Peg-based temperature sensitive nanoparticle synthesis and their use in protein adsorption

In this study, temperature sensitive polymeric nanoparticles were prepared by emulsifi er free emulsion polymerization. N-isopropylacrylamide (NIPA) has been used as main monomer to give temperature sensitivity and poly(ethyleneglycol) ethyl ether methacrylate (PEG-EEM) has been added to recipes as comonomer to aid in particle formation. The effect of NIPA, PEG-EEM and crosslinker content on particle size has been investigated. Temperature sensitivity or lower critical solution temperature (LCST) of the nanoparticles has been followed by Zeta Sizer as change in particle size. In order to determine the protein adsorption capacity of nanoparticles, adsorption experiments have been investigated as a function of temperature and PEG-EEM amount. The results showed that the adsorption capacity of both NIPA and NIPA/PEG-EEM latexes was increased similarly with the increase in temperature. However, amount of BSA adsorbed on NIPA/PEG-EEM particles has been slightly higher than that of for NIPA alone. This study showed that synthesized NIPA/PEG-EEM nanoparticles would be a good candidate for the further studies on protein adsorption. Research Article Peg-based temperature sensitive nanoparticle synthesis and their use in protein adsorption Guldem Utkan* Marmara University, Faculty of Engineering, Chemical Engineering Department, 34722, Istanbul, Turkey Received: 19 December, 2018 Accepted: 30 December, 2018 Published: 31 December, 2018 *Corresponding author: Dr. Guldem Utkan, Marmara University, Faculty of Engineering, Chemical Engineering Department, 34722, Istanbul, Turkey, E-Mail:


Introduction
Stimuli-responsive polymers that respond with dramatic property change to small changes in their environment.
They can be classifi ed according to the stimuli they respond to as: temperature, pH, ionic strength, light, electric and magnetic fi eld sensitive. They are mostly used in the fi eld of biomimetic actuators, immobilized biocatalysts, drug delivery, thermoresponsive surfaces, bioseparation and bioconjugates [1,2]. Important recent advances in poly(N-isopropylacrylamide) (PNIPA) based systems have focused on mechanistic understanding of phase separation, fi ne control of the structureproperty relationship and novel biomedical applications. PNIPA is soluble below 32°C and precipitates above 32°C in water [3].
The adsorption of proteins on monodisperse latex particles has been widely studied in the fi eld of biomedical applications, such as artifi cial tissues and organs, drug delivery systems, biosensors, solid phase immunoassays, immunomagnetic cell separations and immobilized enzymes and catalysts [1][2][3][4].
Various researchers have studied adsorption of proteins. Some of them concluded that hydrophobic interaction is the most important aspect of protein adsorption. [5][6][7][8]. Others claimed that hydrogen bonding is more important then hydrophobic interactions, in which they used the copolymerized particles with acrylate and acrylic acid (carboxylated) [9,10]. Polystyrene latex particles were found quite useful for promoting protein adsorption through hydrophobic interactions [4][5][6]. These particles had long been considered as colloidal polymer models for protein adsorption onto polymeric supports. Protein adsorption onto such latexes exhibited generally a rapid and irreversible process, hydrophobic and electrostatic interactions govern this process, hence the physicochemical properties of two participating components play a predominant role, as well as the external conditions (pH, ionic strength, buffer nature), protein denaturation may be induced by conformation change occurring in the infl uence of hydrophobic interactions.
The main objective of this work was to examine the adsorption behavior of BSA protein, used here as a model, on to well-characterized NIPA/PEG-EEM copolymer particles. BSA adsorption was investigated as a function of temperature and PEG-EEM content.

Preparation of polymeric particles
Polymerizations were conducted in an oil-in-water (o/w) system. Typical recipes are listed in Table 1

Particle characterization
The average particle size and surface charge density

Protein adsorption
In recent years, there is a great interest on the design of smart and intelligent polymeric materials for technological applications and fundamental studies [13][14][15][16][17][18][19][20][21]. These materials can respond with shape and volume changes to small external stimuli, such as temperature, pH, ionic strength, and magnetic fi elds. Among these intelligent polymeric materials, N-isopropylacrylamide is the most widely studied thermosensitive polymer [22]. Shamim et al studied adsorption and desorption behavior of BSA on surface-modifi ed magnetic nanoparticles covered with thermosensitive polymer (PNIPA) was investigated as a function of temperature, pH, and ionic strength. The results showed that the temperature effect on adsorption/desorption behavior was mainly dependent on the properties of the particles' surface. The effect of pH was also investigated and it was observed that a smaller amount of protein was adsorbed at higher pH because of the electrostatic repulsive force between protein molecules and latex particles. The maximum amount of protein was adsorbed near the isoelectric point of BSA. Desorption results showed that more protein was desorbed when adsorption was done at lower temperatures and desorption effi ciency was found to be higher than 80% [23].
In the adsorption experiments, it is required to have low temperatures to keep the active form of the biological molecules. Temperature sensitive polymers, which have ability to hold an enormous amount of water in their body and have high porosity, are very ideal candidates to become a carrier for biological molecules. In this study, adsorption behavior of both poly (NIPA) and poly(NIPA/PEG-EEM) nanoparticles were comparatively examined to determine the change in adsorption capacity of temperature sensitive latexes in the presence of PEG-EEM in the structure. BSA was selected as model protein and the smallest particles synthesized in the last set of the experiments were used (C coded particles) for the adsorption experiments. First of all, temperature effect on adsorption behavior was examined. In this part, temperature was changed from 4-40°C. It has been known that biological molecules denaturate temperatures above 40°C, so temperatures above 40°C did not included in this study. Interaction of polymeric particle (0.1 g) with 2 mg/ml BSA was realized in pH 5 acetate buffer with gentle mixing for 2 hours. Results obtained can be seen in the fi gure 5. It was determined that adsorption capacity of nanoparticles without PEG-EEM (sample C 1 ) increased with increased temperature. In the literature, it was reported that adsorption capacity increases with the increase in temperature