PHEA–PLA biocompatible nanoparticles by technique of solvent evaporation from multiple emulsions

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Abstract

Nanocarriers of amphiphilic polymeric materials represent versatile delivery systems for poorly water soluble drugs. In this work the technique of solvent evaporation from multiple emulsions was applied to produce nanovectors based on new amphiphilic copolymer, the α,β-poly(N-2-hydroxyethyl)-dl-aspartamide–polylactic acid (PHEA–PLA), purposely synthesized to be used in the controlled release of active molecules poorly soluble in water. To this aim an amphiphilic derivative of PHEA, a hydrophilic polymer, was synthesized by derivatization of the polymeric backbone with hydrophobic grafts of polylactic acid (PLA). The achieved copolymer was thus used to produce nanoparticles loaded with α tocopherol (vitamin E) adopted as lipophilic model molecule. Applying a protocol based on solvent evaporation from multiple emulsions assisted by ultrasonic energy and optimizing the emulsification process (solvent selection/separation stages), PHEA–PLA nanostructured particles with total α tocopherol entrapment efficiency (100%), were obtained. The drug release is expected to take place in lower times with respect to PLA due to the presence of the hydrophilic PHEA, therefore the produced nanoparticles can be used for semi-long term release drug delivery systems.

Introduction

The aim of realizing effective pharmacological therapies can be achieved by designing carriers with defined features in terms of biocompatibility, dimensions, high loading, able to carry out a controlled release. Drug delivery systems (DDS), ideally should show a number of properties, for example, having a long residence time in the body; targeting the disease site; releasing a drug by responding to local-stimuli, such as pH, temperature, externally applied heat, magnetic field, or ultrasound; assuring an enhanced intracellular delivery, carrying a contrast agent able to supply a real time information about the DDS biodistribution and target accumulation (Torchilin, 2009). Polymeric nanoparticles based on biocompatible and biodegradable polymers, such as poly(lactic acid) (PLA) or poly(lactide-co-glycolide) (PLGA), have shown in the last years to be good carriers for the vehiculation of drugs, proteins, and DNA.

Surface linkage of these nanoparticles with hydrophilic polymers such as poly(ethyleneglycol) (PEG) and polysaccharides, increases their residence time in the blood stream, moreover the inclusion of tissue-recognition ligands enables targeted delivery (Chen et al., 2014, Craparo et al., 2010). In addition, the use of PLA-based copolymers could allow to obtain nanoparticles with tunable degradation rates compared to PLA alone, whose crystallinity could interfere with controlled degradation (Araque-Monrós et al., 2013, Nampoothiri et al., 2010). It is worth to note that amphiphilic co-polymers form micelle-like aggregates with various morphologies in an aqueous medium by self-assembling, to have the hydrophilic shell, hindering hydrophobic and/or electrostatic interactions, and the hydrophobic core, able to incorporate lipophilic drugs (Chen et al., 2005). Thus, the production of amphiphilic graft copolymers with multi-grafted branches, in which the hydrophilic/hydrophobic balance can be readily controlled, is highly desired. At this aim, polymers, such as poly(N-vinyl-2-pyrrolidone) (PVP), chitosan or α, β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA), can be used (Giammona and Palazzo, 1987). Some of the many recent examples are: micelles composed of PLA and chitosan successfully developed as drug carriers for Rifampin, a lipophilic model molecule (Wu et al., 2009), and for the anticancer drug anthraquinone (Jeevitha and Amarnath, 2013); micelles of PLA and PEG provided interesting release properties of griseofulvin (Pierri and Avgoustakis, 2005) and amphotericin B (Yang et al., 2008), two important antifungal drug, for oral or topical formulations. Moreover, PEG–PLA nanoparticles loaded with the anti-cancer drug paclitaxel (PTX) and functionalized with magnetic Fe3O4 (Yue et al., 2013), and hollow Fe3O4/SiO2-PEG–PLA nanoparticles loaded with cisplatin (Deng and Lei, 2013) showed high potentiality for anti-cancer therapy. Thus, PLA is highly used for its biodegradability, non-toxicity, excellent biocompatibility and in vivo bioabsorbability. Moreover, PLA, due its low hydrophilicity and high crystallinity that gives it a low degradation rate, is often not used alone but in combination with other polymers or properly functionalized (Rasal et al., 2010). Also α,β-poly(N-2-hydroxyethyl)-dl-aspartamide, PHEA, has ideal properties as carrier for drug delivery, with a protein-like structure, a high water solubility, non-toxicity, non-antigenicity, non-immunogenicity (Giammona et al., 1992). The synthesis of hydrophilic or hydrophobic PHEA derivatives suitable to realize polymeric drug systems able to give a prolonged, controlled and/or targeted DDS has been already successfully exploited. By using simple synthetic processes, amphiphilic derivatives of PHEA were synthesized by derivatization of the polymeric backbone with hydrophilic polymers, such as polyethylene glycols (PEG) (Craparo et al., 2006) and hydrophobic residues such as polylactic acid (PLA) (Craparo et al., 2010), phospholipid derivatives (Craparo et al., 2011) hexadecylamine groups (Cavallaro et al., 2003, Craparo et al., 2009) or polymethacrylate chains (Licciardi et al., 2011, Licciardi et al., 2012). Starting from these amphiphilic derivatives of PHEA, polymeric micelles, capable to solubilize and load hydrophobic drugs (Cavallaro et al., 2004) or fluorescent sensors (Diaz-Fernandez et al., 2010), and to coat gold nanostars (Cavallaro et al., 2013) were achieved. Some of these carriers were targeted to the Central Nervous System (CNS) (Craparo et al., 2008a) and to the liver (Craparo et al., 2014, Craparo et al., 2013b). By increasing the lypophilic/hydrophilic balance of these PHEA derivatives and by using several methods (chemical methods, UV and gamma irradiation), polymeric nanoparticles (Craparo et al., 2004, Craparo et al., 2008b, Craparo et al., 2014, Craparo et al., 2013a) and microparticles (Licciardi et al., 2012) were obtained. Moreover, polycations have been successfully prepared and characterized starting from PHEA derivatives bearing carboxypropiltrimethilamonium chloride (CPTA) (Licciardi et al., 2006a, Licciardi et al., 2006b) and spermine (Cavallaro et al., 2010, Cavallaro et al., 2008). A recent example to obtain a target oriented carrier is the synthesis of a copolymer, based on PHEA bearing positively chargeable side oligochains, with diethylamino ethyl methacrylate (DEAEMA) as monomer, representing a novel device for the delivery of both small interfering RNAs (siRNAs) and plasmidic DNAs (pDNAs) (Cavallaro et al., 2014). However, the potential use of these materials in manufacturing application need greater in depth studies.

Preparation techniques of micro and nano-systems are based on both chemical and physical processes or on their combination. Among the used techniques the solvent evaporation from emulsions is the simplest one, since it does not require high temperatures nor phase separation-inducing agents (Barba et al., 2014). In particular, nanometric emulsions are characterized by a great stability due to their very small size: the flocculation, typical for emulsions, is naturally prevented by steric stabilization, due essentially to the composition, density and thickness of interfacial layer of the sub-micrometric size (Anton et al., 2008). Emulsification involves both high mechanical energy methods, where droplet formation depends on formulation parameters such as the energy, the surfactant amount and the components nature, and low-energy methods, based on the intrinsic physicochemical properties of the system. Thus, in the first method, devices such as ultrasound generators and high-pressure homogenizers, formulation parameters can be directly controlled and the process of emulsification seems to be not influenced by the addition of encapsulating molecules because of the high shear rate. In low energy methods, nanoemulsion droplets are obtained by diverting the properties of the surfactants, co-surfactants and excipients. Main methods include solvent displacement method, characterized by a very rapid diffusion of solvent from the oil phase to the water phase, and the phase inversion temperature (PIT) method. This last uses some peculiarities of polyethoxylated surfactants to modify their partitioning coefficient as a function of the temperature, leading to the creation of a bi-continuous systems when the temperature is close to the PIT, broken-up to generate nanoemulsions (Anton et al., 2008).

The reproducible production of particles with the desired properties, especially of suitable particle size and size distribution, can be difficult, due to the large number of factors influencing the outcome, such as solvent composition, total volume and phase volume ratio of the phases, polymer concentration, type of stabilizer, stirring time, stirring speed, etc. (Barakat and Ahmad, 2008). The effect of the increase of some parameters (theoretical drug loading, polymer concentration, stirring time, etc.) on the size of particles obtained by solvent evaporation from emulsions, is analysed in literature and is resumed in Table 1. In effect, a greater theoretical drug loading increases the particles mean diameter because it causes a more viscous dispersed phase, thus the mutual dispersion of the phases becomes difficult giving larger particles (Mainardes and Evangelista, 2005). The same phenomenon (i.e. the increased viscosity of the dispersed phase resulting in a poorer dispersability of the polymer solution into the aqueous phase) is responsible of the increase in particle size with larger amount of polymer. Moreover, a hydrophilic colloid, such as polyvinyl alcohol (PVA), is generally added as stabilizer to the continuous phase to prevent coalescence of emulsion droplets. Particle size is reduced by increasing the stabilizer concentration, however, if the amount is insufficient, particles do not stabilize, and if the stabilizer concentration is excessive, the viscosity solution increases producing larger sized particles (Mainardes and Evangelista, 2005, Sameni et al., 2009). The solvent used for polymer solubilisation influenced the particles size after evaporation: the presence of more hydrophilic solvents causes an easier coalescence between the oil and the water phases causing an enlarged particles size (Barba et al., 2014). Both the stirring power and the stirring time are parameters influencing size particles because they increase the intensity and the duration of shear stresses on droplets, respectively, thus causing a reduced particle size (Sameni et al., 2009). When the amount of the external aqueous phase increases, the average particle size decreases because the solvent evaporates more easily from the emulsion, resulting in a rapid particles structure formation before droplets coalescence. A lower external water volume caused instead a faster droplets coalescence before solidification (Péter Sipos, 2005).

In this work, the technique of solvent evaporation from multiple emulsions, developed in a previous work (Barba et al., 2014), was tested to produce nanovectors based on PHEA derivatives suitable to realize polymeric systems able to give a prolonged, controlled and/or targeted DDS. In particular, α,β-poly(N-2-hydroxyethyl)-dl-aspartamide–polylactic acid (PHEA–PLA) was purposely synthetized to be used as carrier in the controlled release of poorly water soluble active molecules. PHEA–PLA nanovectors were loaded with α-tocopherol (vitamin E) as lipophilic model molecule having relevant functions in biological systems (Renò et al., 2005). Main stages in PHEA–PLA synthesis and optimizing studies in nanoparticles production are analyzed and discussed.

Section snippets

Materials

Poly(ethylene oxide) standards, 1,1′-carbonyldiimidazole (CDI), anhydrous N,N'-dimethylformamide (a-DMF), α-tocopherol (TC), chloroform (CL), were bought from Sigma–Aldrich SrL (Italy). Diethylamine (DEA), ethyl ether, dichloromethane were acquired from Fluka (Italy). d,l-poly(lactic acid) (PLA, 14,000 Da) was purchased from PURAC (Holland). Analytical grade was chosen for all the used reagents, unless otherwise indicated.

Weight-average molecular weight (M¯w) of each copolymer was assayed by

PHEA-PLA graft copolymer synthesis

Derivatization of PHEA with PLA to obtain the PHEA–PLA graft copolymer was achieved by using CDI as coupling agent to activate the terminal carboxyl group of PLA (Craparo et al., 2010).

A calculated amount of CDI dissolved in a-DMF (123 mg/ml) under argon atmosphere, was added drop-wise to PLA solution in a-DMF (171 mg/ml), using a ratio of R1 = 2, in which R1 is:R1=CDI molesPLA molesThe solution was stirred at 40 ± 0.1 °C for an activation time of 4 h under inert atmosphere by argon. Simultaneously,

Copolymer synthesis and characterization

This work was focused on the application of the method of solvent evaporation from multiple emulsion to produce nanovectors based on a new amphiphilic copolymer such as α,β-poly(N-2-hydroxyethyl)-dl-aspartamide–polylactic acid (PHEA–PLA) purposely synthesized. The use of PLA-based copolymers instead of PLA alone for production of nanovectors could allow to obtain systems with tunable properties such as size, surface charge and hydrophilicity, and degradation rate (Saulnier et al., 2006, He et

Conclusions

In this work, a new copolymer of PHEA and PLA was synthesized and characterized. The new material has an amphiphilic nature, in principle useful in order to produce drug delivery systems for hydrophilic as well as lipophilic drugs. Then, the new material has been adopted in a preparation technique based on solvent evaporation from multiple emulsions assisted by ultrasonic energy, using the α tocopherol (TC, vitamin E) as model lipophilic drug. The process has produced nanoparticles with uniform

Conflict of interest

The authors declare that they have no conflict of interest.

For reference reported only in Table 1: (Meissner et al., 2007, Mastiholimath et al., 2007, Rizkalla et al., 2006, Mahdavi et al., 2010, Abismaı̈l et al., 1999).

Acknowledgements

This study was financed by the Ministero dell’Istruzione dell’Università e della Ricerca (PRIN 2010/2011, contract grant number 20109PLMH2).

Annalisa Dalmoro’s research grant was supported by “POLIFARMA”—PON R&S 2007–13 n.02_00029_3203241.

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