer is clearly visible. The SANS profile was consistent with the scattering of hollow and relatively monodisperse spheres, as indicated by the oscillation around 0.0056 Å-1 followed by a q-2 decrease in the middle q domain. A more detailed scattering profile involving a core-3shells model (see Supporting Information for equation details), taking account the PEG layer, slightly improve the data fitting in the middle q-range. From the SANS data analysis, an inner core radius of 400 Å (σ=0.35) and a total bilayer thickness of 36 Å (σ=0.15) are obtained. The PEG layer extend over 40 Å, but the exact value cannot be obtained from the fits due to the poor contrast with the solvent. It is however consistent with an extended layer of the PEG2000 on the liposome surface. X-rays, which interact with the electronic cloud, probe the profile of the membrane, and are especially sensitive to the difference of SLD between the tail and head groups. The PEG polymer layer also contributes to an additional contrast giving raise to the large and asymmetric oscillation observed here. The SANS size parameters were used to model the SAXS data with a core + 5 shells model to distinguish the head and tail groups of the bilayer. The fit obtained is shown in Figure 1 and the fit parameters are summarized in Table 1. Although poorer than the SANS fit, a reasonable adjustment was obtained with SLDs values close to the theoretical ones. An improvement of the data fitting could be obtained by considering some asymmetry for the PEG shell between the inner and outer layers and a parabolic profile of the SLD along the PEG


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shell as reported by other authors.26 However, this kind of ultra-detailed analysis would be beyond the scope of this paper. According to previously reported data23, the average hydrodynamic radius obtained by DLS is ~ 60-70 nm, which is appreciably higher than the value extracted from SANS fitting and from cryo-TEM micrographs (Figure 2). This difference can be attributed to a thick hydrodynamic layer which also containing loosely bound chloride counterions, weighted more by Brownian motion and DLS modeling than by SANS. Direct spatial images of soft matter nano-objects can be obtained by cryo-TEM. A representative micrograph obtained from DOTAP/Chol/DSPE-PEG2000 liposomes is reported in Figure 2 and shows monolamellar vesicles with diameter of 900-1000 Å. This size is in excellent agreement with the SANS data analysis and definitely confirms our picture of the pure liposome structure.

As a first conclusion, it is important to point out the role exerted by the cationic component DOTAP in determining the liposome structure24 since the repulsion among surface charges, characterized by a highly positive zeta potential (37.5±1.5 mV), is able to prevail on the tendency to bilayer stacking. The presence of PEGylated lipids and the consequent steric hindrance on the liposome surface could also contribute to avoid vesicle aggregation.

Figure 1: SANS (A) and SAXS (B) scattering profiles of pure PEGylated liposome. The full line is the data fitting. In insert, the scattering length density profile is shown.


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Table 1: SANS and SAXS best fit parameters for the pure PEGylated liposome.

LPEG 25 °C

LPEG 25 °C

(SANS)

(SAXS)

Rc (Å)

400

400

e2 (Å)

36

e1,3 (PEG layer) (Å)

40

40

ρc (cm-1)

6.36 1010

9.33 1010

ρ2 (cm-1)

1 1010

ρ1,3,peg (cm-1)

5.8 1010

9.65 1010

PD (Rc)

0.35

0.35

PD (e)

0.15

0.15

head: 5.5 tail : 24

head: 12.5 1010 tail : 7.1 1010

Rc and ρc are radius and scattering length of the core, respectively; ei is the thickness of the i-th layer in the model and ρi the corresponding scattering length (see figure SI1 for more details). PD is the polydispersity index of the different shells expressed as standard deviation in a Gaussian distribution

Figure 2: Cryo-TEM image of DOTAP/Chol/DSPE-PEG2000 pure liposomes.


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3.2 Calcium phosphate nanoparticles complexed with zoledronic acid (CaPZ) The scattering (SANS and SAXS) diagrams of CaPZ nanoparticle are simple intensity decay, with no features typical of a particular shape or scattering object distribution (Supplementary figure SI3), thus not requiring a detailed structural investigation. These curves suggest that large objects, or nanoparticle aggregates, are present in the dispersion. The corresponding TEM images (Figure 3), in agreement with these finding, show an extended network of globular particles connected to each other, suggesting the occurrence of strong attractive electrostatic interactions, possibly mediated by the presence of ZOL on the particle surface.

Figure 3: Cryo-TEM image of calcium phosphate nanoparticles complexed with zoledronic acid (CaPZ).

3.3 Hybrid nanoparticles PLCaPZ PLCaPZ NPs have been developed and visualized by Cold Field Emission Gun Scanning Electron Microscopy (cFEG-SEM) analysis.18 cFEG-SEM images showed an homogeneous population of NPs, while any information was available on how the different components forming the NPs arrange to form the PLCaPZ NPs. In the same work, we hypothesized that positively charged liposomes interact with CaPZ thus inducing the rearrangement of the components with consequent formation of hsaNPs. This study for the first time provides information on the architecture of hsaNPs. The SANS and SAXS scattering curves of the systems


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containing both CaPZ particles and PEGylated liposomes (PLCaPZ) are shown in Figure 4. We observed here that the addition of CaPZ nanoparticles produced a strong modification of the scattering profiles with respect to plain liposomes. In the SANS profiles (Figure 4a), a strong ondulation of the scattered intensity appeared in the middle q region, while in SAXS profiles (figure 4b) a series of well defined oscillations showed up in the middle-large q region, together with a strong enhancement of the scattered intensity at low q compared to the scattering of pure liposomes. By fitting these new patterns hybrid double layer structures were clearly shown to form in solution upon mixing of liposomes and CaPZ nanoparticles. In order to keep the number of parameter reasonable, both for neutron and x-ray experiments, the PEG shell is ignored. With neutron, the membrane is considered as homogenous and a core + 3shells model is used; for xray, head groups and tails are distinguished which involves a core + 7shells model. The parameters are summarized in Table 2. In a first approach, the curve analysis represents correctly the main features of the experimental SANS and SAXS data. We obtained a water core of 250 Å with a relative high polydispersity (σ = 0.4) and a water layer of 125 Å (σ = 0.15) between the two bilayers of total thickness 36 Å (σ = 0.10) similar to the pure liposomes. The total average radius is 450 Å (without taking account the outside PEG layer). For SANS, the amplitude of the oscillation was better fitted when assuming that unilamellar and bilamellar liposomes are in coexistence (Figure left, full line). We find that around 20% unilamellar liposomes are still present. The presence of bilamellar structures was confirmed by cryo-TEM measurements, since micrographs of the dispersion obtained by mixing CaPZ NPs and liposomes (PLCaPZ) showed the presence of new nanostructures, with enhanced lamellarity compared to the plain liposome system (Figure 5). Electron microscopy also confirms the existence of unilamellar liposomes. In agreement with the scattering, we observed a relative polydispersity in the core whereas the distance between the bilayer is well defined. These experiments do not allow to assert undisputably the presence of the particles within the core of the hasNPs. However, the SAXS data suggest that the increase of the SLD of the core found from the data analysis compared to the SLD of water could be due to the presence of CaP aggregates and ZOL. It is worthy of note that the globular particles evidenced in the Figure 3 are not evident in the hsaNP core, while the latter is characterized by a higher electron density, compared to the surrounding aqueous medium. This could be explained hypothesizing that, once mixing CaPZ NPs with the cationic


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liposomes, non only the unilamellar liposome reorganize in a bilamellar shell structures, but also CaP and ZOL reorganize swiching from a pearl-like structure to more uniform matrix within the hsaNP core. CaPZ NPs separated from the hsaNPs were also found in the sample (figure SI 4), suggesting that a further optimization of the experimental conditions, especially the time of incubation and separation of free CaPZ NP, could be still taken into account.

1000

100

100

10

1

x1010

7

I ( cm-1)

10

I (cm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cm-2

6

0.1

1

0.1

5

x1010 cm-2

12 11 10

4

9

3 2

0.01

13

0.01

1

8 7

d (Å)

0

d (Å)

6 0

0.001 0.001

100

200

300

400

0.01

500

0

0.1

q (Å-1)

100

0.001 0.001

200

300

0.01

400

500

0.1

q (Å-1)

Figure 4: Effect of CaPZ nanoparticles on the scattered intensity of PEGylated liposomes. Left: SANS profile; the black dotted line is obtained with the model of bilayer liposome, with the corresponding SLD profile in insert. The grey dotted line represents the unilamellar liposomes. The combination of the two models yields to the black full line. Right: SAXS profile. The lines are the fitted curves and the insert shows the scattering length density profiles.


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Table 2: SANS and SAXS best fit parameters for the PLCaPZ hybrid liposomes

Rc (Å)

Core + 3 shells SANS 250

Core + 7 shells SAXS 250

e1,3(Å)

36

head:5.5 tails: 24

e2 (water) (Å)

125

125

ρc (cm-2)

6.36 1010

12 1010

ρ1(cm-2)

2 1010

head:12.5 1010 tails: 7.0 1010

ρ2(cm-2)

5.8 1010

9.33 1010

ρ3(cm-2)

1 1010

head:12.5 1010 tails: 7.9 1010

6.36 1010

9.33 1010

PD (Rc)

0.4

0.4

PD (e1 , e3)

0.1

0.1

PD (e2)

0.15

0.15

PLCaPZ

ρsolvent (cm-2)

Rc and ρc are radius and scattering length of the core, respectively; ei is the thickness of the i-th layer in the model and ρi the corresponding scattering length (see figure SI1 for more details). PD is the polydispersity index of the different shells expressed as standard deviation in a Gaussian distribution


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A

Figure 5: Cryo-TEM images of the hybrid self-assembling or PLCaPZ NPs.

3.4 Characterization of Tf in solution Prior to studying the formation of adducts between the protein Tf and the hybrid nanoparticles LCaPZ, we investigated the structural properties of transferrin in solution with and without ZOL. The SANS and SAXS scattering data from the pure transferrin are presented in Figure 6 together with the data analysis. In a first approach, assuming a tri-axis ellipsoid model (see Supporting Information for details), we obtained semi-axes of 12, 40 and 84 Å. The upturn at low angle in SANS might be due to aggregation often observed for proteins in D2O. The structural data reported in the literature for free transferrin in solution are rather sparse. Martel et al.26 described the transferrin molecules as oblate spheroids with semi-axis 46.6, 46.6 and 15.8 Å, while Kilar et al.28 proposed a model of cylinders with 48 Å diameter of and of 96 Å length. In the formulation of has NPs encapsulating ZOL, the molar ratio of transferrin and zoledronic acid is Tf : ZOL ∼ 100:1; here, we decreased this ratio to 50:1 and to 5:1, without observing any change in the corresponding SANS scattering profile (Figure SI 5) where all the curves are superimposed. The decrease of intensity observed from pure Tf to the Tf-PLCaP NPs could be due to a dilution by a factor of 1.5.


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I (cm-1)


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1

0.1

0.01

0.001 0.001

0.01

0.1

q (Å-1)

Figure 6: SANS (blue diamond) and SAXS (red square) scattering profiles of Tf. The full line is the data fitting considering a three axis ellipsoid model.

3.5 Hybrid nanoparticles PLCaPZ modified with Tf The SANS and SAXS scattering data of the hybrid liposomes with transferrin are shown in Figure 7 together with the best fits. The curves exhibit an additional signal in the high q domain which is characteristic from the form factor of the Tf. The data are then analyzed with two populations corresponding to the hybrid double bilayer-containing NPs in coexistence with the proteins. The radius of the core - 250 Å- is unchanged compared to the hsaNPs without transferrin but the distance between the two bilayers is significantly reduced (85- 90 Å). The core and inter-water SLDs are similar to what was found previously and this indicates that the protein is not encapsulated inside the NPs. Indeed, with a typical SLD of 3 1010 cm-2 for protein in D2O (SANS)27 the presence of transferrin in the core or in the interbilayer gap would induce a decrease the average SLD of water compartments. The cryo-TEM picture (Figure 8) support well the structure extracted from the scattering data, with a clear decrease of Dw.


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1000

13

x1010 cm-2

7

11

5

100

10

4

9

3

10

2

0

100

200

300

400

500

1

0

0.1

0.01

0.01

0.1

q (Å-1)

100

200

300

400

500

1

0.1

0.01

d (Å)

6

d (Å)

0

0.001 0.001

8 7

1

I ( cm-1)

10

x1010 cm-2

12

100

6

I (cm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.001 0.001

0.01

0.1

q (Å-1)

Figure 7: Effect of Tf on the PEGylated liposomes with the nanoparticles. Left: SANS; Right: SAXS profile. In both plots, the grey dotted line is obtained with the model of bilayer liposome, with the corresponding SLD profile in insert. The black dotted line represents the scattering from Tf. The combination of the two models yields to the black full line.

Table 3: SANS and SAXS structural parameters for the PLCaPZ hybrid liposomes

Rc (Å)

Core + 3 shells SANS 250

Core + 7 shells SAXS 250

e1,3(Å)

36

head:5.5 tails: 24

e2 (water) (Å)

90

85

ρc (cm-2)

6.36 1010

12 1010

ρ1(cm-2)

3 1010

head:12.5 1010 tails: 7.0 1010

ρ2(cm-2)

5.8 1010

9.33 1010

ρ3(cm-2)

1 1010

head:12.5 1010 tails: 8 1010

6.36 1010

9.33 1010

PD (Rc)

0.4

0.4

PD (e1 , e3)

0.1

0.1

PD (e2)

0.15

0.15

PLCaPZ

ρsolvent (cm-2)

Rc and ρc are radius and scattering length of the core, respectively; ei is the thickness of the i-th layer in the model and ρi the corresponding


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scattering length (see figure SI1 for more details). PD is the polydispersity index of the different shells expressed as standard deviation in a Gaussian distribution

Figure 8: Cryo-TEM image of hsaNPs modified with transferrin (Tf-PLCaPZ NPs).

From scattering data analysis it resulted that the formation of adducts with transferrin didn’t alter significantly the global size and the layer arrangement of the hsaNPs. However, a marked decrease in the interbilayer thickness was observed in the presence of transferrin, suggesting that a fraction of protein molecules could be intercalated in the inner aqueous compartment comprised between the two bilayers of the hsaNPs. This evidence was also supported by the fact that the transferrin isoelectric point is 5.2 – 5.7, which imply that the protein molecules are negatively charged at our working experimental conditions. This suggested that Tf mainly interacts with the cationic heads of DOTAP, although non-electrostatic interactions, e.g. van deer Waals interactions among lipids and the hydrophobic residues of transferrin cannot be excluded. This finding was in agreement with the Cryo-TEM images which showed a reduced inter-layer spacing. It is worthy of note that in a previous work23 we found that about 54% of the Tf was found free in solution upon Tf-PLCaPZ NPs. In this study, we demonstrated that a percentage lower that the 46% of the Tf used in the preparation should be present on the NP surface. This is not surprising when considering the large amount of Tf used in the preparation control, e.g. 10


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mg/ml before mixing with liposomes, that is certanly enough to saturate the NPs surface. Here, we also found that a fraction of Tf is also located between the two bilayers and should have only a “structural” role, presumably stabilizing the observed core-shell structure. On the other hand, a “biological” role of the Tf located between the bilayers can be excluded, due to the physiological role of Tf as transporter of iron into the cells with following recycle back to the exterior of the cell and release in the extracellular matrix. Thus, only Tf molecules located on the hsaNPs surface could be involved in the binding the Tf receptors on the endothelial cells of blood brain barrier and on the surface of cancer cells, consequently contributing to the higher cytotoxicity of the Tf-modified hsaNPs containing ZOL.


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4. CONCLUSIONS The combination of scattering and microscopy techniques together with the in-depth analysis carried out in this work allowed to define the architecture of the hsaNPs encapsulating ZOL at high resolution. Specifically, a transition from monolamellar liposome to bilamellar NPs was found upon addition of CaP NPs. The interbilayer thickness markedly decreased in presence of Tf, suggesting that a fraction of the protein molecules were located in the inner compartment of the layered aggregates, where they could act as a bridge between lipid bilayers. Previous studies demonstrated that mixing of CaPZ and PEGylated liposomes results in NPs with a mean diameter lower than liposomes and a polydispersity index lower than 0.2. This homogeneous population of NPs was also observed by SEM18. Although hsaNPs encapsulating ZOL have been successfully used to deliver ZOL into different cancer cells and animal models of tumor, the precise structure of hsaNPs was still unknown. We believe that the findings provided in this work fill a gap in the characterization of hsaNPs. The knowledge on the architecture of these NPs should therefore be of guidance for further developments of these engineered hybrid nanoparticles toward clinical and industrial applications. Moreover, this study could provide a strategy to investigate the structural features of other hybrid nanosystems.

5. ACKNOWLEDGMENTS. Lipoid GmbH is acknowledged for the endowment to the University of Jena (P.L.). We thank the Institut Laue-Langevin for allocation of neutron beam time (experiment 9-13-505, doi:10.5291/ILL-DATA.9-13-505). We are also grateful to Dr Michael Sztucki of the ESRF ID02 staff for assistance during SAXS experiments. This work benefited from the SasView software developed by the DANSE project under NSF award DMR-0520547. SUPPORTING INFORMATION Expression to calculate the scattering intensity per unit volume of spherically symmetric monodisperse particles. Expression to calculate the amplitude of form factor for a sphere with radius R.


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Fit of conventional (i.e. non pegylated) DOTAP/chol liposomes. Figure SI 1: : Schematic representation of a pegylated liposome with the different layer thicknesses. Fit of the bilayer liposomes. Figure SI 2: Schematic representation of a DOTAP/chol liposome. Form factor model used for transferrin. Table SI 1: Calculated neutron and x-ray scattering length density of the lipids. Figure SI 3: Scattering intensity diagram of the CaP particles. Figure SI 4: Cryo-TEM images of the hybrid self-assembling or PLCaPZ NPs. Figure SI 5: SANS scattered intensity from Tf and its complex with ZOL for different Tf: ZOL molar ratios. 6.REFERENCES 1. Maeda, H.; Nakamura, H.; Fang, J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev 2013, 65, 71-9. 2. Salmaso, S.; Caliceti, P. Stealth properties to improve therapeutic efficacy of drug nanocarriers. J Drug Deliv 2013, 2013, 374252. 3. Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V. ; Wurm, F. R. Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers. Nat Nanotech. 2016, 11, 372-7. 4. Bulbake, U.; Doppalapudi, S.; Kommineni, N.; Khan, W. Liposomal Formulations in Clinical Use: An Updated Review. Pharmaceutics 2017, 9, 12. 5. Trindade, T.; Daniel da Silva, A. L. Nanocomposite Particles for Bio-Applications: Materials and Bio-Interfaces. CRC press, 2011, 255-256. 6. Lindner, P.; Zemb, Th. Neutron, X-rays and Light. Scattering Methods Applied to Soft Condensed Matter. Lindner, P., Zemb, Th., Eds.; Amsterdam ; Boston: Elsevier 2002, pp.541.


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7. Mehan, S.; Chinchalikar, A. J.; Kumar, S.; Aswal, V.K.; Schweins R. Small-Angle Neutron Scattering Study of Structure and Interaction of Nanoparticle, Protein, and Surfactant Complexes. Langmuir 2013, 29, 11290-11299. 8. Angelov, B.; Garamus, V.M.; Drechsler, M. Angelova, A. Structural analysis of nanoparticulate carriers for encapsulation of macromolecular drugs. Journal of Molecular Liquids 2017, 235, 83-89. 9. Di Cola, E.; Grillo, I.; Ristori, S. Small Angle X-ray and Neutron Scattering: Powerful Tools for Studying the Structure of Drug-Loaded Liposomes. Pharmaceutics 2016, 8, 10. 10. Feigin, L. A.; Svergun, D. I. Structure Analysis by Small-Angle X-Ray and Neutron Scattering;Taylor G.W. Eds; Penum Press: New York and London, 1987; pp. 333. 11. Ristori, S.; Di Cola, E.; Lunghi, C.; Richichi, B.; Nativi C. Structural study of liposomes loaded with a GM3 lactone analogue for the targeting of tumor epitopes. Biochim Biophys Acta. 2009, 1788,2518-25. 12. Stewart, P. L. Cryo-electron microscopy and cryo-electron tomography of nanoparticles. WIREs Nanomed Nanobiotechnol, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2017, 9, 1417. 13. Giger, E.V.; Castagner, B.; Leroux, J.C. Biomedical applications of bisphosphonates. J Control Release 2013, 167,175-88. 14. Caraglia, M., Budillon, A., Tagliaferri, P., Marra, M., Abbruzzese, A., Caponigro, F. Isoprenylation of intracellular proteins as a new target for the therapy of human neoplasms: preclinical and clinical implications. Curr Drug Targets. 2005, 6(3), 301-323. 15. Caraglia, M., Marra, M., Naviglio, S., Botti, G., Addeo, R., Abbruzzese, A. Zoledronic acid: an unending tale for an antiresorptive agent. Expert Opin Pharmacother 2010, 11(1), 141-154. 16.Marra, M.; Salzano, G.; Leonetti, C.; Tassone, P.; Scarsella, M.; Zappavigna, S.; Calimeri, T.; Franco, R.; Liguori, G.; Cigliana, G.; Ascani, R.; La Rotonda, M.I.; Abbruzzese, A.; Tagliaferri, P.; Caraglia, M.; De Rosa, G. Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes. Nanomedicine 2011, 7, 95564. 17. Shmeeda, H.; Amitay, Y.; Tzemach, D. ; Gorin, J;. Gabizon A. Liposome encapsulation of zoledronic acid results in major changes in tissue distribution and increase in toxicity. J Control Release 2013, 167,265-75. 18. Salzano, G.; Marra, M.; Porru, M.; Zappavigna, S.; Abbruzzese, A.; La Rotonda, M.I.; Leonetti, C. Caraglia, M.; De Rosa. G. Self-assembly nanoparticles for the delivery of bisphosphonates into tumors. Int. J. Pharm. 2011, 403, 292.


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19. A G.; Chieffi, P.; Lamberti, M.; Vitale, G.; Abbruzzese, A.; La Rotonda, M.I.; De Rosa, G.; Caraglia, M. New self-assembly nanoparticles and stealth liposomes for the delivery of zoledronic acid: a comparative study. Biotechnol Adv 2012, 30, 302–309. 20. Porru, M.; Zappavigna, S.; Salzano, G.; Luce, A.; Stoppacciaro, A.; Artuso, S.; Lusa, S.; De Rosa, G.; Leonetti, C.; Caraglia M. Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid. Oncotarget 2014, 5,1044659. 21. Kopecka, J.; Porto, S.; Lusa, S.; Gazzano, E.; Salzano, G.; Giordano, A.; Desiderio, V.; Ghigo, D.; Caraglia, M.; De Rosa, G.; Riganti, C. Self-assembling nanoparticles encapsulating zoledronic acid revert multidrug resistance in cancer cells. Oncotarget, 2015, 6, 31461-78. 22. Borghese, C.; Casagrande, N.; Pivetta, E.; Colombatti, A.; Boccellino, M.; Amler, E.; Normanno, N.; Caraglia, M.; De Rosa G.; Aldinucci, D. Self-assembling nanoparticles encapsulating zoledronic acid inhibit mesenchymal stromal cells differentiation, migration and secretion of proangiogenic factors and their interactions with prostate cancer cells. Oncotarget 2017, 8, 42926-38. 23. Salzano, G.; Zappavigna, S.; Luce, A.; D’Onofrio, N.; Balestrieri, M.L., Grimaldi,A.; Lusa, S.; Ingrosso, D.; Artuso, S.; Porru, M.; Leonetti, C.; Caraglia, M.; De Rosa.G. Transferrintargeted nanoparticles containing zoledronic acid as a potential tool to inhibit glioblastoma growth. J Biomed Nanotechnol 2016,12:811-30. 24. Ristori S.; De Rosa G.;Grillo I. Structural investigation of bisphosphonate self-assembling nanoparticles used in bone cancer treatment. Institut Laue-Langevin (ILL), 2013 doi:10.5291/ILL-DATA.9-13-505. 25. Efimova, Y.M.; van Wella, A.A.; Hanefeld, U.; Wierczinski, B.; Bouwman, W.G. On the neutron scattering length density of proteins in H2O/D2O. Physica B 2004, 350, e877–e880. 26. Varga, Z.; Wacha, A.; Vainio, U.; Gummel, J.; Bóta A. Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study. Chem Phys Lipids 2012, 165,387-92. 27. Martel, P.; Kim, S.M.; Powell, B.M. Physical characteristics of human transferrin from small angle neutron scattering. Biophys J 1980 ,31,371-80. 28. Kilár, F.; Simon, I. The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study. Biophys J 1985,48, 799-802.


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Poly(ethylene glycol) 1000 100

Zoledronic acid

I (cm-1)

10 1

7

x1010 cm-2

6

0.1

5 4 3

0.01

2 1

d (Å)

0

0

100

0.001 0.001

200

300

400

500

0.01

0.1

q (Å-1)

100

Lipid bilayers

10 I ( cm-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38


1

0.1

13

x1010 cm-2

12 11 10 9

0.01

8 7

d (Å)

6

0.001 0.001


0

100

200

300

0.01

400

500

0.1 q (Å-1)

Structural Characterization of Self-Assembling Hybrid Nanoparticles

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