Preparation of PVP hydrogel nanoparticles using lecithin vesicles

ARTIGO

Preparation of PVP hydrogel nanoparticles using lecithin vesicles^#

Vânia Blasques Bueno^I; Luiz Henrique CatalaniI, ^*; Katia Regina Perez Daghastanli^II; Iolanda Midea Cuccovia^II; Hernan Chaimovich^II

^IDepartamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo - SP, Brasil

^IIDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo - SP, Brasil

ABSTRACT

Hydrogels micro, sub-micro and nanoparticles are of great interest for drug encapsulation and delivery or as embolotherapic agents. In this work it is described the preparation of nano and sub-microparticles of pre-formed, high molecular weight and monomer free poly(N-vinyl-2-pyrrolidone) encapsulated inside the core of lecithin vesicles. The hydrogel particles are formed with a very narrow diameter distribution, of about 800 nm, and a moderate swelling ratio, of approximately 10.

Keywords: PVP hydrogel nanoparticle; lecithin vesicle; photo-Fenton reaction.

INTRODUCTION

Hydrogel nanoparticles (nanogels) are of interest due to their ability to combine the advantages of biocompatibility, inherent to most hydrogels,¹ and small size.² Potential applications of nanogels include controlled drug delivery systems³ (oral⁴ and/or parenteral⁵ delivery) and alternative therapies, like embolotherapy.⁶

Nanogels can be obtained from monomer polymerization in presence of difunctional monomers, either in w/o emulsions^7,8 or using the core of reverse micelles as formatting system.⁹ Alternatively, nanogels can be prepared by inclusion in reverse micelles of pre-formed polymers, followed by crosslinking.¹⁰ Vesicles also can be used to obtain hydrogels nanoparticles, by monomer encapsulation followed by polymerization¹¹ or gelation of encapsulated polymers, generally induced by sol-gel temperature transitions¹² or ionic crosslinking,¹³ typically without removal of the lipid bilayer.¹⁴ These gel-like vesicles work as cell models, since they have elastic modulus comparable to that of cell cytoplasm¹⁴ and are considered artificial cytoskeletons.^14,15 Vesicles containing poly(N-isopropylacrylamide) are good examples. This polymer responds to temperature changes forming physical gels on a reversible process, even within vesicles.¹⁶ This temperature induced sol-gel transition within the vesicle mimics cell stiffening.¹² Hydrogel-liposome assemblies (lipobeads) can also be used as drug delivery systems.¹⁷ The lipid bilayer is often left intact and the encapsulated polymer is not crosslinking.

Vesicles have advantages compared to other systems for producing nanogels, since these assemblies permit more diameter control alternatives.¹⁸ The particle diameter can be controlled varying the vesicles diameter from a few nanometers to millimeters and can be obtained with a narrow size distribution. Vesicles can be prepared using low amounts of non-toxic solvents and this compares favorably with other methods such as emulsion polymerization. The major component of a vesicle preparation is water and a non-toxic surfactant can be chosen, thereby resulting in a biocompatible preparation.

Poly(N-vinyl-2-pyrrolidone) hydrogels can be prepared by several straightforward methodologies.^19-22 One of these methods involves the use of photo-Fenton reaction²¹, using ferric ions, hydrogen peroxide and UVA radiation.²²

Here we describe the use of lecithin vesicles as formatting system to obtain poly(N-vinyl-2-pyrrolidone) hydrogel particles, using photo-Fenton or Fenton reactions for crosslinking.

EXPERIMENTAL

Materials

Egg phosphatidylcholine was purified from egg yolks as described by Maximiano et al..²³ Soy lecithin was obtained from crude soy lecithin capsules by purification with the same method used for egg phosphatidilcholine.²³ Dioctadecyldimethylammonium chloride (DODAC) was obtained from the bromide salt (DODAB; Aldrich) after ion exchange with a Dowex-21K resin (Fluka) in the chloride form in methanol. 1,2-Dipalmitoyl-3-trimethylammonium-propane chloride (DPTMA) was synthesized as described previously²⁴ as bromide salt, which was exchanged for chloride as indicated for DODAC. Poly(N-vinyl-2-pyrrolidone) (PVP) (Plasdone K90, ^-M_w = 1,300,000) was kindly donated by BASF. FeCl₂, FeCl₃ and H₂O₂ 30% (Aldrich), NaCl (Merck) and CH₂Cl₂ (Synth Brazil) were analytical grade and used as received. Water was deionized.

Methods

PVP encapsulation in liposome

PVP containing vesicles were obtained as follows (Figure 1): 0.5 mL of a CH₂Cl₂ solution containing 50 mg of egg or soy phosphatidylcholine (100 mg mL^-1) were injected into 5 mL of PVP aqueous solution (80 g L^-1), containing 17 mmol L^-1 FeCl₂ maintained at 50 ºC. CH₂Cl₂ solution was injected with a syringe adapted in a KD Scientific Inc. Model KDS120 Push-Pull Pump, equipped with a fine-gauge needle (No 3D). During injection, a N₂ stream was bubbled into PVP solution, which continued after liposome formation until removal of residual solvent. Liposome suspension was centrifuged at 22,800 × g for 30 min (Hitachi Himac CR20B2 centrifuge; Hitachi Ltd.) at 25 ºC to remove non-entrapped polymer from the external phase. The liposome-containing pellet was washed twice by centrifugation under same conditions in saline solution (51 mmol L^-1). The washed pellet was suspended on 20 mL of NaCl_(aq).

PVP concentration inside vesicles was estimated using Lugol method.²⁵ Resulting PVP complex with Lugol Reagent (PVP-I₃^-) absorbs at 400 nm, and the PVP concentration was obtained using appropriate calibration curve. The encapsulation yield (EY) was calculated using Equation 1.

where is the mass of encapsulated PVP in the vesicles pellet and 0.4 is the mass of PVP in the initial solution (in g).

PVP entrapped polymerization

To crosslink PVP inside the vesicles, 4 mL of H₂O₂ diluted on NaCl_(aq) (51 mmol L^-1) was added to the PVP-containing vesicles ([H₂O₂]_final = 200 mmol L^-1).²² As Fe²⁺ can be oxidized to Fe³⁺ inside vesicles before H₂O₂ addition, to promote crosslinking by photo-Fenton reaction²¹ H₂O₂ containing vesicles suspension was submitted to 12 h of UV radiation (360 nm). The hydrogel particles were purified by lysing vesicles washing 3× with ethanol 50%. The resulting pellet was resuspended in water and freeze-dried.

Dynamic light scattering measurements

The determination of particles size distribution was carried out by dynamic light scattering (DLS), using a spectrometer from Brookhaven Instruments. The average hydrodynamic diameters were obtained from the unimodal distribution of particles analyzed by Zeta PALS Particle Sizing Software Version 2.29.

Scanning electron microscopy (SEM)

Particles were analyzed by SEM (FEG-SEM, model FEG 7401F, from Jeol). Samples were prepared by fixing the particles (freeze-dried powder) on a double-face copper tape and the specimens were analyzed without coating.

Swelling ratio determination (Q)

Swelling ratio (Q) determination was done by weighing the swollen particles pellet in an analytical balance (m_swo), followed by freeze drying and weighing the dry pellet (m_dry). Q was calculated directly by Equation 2.

RESULTS AND DISCUSSION

Polymer encapsulation

The efficiency of vesicle encapsulation depends on polymer size and decreases with the increase of polymer molecular weight.^26,27 Szoka and Papahadjopoulos²⁸ found that encapsulation efficiency decreases with the increase of molecular weight of the encapsulated protein. Dominak et al.²⁶ observed the same inverse relationship between encapsulation efficiency and molecular weight studying polyethylene glycol and dextran encapsulation in giant vesicles. To encapsulate PVP, various surfactants were tested, including lecithin from egg yolk and soybeans and cationic surfactants. Table 1 shows the surfactants and their respective PVP encapsulation yield. As each vesicle in solution can have different encapsulation values,²⁶ it is important to emphasize that PVP inclusion was measured by estimating the total amount that was incorporated and therefore the incorporation represents an average over all vesicles.

The best encapsulation efficiency was obtained with soy lecithin vesicles. Lecithins of different sources exhibit distinct properties.^23,29 In particular the length of the apolar portion of the molecule and the insaturation of the alkyl chain will result in vesicles with different properties.³⁰ The method used to obtain vesicle encapsulated PVP also contributes to high encapsulation efficiency.¹⁸

Since the method used to crosslink the encapsulated polymer requires long irradiation times (photo-Fenton reaction), the addition of cationic surfactants decreases vesicle aggregation during irradiation by increasing vesicle repulsion. Two saturated surfactants where tested: DODAC and DPTMA. Encapsulation efficiency was higher with DPTMA than with DOCAC. DODAC contains two octadecyl chains linked to the tetraalkylammonium group; in DPTMA the acyl chains are attached to a propanediol moiety linked to tetraalkylammonium group (Figure 2). Bilayer packing defects are expected^31,32 upon incorporation of DODAC to lecithin causing a decrease in polymer entrapment. Measurements of polymer leak showed that entrapped PVP was retained after several washes (supernatant PVP concentration was measured as <10^-4 g L^-1 for DPPC vesicles and as 0.02 g L^-1 for DODAC/lecithin vesicles, after 5 wash cycles).

Crosslinking reaction

Two methods were used to crosslink the polymer. The Fenton reaction, an efficient method to obtain PVP hydrogels with good mechanical properties,²² has been used to obtain PVP nanogels with superabsorbent properties.¹⁰ Photo-Fenton reaction, a photochemical analog of Fenton reaction, also produces PVP hydrogels²¹ (Scheme 1).

The Fenton reaction is interesting because it allows a fast route to obtain PVP hydrogels with any desired format.³³ However, this reaction requires Fe²⁺ which autoxidizes. On the other hand, photo-Fenton reaction uses Fe³⁺ ions, H₂O₂ and UV radiation (λ = 365 nm). Since the vesicles can scatter and react with H₂O₂ (lipoperoxidation), the UV dose and H₂O₂ concentrations were higher than those described.²¹

Fenton and photo-Fenton reactions did not change significantly the vesicles diameter distribution (Figures 3 and 4). This finding does not imply that the lipid bilayer remains unchanged during crosslinking reaction, since hydroxyl radical is known to react with unsaturated acyl chains.³⁴

Particle characterization

When conventional Fenton reaction was used no pellet was obtained after vesicle´s lysis, evidencing absence of crosslinking, probably due Fe²⁺ depletion via its autoxidation. A phosphorus-free yellow pellet was obtained after crosslinking by photo-Fenton reaction and vesicles lyses.³⁹ This pellet was resuspended in water and analyzed by SEM and DLS. Results demonstrated the presence of a formless particulate material with dimensions comparable to vesicles (Figures 5 and 6).

The diameter of nano and sub-microgel and nanogel particles can be controlled by choosing the vesicle preparation method. Polyacrylamide nanogels (from monomers) covered by phospholipids bilayers obtained by detergent depletion³⁵ or freeze-thaw cycles followed by extrusion³⁶/sonication^11,17 presented diameters of 100 and 200 nm, respectively. Larger nanogels (about 450 nm) are obtained from encapsulation of 20% dextran hydroyethylmethacrylate solution (-Mn= 19000) inside vesicles obtained by phospholipids film hydration, followed by extrusion.³⁷ Poly(ethylenedioxythiophene)/poly(styrenesulfonate)¹³ and poly(N-isopropylacrylamide)^12,16 microgels, in conjunction with lipid bilayers, are obtained by injecting a polymer solution inside GUVs, followed by freeze-thaw cycles and electroporation. The vesicle diameters, in these latter cases, vary from 5 to 100 μm.

The hydrogel particles obtained here showed no defined morphology, in spite of being obtained from spherical vesicles. Vesicles may be destabilized during crosslinking by H₂O₂ oxidation and ensuing lipoperoxidated³⁸ (Figure 7). Fluctuation of crosslinking density, known to influence hydrogel particle morphology, may also account for the lack of defined shape.¹³ Non spherical particles have also been reported with other nanogels prepared using vesicles.^17,37

Particle swelling was estimated directly and the swelling ratio (Q) was 10, i.e., particles absorb only 10 times their mass in water. This is a strong indication that the crosslinking density is high since Q is inversely proportional to the ability of absorbing water.

Swelling of nanogels formatted from vesicles has seldom been discussed in literature. In the case of poly(N-isopropyl acrylamide) hydrogels and copolymers, coagulation of particles was observed instead of the variation in swelling. Kasakov et al. have extensively studied this systems^11,17,36 mainly in regard to the interaction between particle and liposome. Since the hydrogel used is thermo- and pH-responsive, coagulation was observed upon heating, including coagulation of the lipid bilayer, which, after cooling, surround the entire set of nanoparticles in a reversible process.³⁶ The authors also observed an increase in diameter with pH change: 100 nm at pH 6.5 to 300 nm at pH 3.0, with dimerization of particles.¹⁷

CONCLUSION

Injected lecithin vesicles can encapsulate high molecular weight PVP. The encapsulated polymer can be crosslinked by photo-Fenton reaction and hydrogel nano and sub-microparticles can be isolated. Spherical morphology was not achieved, but the produced hydrogel particles have a narrow diameter distribution compatible with the vesicles diameter distribution.

Recebido em 21/6/10; aceito em 20/8/10; publicado na web em 15/10/10

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Publication Dates

• Publication in this collection
  15 Dec 2010
• Date of issue
  2010

History

• Accepted
  20 Aug 2010
• Received
  21 June 2010