High-pressure gas-assisted absorption of protein within biopolymeric micro-patterned membrane

Abstract

The loading of bio-molecules within polymeric matrices, such as particles, sponges or thin membranes, by completely solvent-free processes has an enormous interest in biomedical engineering applications, in particular, for the production of drug carriers and biomimetic scaffolds. In this research, we developed a process based on the use of high-pressure carbon dioxide (CO₂) in order to absorb bioactive molecules within poly(lactic-co-glycolic acid) (PLGA). A dye, methylene blue, was used to optimize process conditions, whereas lysozyme was used to evaluate the protein-loading efficiency. The in situ UV–vis measurements of the gas-assisted absorption of methylene blue showed that the dye concentration within the polymer increased tenfold with respect to the control sample, after 35 min at a relatively low pressure of 2.0 MPa. Experimental measurements in the range of 0.1–6.7 MPa, and at 308 and 313 K, showed that the protein loading was highly enhanced by CO₂ assisted absorption, whereas no detectable amount of protein was observed in the control. This gas-assisted technique is a completely solvent-free process, operates at mild temperature conditions (<313 K), and is particularly promising for the absorption of thermo-degradable molecules within PLGA membranes without affecting their morphology at macro- and micro-scale.

Introduction

In recent years, biocompatible and biomimetic polymeric three-dimensional structures have been of enormous interest in biomedical engineering applications, in particular, for the production of cell carriers and scaffolds [1], [2]. The properties of scaffolds are of fundamental importance for controlling cell growth, proliferation, and differentiation [1]. These basic cell activities can be regulated by proteins permanently adsorbed on scaffold surfaces that interact with the cell through the membrane receptors, or by the release of soluble growth factors from a polymeric matrix [3]. In both cases, a process suitable for the absorption of bioactive principles within a biodegradable polymeric matrix without altering the scaffold properties at macro- and micro-scale is of great importance [1], [4].

Various methods have been investigated in order to produce scaffolds with different chemico-physical properties of bulk and surface, morphology, and structure [5], [6], [7]. For example, biopolymers have been used successfully in gel casting [8], solvent casting, and salts leaching [9], or in micro-fabrication techniques [1], [2], [3]. In these techniques, the incorporation of labile proteins during the scaffold fabrication procedure is not straightforward because the operative conditions often deal with high temperatures [10] and the use of organic solvents [8], [9], [10]. On the other hand, downstream operations required for scaffold purification may cause a premature protein burst and release. For example, in porogen leaching techniques [8] it is possible to lose protein during the leaching step [9], [11]. For these reasons, it would be particularly valuable to develop a new methodology for absorbing bioactive compounds within a polymeric matrix of “ready to use” scaffolds. Therefore, this ideal methodology should preserve the activity of loaded compounds; and it has to be non-destructive, i.e., to preserve the macro- and micro-morphology, the chemico-physical, mechanical, and structural properties of the processed scaffolds.

A technological solution for these problems is represented by the polymer processing techniques that use supercritical fluids or compressed gas, and which avoid the use of organic solvent and high process temperatures. In other words, these processes can be successfully used to modify the chemical/physical properties of polymeric structures [11], [12], [13]. The absorption of supercritical fluids or compressed gases that show liquid-like density and gas-like diffusivity cause a swelling in volume, a reduction of glass transition temperatures (T_g) and melting points (T_m), and a reduction of viscosity in the bulk polymeric-rich phases [14]. Among many chemical substances, carbon dioxide (CO₂) is used as compressed gas in consequence of its relatively low critical point (304 K, 7.4 MPa), and because it is a non-toxic, inexpensive and environmentally friendly compound. Recent studies by Sicardi et al. [15] have shown that the diffusion coefficient values of adsorbed substances (dispersed yellow and blue) in the PET films were about from 1 to 3 orders of magnitude higher when compressed or when supercritical CO₂ was used instead of other classical treatments. These phenomena were also applied to load dyestuff within a polymeric substrate [16] using an impregnation process, which consists of two steps: first, the dyestuff is dissolved in the supercritical gas, and then it is loaded into the chamber, where the polymeric substrate is located. However, this technique is greatly limited by the low dissolution of compounds in a supercritical or high-pressure gas media; only low molecular weight and mainly hydrophobic substances show reasonable solubility [17], [18].

On the other hand, the ability of CO₂ to depress the glass transition temperature and the swelling of treated materials is often associated with foaming during the depressurization process [19]. In particular, the dissolution of CO₂ within the hydrophobic poly(lactic-co-glycolic acid) results in macro- and micro-bubble foam during the depressurization step and the subsequent release of CO₂ [17]. This effect was used by Mooney et al. [19] and Hile et al. [20] to produce porous sponges that were employed as growth factor delivery systems in tissue engineering applications [11]. Hile et al. [20] observed that this foam released more protein than that obtained by solvent casting-salt leaching. In order to eliminate the influence of the CO₂ treatment on the processed polymeric matrices, Sproule et al. [17] reduced the swelling/foaming of the final product by applying a slow depressurization step after the impregnation treatment. In their work, the poly(methyl methacrylate) films were loaded with fluorescent protein by using a supercritical CO₂ impregnation process, where the protein aqueous solution absorbed in cotton swabs was placed in contact with a polymeric film surface. However, this process required a stable protein solution during the entire impregnation process. In particular, the use of water leads to protein denaturation and/or its structural modification because the solubilization of CO₂ caused intense acidification of the medium [21]. Protein degradation depends a great deal on its physico-chemical structures [22], [23].

For these reasons, we aimed at developing a gas-assisted impregnation technique of biodegradable polymeric scaffolds operating at room temperature, and without the use of organic solvents. In particular, we developed an impregnation process that operates at “dry” conditions where operative variables for the protein deposition and polymer absorption procedure were decoupled. Thus, the process can be summarized in two phases: (1) protein deposition onto a scaffold surface achieved by rapid evaporation of an aqueous buffer solution, and (2) protein absorption within a biodegradable polymeric matrix enhanced by high-pressure CO₂. In addition, we demonstrated that it is possible to adjust operative process conditions in order to obtain a non-destructive scaffold treatment. Low density CO₂ at moderate pressure (3.0 MPa) was sufficient to reduce the glass transition of the polymer, to swell the polymeric matrix, and to allow the dye or the protein loading within the polymeric matrix. The enhancement of the high-pressure CO₂ was evaluated by following in situ the absorption phenomena by UV–vis spectroscopic analysis. It is worth noting that high-pressure CO₂ promotes low temperature microbial inactivation, which is particularly useful for scaffold production processes [24]. The use of micro-patterned membranes as scaffold models showed that the process does not alter the morphology for either macro- or micro-scales. Biodegradable PLGA was used because it is suitable in producing controlled drug release systems [25], [26] and scaffolds [20], [27], [28] for pharmaceutical and biomedical applications [20], [25], [26], [27], [28]. The protein loading was greatly increased with respect to the classical contact method also at moderate temperatures. These results are very promising for the development of an impregnation process for loading bioactive molecules in “ready to use” scaffolds.