Acrylic hydrogels modified with bee pollen for biomedical applications

Article history: Received on: 02/09/2015 Revised on: 26/09/2015 Accepted on: 20/10/2015 Available online: 27/11/2015 The focus of this paper is to present the effect of bee pollen on the structure and properties of acrylic hydrogels, often used as materials for wound dressings. Honey, propolis and bee pollen have been very well known for their extraordinary healing and medical properties for thousands of years. Ancient Egyptians, Greeks and Romans have reportedly used bee pollen as a drug, especially for a number of skin diseases and lesions. Acrylic hydrogels are, on the other hand, rather recently discovered materials used for novel wound dressings. The idea of our research was to combine a synthetic polymer matrix with bee pollen, in order to obtain innovative materials which could be used as wound dressings exhibiting new properties. The obtained acrylic hydrogels containing bee pollen were investigated towards swelling ability in distilled water and different fluids, including pseudoextracellular fluid, simulated body fluid and Ringer’s solution. Moreover, incubation in water and simulated body fluids (SBF) and Ringer’s solution was carried out. The morphologies of bee pollen and obtained acrylic hydrogels modified with bee pollen were characterized by means of scanning electron microscopy (SEM). The chemical structure of the polymer matrix was confirmed by ATR-FT-IR spectra.


INTRODUCTION
Acrylic hydrogels are polymeric matrices with a threedimensional structure, which is characterized by an extraordinarily high swelling degree in water and other fluids.Moreover, appropriate synthesis allows them to be biocompatible, biodegradable and bioactive -all are traits of good biomaterials (Peppas and Hilt 2006;Serra et al., 2009).Although these materials have a vast number of possible applications, we focus solely on their use as wound dressings, which are especially used in treatment of slowly healing and chronic wounds, such as decubitus ulcers, venous ulcers, diabetic foot ulcers, as well as second-degree burns and post-operative wounds (Queen et al., 2004).Introduction of an active component into the polymer matrix would allow the hydrogel to exhibit additional properties, rendering it an even more suitable material for biomedical applications.Natural products are a promising source of new discoveries in the field of pharmacy and medicine, though a majority of them has been known for centuries.An excellent example is provided by apiproducts, such as honey, propolis and bee pollen, which have been known since the ancient times because of their unique properties.Bee pollen and propolis both exhibit a vast spectrum of biological and pharmacological properties, such as immunomodulatory, anti-inflammatory and antioxidant (Burdock, 1998;Catchpole et al., 2004;Kashkooli et al., 2011;Leja et al., 2007;Sforcin 2007;Sforcin and Bankova 2011).Bee pollen contains a variety of nutrients, such as amino acids, minerals, including phosphorus, potassium, magnesium, calcium, copper, manganese, iron and zinc, some trace minerals, all B vitamins, including B 12 , folic acid, panthothenic acid, rutin and lecithin (Almeida-Muradian et al., 2005;Human and Nicolson 2006;Kashkooli et al., 2011;Kroyer and Hegedus, 2001;Sforcin, 2007).It appears highly beneficial for human organism to profit from these bee products, and one may see a great potential of their use especially in healing and wound dressing materials, as they can provide additional anti-inflammatory activity, thus leading to shorter healing time.We have followed this lead and we have focused on modifying hydrogels with bee pollen, hoping that these would constitute very interesting materials for biomedical applications.
The reacting mixture was mixed intensively on a magnetic stirrer at 60°C for 30 min.Afterwards the mixture was poured into Petri dishes, which were left at 37°C for 24h.The compositions of formed acrylic hydrogels modified by appropriate amounts of bee pollen 0, 2.5 and 5 %wt.respectively are shown in Table 1 presented below.
The swelling capacity (Q) was calculated according to the following formula: where: W 1 and W 0 refer to the weights [g] of the hydrogel in swollen and dried states, respectively.(Kokubo and Takadama, 2006;Vallet-Regí and Arcos, 2008).

Incubation studies
In the incubation studies 0.5 g of prepared acrylic hydrogels were placed in 50 ml of an appropriate medium: water, SBF and Ringer's solution for a period of 32 days.During this time pH values were measured periodically.

Attenuated total reflectance FT-IR spectroscopy analysis
All Attenuated Total Reflectance Fourier-Transform Infra-Red (ATR-FT-IR) spectra were recorded using Spectrum 65 (Perkin Elmer) spectrometer.The ATR probe was equipped with a diamond crystal.All spectra consisted of 32 scans at 4.0 cm −1 resolution and were recorded at 25 0 C.

Scanning Electron Microscopy
In order to investigate the structure of the obtained hydrogels modified with bee pollen, as well as bee pollen alone, images using scanning electron microscopy were taken and analyzed (JEOL JSM 7500F with a Back Scattered Electrons detector).

SEM analysis
In order to assess whether and how the hydrogel and bee pollen interact, SEM images of both bee pollen and the hydrogel modified with bee pollen were taken.Fig. 1 shows bee pollen in its raw form, as purchased.As it can be seen, bee pollen grains occur mostly in elliptic forms with two or three grooves.Moreover, these particles have a very uniform size distribution.This is due to the fact that the pollen grains were all collected from the same plant species.
Enlargement of a single particulate of bee pollen clearly shows a porous surface of the grain, with a very uniform distribution of the pores.Since the surface is not smooth but exhibits certain porosity, it is highly likely that adhesion to such a surface ought to be feasible and allow good bonding between the acrylic hydrogel matrix and the natural substance (see Fig. 1).
Images shown in Fig. 2, were collected using SEM imaging and show hydrogel modified with bee pollen.The image to the left shows fragments of the gel in reasonably small magnification, while the image in the centre shows clearly how the bee pollen particles are bound to the hydrogel.As expected, it appears that the gel at least partially coats the bee pollen particles.

Swelling behavior
Hydrogels are very well known for their excellent moisture sorption abilities which allow them to be used in a variety of biomedical and personal hygiene applications.Addition of any substance, such as bee pollen, may affect these properties therefore sorption capacity of modified hydrogel was tested using a variety of solutions, as it is shown in Fig.
The results show that the polymer matrix and modified pollen hydrogels exhibit the lowest sorption capacity in CaCl 2 solution.This is a consequence of presence of Ca 2+ , a multivalent cation which contributes to further cross-linking of the gel, leaving less void spaces in the macromolecule three-dimensional structure available for the solution to penetrate.The highest sorption values are achieved upon sorption of water, as expected.The water molecules can easily penetrate to the interior of the hydrogel and since they are not charged and are inert, they do not meet any hindrances.
It is impossible to define an overall relationship between the amount of added bee pollen and general sorption capacity.In case of swelling in SBF, addition of bee pollen caused a dramatic decrease, while other sorption capacities changed only insignificantly and could be both positive (PECF, Ringer's solution, water) and negative (0.9%wt.NaCl).It is possible that the worsened sorption capacities are caused by the fact that there is less volume available to penetrate in the hydrogel matrix, as it is partially occupied by the bee pollen particles.Nevertheless, since sorption properties are not severely affected, there is a good possibility that hydrogel modified with bee pollen could be used in the same applications as the non-modified material.

Incubation studies
In order to assess stability of the obtained hydrogels modified with bee pollen, pH measurements were carried out in the course of 32 days of incubation in the fluids mentioned in the Experimental section above.The results of these measurements performed on hydrogel modified with 2.5 %wt.and 5 %wt. of bee pollen are shown in Fig. 4 a and b, respectively.
The conducted pH measurements during the incubation period show significant changes in pH values only for the first four days, after which, regardless of the type of solution used, a plateau is reached.In case of Ringer's fluid and SBF, this initial change which took place over the first four days is far more significant than for samples immersed in water.This is certainly associated with presence of metal cations in both SBF and Ringer's solution and their absence in de-ionized water.As the results show, amount of introduced bee pollen has no significant effect on the pH changes during the incubation.The results for both polymer matrices containing 2.5% wt. and 5% wt. of bee pollen, are very similar.Lack of changes over an extended period of time appears to confirm that the hydrogel matrix would not degrade in conditions typical for biomedical applications.

Fig. 1 :
Fig. 1: SEM images of bee pollen where A denotes raw bee pollen magnified250x, B shows single particulate of raw bee pollen magnified 2500x and C-shows enlargement of image A magified1000x.

Fig. 2 :
Fig. 2: SEM micrographs of acrylic hydrogels modified with bee pollen where A denotes acrylic hydrogel modified with bee product magnified 150x, B-and C-single particulate of modified hydrogel magified 1000x.

Table 1 :
The compositions of acrylic hydrogels modified by bee pollen.

Table 2 :
Human plasma and ions concentration [mM] of SBF and PECF