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Behavior of PPI-G2 Dendrimer in a Microemulsion Shifra Rokach, Maria Francesca Ottaviani, Alexander I. Shames, Abraham Aserin, and Nissim Garti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10237 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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The Journal of Physical Chemistry

Behavior of PPI-G2 Dendrimer in a Microemulsion Shifra Rokach 1,2§, Maria Francesca Ottaviani3*, Alexander I. Shames4, Abraham Aserin1 and Nissim Garti1* 1

The Ratner Chair of Chemistry, Casali Institute of Applied Chemistry, The Institute of

Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 9190401, Israel. 2 3

Judea Regional Research & Development Center, Carmel 9040400 Israel

Department of Earth, Life and Environment Sciences, University of Urbino, Loc. Crocicchia, Urbino 61029, Italy. 4

Department of Physics, Ben-Gurion University of the Negev, P.O. Box 653, Be’er-Sheva 84105, Israel.

§

The results presented in this manuscript are part of S.R.'s fulfillment of the requirements for the

M.Sc. degree in Applied Chemistry, The Hebrew University of Jerusalem. *Authors to whom correspondence should be addressed Tel:+39-0722-304320; +972-2-658-6574/5 Fax:+39-0722-304222; +972-2-652-0262 e-mails: [email protected]; [email protected]

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Abstract Dendrimer nanostructures are of eminent interest in biomedical applications because of their uniform and well-defined molecular size and shape, and their ability to cross cell membranes and reduce the risk of premature clearance from the human body. Dendrimers perform as gene and drug carriers and have also shown significant therapeutic properties for treating cancer and neurodegenerative diseases. A complex drug delivery system, based on a dendrimer solubilized in the aqueous core of a water-in-oil (W/O) microemulsion (ME) along with the drug may combine the advantages of both dendrimers and MEs to provide better control of drug release. We propose a new microemulsion composed of drug-permitted surfactants and dendrimer that can be used as a potential controlled drug delivery nanosystem. The influence of second generation poly(propyleneimine) (PPI-G2) dendrimer, solubilized in (W/O) ME with a capacity of up to 25 wt% PPI-G2 at various pHs, and their interactions with the surfactant phosphatidylcholine (PC), cosurfactant (butanol), and water was studied. SAXS and EPR measurements indicated that increasing PPI-G2 concentration reduces droplet curvature and increases droplet size thus increasing macro- (SAXS) and micro- (EPR) order degree. Furthermore, SD-NMR and ATR-FTIR show stronger interactions between PPI-G2 and water molecules at the expense of PC and butanol headgroups hydration, which increases microviscosity (EPR). PPI-G2's effect is somewhat opposite to the increasing water phase effect, thus reducing the amount of free water (DSC) and slowing the mobility of all ME components (SD-NMR).

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1. Introduction Since the beginning of the 1990s, when the first dendrimers were synthesized, the incidence of dendrimer research has greatly increased. There has been an increasing interest in the biological, (bio)medical, and pharmaceutical applications of so-called biodendrimers.1-8 Dendrimers are globular macromolecules that have a treelike hyperbranched structure, a welldefined molecular weight, and large number of peripheral groups.4,9 Dendrimers are synthesized in a layer-by-layer fashion around a core unit.10 The narrow polydispersity of dendrimers and their nano-scale facilitates passage across biological barriers and the ability to mimic biomolecules. Dendrimers are suitable hosts for drugs.5,11 Drugs are either entrapped in the interior of the dendrimer architecture (by electrostatic, hydrophobic, and hydrogen bond interactions) or conjugated to the peripheral groups (by electrostatic interactions and covalent bonding).4,12,13 The presence of multiple terminal groups on the exterior of the dendrimer offers an excellent platform for the attachment of drugs,14 cell-specific targeting groups,15,16 solubility modifiers, and stealth moieties and genes.17-20 Dendrimers themselves can serve as therapeutic agents by virtue of their activities21 against prion diseases,22 Alzheimer disease,23 inflammations,24 human immunodeficiency virus (HIV),25 herpes simplex virus (HSV),26 bacteria,27 cancer,28 and others.29 Dendrimers prevent formation of amyloid fibrils and disaggregate previously formed fibrils,23,30,31 thus preventing viral adhesion and replication.25 Dendrimers are being considered as additives in several routes of administration, including intravenous, oral, transdermal, and ocular.10,32 Water-in-oil (W/O) microemulsions (MEs) are self-assembled and thermodynamically stable, macroscopically homogenous, and optically transparent systems of water nanodroplets

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dispersed into apolar solvents (mainly water) in the presence of adequate surfactants.33-38 MEs are formed by reducing the oil/water interfacial tension to ultra-low values by means of a surfactant, or, more commonly, a mixture of surfactants and cosurfactants, thereby allowing thermal motions to spontaneously disperse the two immiscible phases. ME droplets’ typical size is in the range of ~5–50 nm.39,40 The ability to sustain delivery of the encapsulated drugs in a dendrimer is much lower compared to colloidal carriers such as microemulsions or emulsions. A complex drug delivery system based on a dendrimer solubilized in the aqueous core of a W/O ME, was proposed in our lab. This approach may combine the advantages of both dendrimers and ME nanostructures, to provide better control of drug release. In this study, a second generation poly(propyleneimine) (PPI-G2) dendrimer (5–25 wt%) was solubilized into the water core of a four-component W/O ME based on phosphatidylcholine (PC), butanol, isopropyl myristate (IPM), and water as shown by Aboofazeli et al.,41 all of which are drug permitted. Poly(propyleneimine) (PPI) polycationic dendrimers are of particular interest since they are biocompatible and commercially available.5,42-47 The PPI-G2 possesses eight primary amine groups on the surface and has a spherical shape with a radius of gyration between 6 and 7 Å.48,49 The localization of PPI-G2 within the ME, its specific interactions with the components of the carrier, and its effect on the ME structure were explored by spectroscopic techniques such as small angle X-ray scattering (SAXS), self-diffusion nuclear magnetic resonance (SD-NMR), electron paramagnetic resonance (EPR), attenuated total reflectance–Fourier transform infrared (ATR-FTIR), and differential scanning calorimetry (DSC).

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2. Materials and methods 2.1. Materials n-Butanol, isopropyl myristate (IPM), and 5-doxylstearic acid [2-(3-carboxypropyl)-4,4dimethyl-2-tridecyl-3-oxazolidinyloxy] (5-DSA), free radical, were purchased from SigmaAldrich (St. Louis, MO, USA). PPI-G2 (>95% purity), was obtained from SyMO–Chem, The Netherlands. Epikuron 200 phosphatidylcholine (PC) (92%) was purchased from Degussa BioActives KGaA (Hamburg, Germany). Hydrochloric acid (37%) was purchased from Bio Lab Ltd. Jerusalem, Israel. Water was double distilled. All ingredients were used without further purification.

2.2. Preparation of the microemulsions The PC-based MEs were prepared by adding the aqueous phase (solution without or with PPI-G2 in water) to a ternary mixture of PC, butanol, and IPM (3.6/2.4/2 weight ratio). In some measurements, the aqueous phase content in the ME samples was kept constant (20 wt%) while the PPI-G2 concentration was 0–25 wt% (from the aqueous phase) at different pH values (6, 7, 8, and 13). In other samples the PPI-G2 concentration was kept constant (5 wt%, from the aqueous phase) and the total aqueous phase varied from 0 to 35 wt%. Each sample was mixed by vortexing until a clear solution was obtained. All measurements were done a day after preparation in order to ensure an equilibrium state was reached.

2.3. Small angle X-ray scattering (SAXS) Scattering experiments were performed using Ni-filtered Cu Kα radiation (0.154 nm) from a Genix 3D X-ray generator that operated with a voltage of 50 kV and a current of 0.6

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mAmp. The X-ray radiation was further monochromated and collimated by a single Franks mirror and a series of pinholes and height limiters. The direct beam then went through a vacuum of 3×10–3 mbar. Once the beam hit the sample, the scattering was measured by a twodimensional Pilatus 300K detector; q range was 0.005 to 7 Å–1. The samples were held in 1.5 mm quartz X-ray capillaries inserted into a copper block sample holder. The measurements were performed at a constant temperature of 25±0.5°C and an exposure time of 15 minutes.

2.4. Self-diffusion nuclear magnetic resonance (SD-NMR) NMR measurements were performed with a Bruker AVII 500 spectrometer equipped with GREAT 1/10 gradients and a 5 mm BBI probe with a z-gradient coil with a maximum gradient strength of 0.536 T m−1. The components were identified by their chemical shift in 1H NMR. Diffusion was measured using an asymmetric bipolar LED, ramping the strongest gradient from 2% to 95% of maximum strength in 32 steps. The spectrum was processed with the Bruker TOPSPIN software. NMR spectra were recorded at 298±0.5 K.

2.5. Differential scanning Calorimetry (DSC) A Mettler Toledo DSC822 (Greifensee, Switzerland) calorimeter was used to monitor the thermotropic behavior of the microemulsions. The DSC measurements were carried out as follows: 10–13 mg samples were weighed, using a Mettler M3 microbalance, in standard 40 μL aluminum pans and immediately sealed by a mechanical press. The samples were cooled in liquid nitrogen from +25 to −100°C at a rate of −6°C min−1. The samples remained at this temperature for 20 min and then were heated at a rate of 5°C min−1 to 25°C. An empty pan was used as reference.

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2.6. Electron paramagnetic resonance (EPR) 2.6.1. Insertion of the probe The 5-DSA probe was first dissolved in chloroform at a concentration of 2.5×10–3M. An appropriate quantity of the probe solution was placed in tubes, and the solvent then evaporated before preparing the MEs within these tubes. A low probe concentration of 4×10–5M in the examined MEs systems has already been demonstrated to not perturb similar nanostructures.50-52

2.6.2. EPR instrumentation and method Room temperature (T = 2951 K) EPR measurements were carried out using a Bruker EMX-220 X-band (ν ~ 9.4 GHz) equipped with Oxford ESR 900 temperature accessories and Agilent 53150A frequency counter. EPR experiment setup includes a non-saturating MW power of 20 mW, modulation amplitude of 1 G, MW frequency of 9.465 GHz, center magnetic field of 3369.85 G, sweep width of 200.00 G, resolution of 1024 points, time constant of 1.28 ms, and conversion time of 20.48 ms, with a coherent acquisition of 49 scans per EPR spectrum.

2.6.3. Computation of the EPR spectra The computer aided analysis of the EPR spectral line shape was performed by means of the well-established procedure of Budil et al.53 and Schneider and Freed.54 The EPR spectra of 5DSA in solution was constituted by the three hyperfine lines (due to the coupling between the unpaired electron spin, S = 1/2, and the nitrogen

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N nuclear spin, I = 1) at almost the same

intensities. The main parameters that monitor the structural and dynamic modifications of 5-DSA environment in the different experimental conditions are: (a) The correlation time for the

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rotational motion (). This parameter increased with the decrease in 5-DSA mobility; that is, with the increase in the microviscosity of the probe-environment. The jump or the Brownian rotational diffusional models were used, for which  = 1/D and 1/6D, respectively, where D is the rotational diffusion coefficient. (b) The Azz component of the A tensor of the hyperfine coupling between the electron spin and the nuclear spin changed as well from system to system, monitoring the variation in the environmental polarity of the probe (the Axx and Ayy components were assumed to be the same and fixed as 7 G. (c) When the probe is inserted in an ordered lipid layer, the molecular order parameter (S) extracted from computation monitors the order degree, being S = 0 for a completely disordered system and S = 1 for an ordered one. (d) When the spectra are constituted by two components due to the distribution of the probes in two different environments, subtraction of the spectra in different conditions allowed extraction of the two components, to compute them, and to evaluate the relative percentages of them. The accuracy in the parameters, as obtained from computation, was in the range 1–3%, depending on the resolution of the spectra.

2.7. Attenuated total reflectance – Fourier transform infrared (ATR-FTIR) An Alpha T model spectrometer, equipped with a single reflection diamond ATR sampling module, manufactured by Bruker Optik GmbH (Ettlingen, Germany), was used to record the FTIR spectra. The spectra were recorded with 100 scans at 25°C; a spectral resolution of ±1 cm–1 was obtained. Multi-Gaussian fitting has been utilized to resolve individual bands in the spectra. Several measurements were conducted with D2O replacing the water.

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3. Results and discussion Nir et al.49 proposed a complex drug delivery system based on a PPI-G2 dendrimer solubilized in the aqueous core of a ternary AOT/heptane/water W/O ME based on AOT surfactant (AOT-ME). The disadvantage of this system was the possibility to solubilize up to 25 wt% PPI-G2 (from 24 wt% aqueous phase) into AOT-ME under highly alkaline conditions (pH 13). Only low PPI-G2 concentrations (up to 5 wt%) could be solubilized in low aqueous contents (

Behavior of PPI-G2 Dendrimer in a Microemulsion - The Journal of

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