A Freezing and Thawing Method for Fabrication of Small Gelatin Nanoparticles with Stable Size Distributions for Biomedical Applications

Abstract

Background:

Gelatin, a natural polymer, has a number of advantages as a material for fabricating nanoparticles, such as its hydrophilicity, biodegradability, nontoxicity, and biocompatibility, as well as low cost. Despite these various advantages, gelatin-based nanoparticles still have critical limitation for biomedical applications due to their relatively larger size than those of other materials.

Methods:

In this study, a new strategy to design and fabricate small gelatin nanoparticles (GNPs) was proposed. The technique was based on the natural phenomenon where with decreasing temperature, the compression between the molecules of substances increases and the volume shrinks.

Results:

The average size of the fabricated small GNPs was less than 100 nm and their gelatin properties (including non-cytotoxicity) were well maintained. The drug release profiles of the GNPs were confirmed, for which a simple mathematical model based on the conventional diffusion equation was proposed. There was a burst of drug release in the first 3 days, with different release profiles according to the concentration of model drugs loaded onto the GNPs. It was also demonstrated that the drug release profiles of the proposed mathematical model were consistent with the experimental results.

Conclusion:

Our work proposes that these small GNPs could be used as efficient drug and gene delivery and tissue engineering platforms for various biomedical applications.

Introduction

In recent decades, nanoparticles have attracted much attention in various biomedical fields, including as drug delivery systems and tissue engineering, owing to their small nanoscale size, stability, and biocompatibility [1]. In particular, polymer-based nanoparticles have shown higher efficiency in the targeting of diseases, because the encapsulation process has been more developed compared with other nanoparticle systems [2]. The targeting efficiency of these nanoparticles is mainly influenced by their particle size, surface charge, and surface modification. In particular, the size of nanoparticles is known to be one of the most important factors that determine their interaction with the cell membrane and their penetration across the biological barriers [3]. For example, small-sized nanoparticles (lees than 100 nm) could penetrate various biological barriers, such as the brain–blood barrier.

Gelatin, a natural polymer, has a number of advantages as a material for fabricating nanoparticles, such as its hydrophilicity, biodegradability, nontoxicity, and biocompatibility, as well as low cost [4]. Because of these various advantages, many researchers have studied the use of gelatin-based nanoparticles for various biomedical applications [5]. Although previous studies have developed small gelatin nanoparticles (GNPs) using general desolvation methods, the fabricated GNPs tended to be unstable and prone to aggregate [6]. To overcome these limitations, a two-step desolvation method was reported that could fabricate stable GNPs that did not aggregate. However, the average size of GNPs fabricated through the conventional two-step desolvation method was 150–300 nm [7–9], which may be a major limitation to their use. It is known that the size of nanoparticles is the most important factor determining their biodistribution [10]. For example, nanoparticles of larger than 100 nm accumulate in a large amount in the liver and spleen.

In this study, the new method was proposed for the fabrication of GNPs of small sizes (i.e., < 100 nm) through a freeze–thawing process. The fabricated small GNPs were characterized in terms of their size distribution and subjected to chemical and cytotoxicity analyses. In addition, the properties of the drug release profiles from the small GNPs were investigated both experimentally and mathematically. Finally, we compared our small GNPs (less than 100 nm) with conventional GNPs (more than 100 nm).

Materials and methods

Freezing and thawing method for the fabrication of small nanoparticle

The new freezing-thawing method was proposed as follows: Gelatin type A (Sigma, St. Louis, MO, USA, 1.25 g) was dissolved in 25 mL of water under 50 °C heating. After the gelatin had been sufficiently dissolved, 25 mL of acetone was added, and the mixture was stirred rapidly for quick sedimentation of the gelatin. The supernatant was discarded, and the sediment was left in order to leave only a high molar mass of gelatin. The sediment was redissolved in 25 mL of water under 50 °C heating, and the pH of the mixture was adjusted to 3 by the addition of HCl. Then, 75 mL of acetone (Sigma) was added with stirring at 40 °C and 600 rpm [11], following which the gelatin solution was frozen rapidly in the deep freezer for compression of the gelatin molecules. After 1 h, the frozen gelatin solution was melted and stirred at 4 °C in a cold laboratory chamber. Then, 280 μL of glutaraldehyde (25%, v/v) was added for crosslinking of the gelation particles. The GNPs were purified by dissolution in 75% acetone and centrifugation at 4 °C and 6500 rpm, carried out 3 times. Finally, the particles were lyophilized for 3 days.

Characterization of the gelatin nanoparticles

The size of the GNPs was measured by scanning electron microscopy (JSM-7500F, Tokyo, Japan) using ImageJ software. Fourier-transform infrared spectroscopy (FT-IR) was carried out on a PerkinElmer Spectrum 400 apparatus (Waltham, MA, USA).

Cytotoxicity assay

The toxicity of the GNPs toward MG-63 cells and tenocytes was evaluated by water soluble tetrazolium salts (WST)-1 assay. In brief, the cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells/well and incubated for 24 h in culture medium (Cellgro, CORNING, NY, USA). After 24 h, the culture medium was replaced with cell culture medium containing 0.05%, 0.1%, 0.5%, 1%, or 2% (w/v) of the GNPs and the cells were again incubated for 24 h. The number of viable cells was quantified using the WST-1 assay.

In vitro drug release

Trypan blue was used as a model drug to evaluate the drug release profile of the GNPs. In brief, the GNPs were impregnated with a 0.05%, 0.1%, or 0.2% (w/v) solution of trypan blue at a ratio of 5 μL/mg and cured for 2 h at room temperature. Thereafter, the GNPs were immersed in phosphate-buffered saline at a ratio of 1.5 mL/5 mg and then inverted at 20 rpm in a 37 °C incubator. The trypan blue concentration in the supernatant was determined by measuring the absorbance at 595 nm using a spectrometer [12].

Mathematical model

The proposed model is a simple one-dimensional model based on Ficks second law. The equation for a general diffusion equation is as follows:

$$\frac{\partial C_A}{\partial t}=D_{\mathit{AB}}\frac{{\partial }^2{C}_A}{\partial x^2}$$ 1

where C_A is the time-dependent concentration of the drug, A is the position inside of the particles, B is the position outside of the particles, t is the time, and D_AB is the diffusion coefficient of the drug through the particles. The determination of the diffusion coefficient from Eq. (1) was achieved by modeling the first 3 days of drug release from the GNPs.

Results

To fabricate GNPs of a small size, a new two-step desolvation method was established based on the freeze–thawing process. This method was based on a natural phenomenon where with decreasing temperature, the compression between molecules of substances increases and the volume shrinks [13, 14]. Figure 1 shows a schematic of the GNP fabrication by the freezing-thawing method. In the conventional two-step method, the gelatin is first precipitated, and glutaraldehyde is then added when the particles have been formed (Fig. 1A) [11]. In contrast, in the modified method, the compression between the GNP molecules was facilitated by the rapid freezing after gelatin precipitation, resulting in the fabrication of small-sized GNPs (i.e., average size < 100 nm) (Fig. 1B). Figure 2A shows the representative SEM images of the GNPs fabricated using conventional method, where more aggregated nanoparticles were observed. Figure 2B shows the representative SEM images of the GNPs fabricated using freezing-thawing method, where non-aggregated nanoparticles of spherical shape and small size were observed compared to GNPs fabricated using conventional method. the sizes of the GNPs fabricated using conventional method were more than 150 nm on average (Fig. 2C) and the sizes of the GNPs fabricated using freezing-thawing method were 40–60 nm at a minimum and less than 100 nm on average (Fig. 2D). To compare the chemical structure of gelatin and the fabricated GNPs, their functional groups were investigated by FT-IR analysis (Fig. 3). The amide-A peak arising from N–H stretching was distributed at 3466 cm⁻¹ relatives to the degree of cross-linking, with C=O stretching at 1630 cm^—1 for amide I and N–H deformation and C–N stretching at 1565 cm⁻¹ for amide II [15]. These results indicated that the natural functional groups of gelatins were well maintained in the small GNPs.

Fig. 1.

Fabrication of small gelatin nanoparticles (GNPs). Schematic illustration of the GNP fabrication process, using a conventional method and freezing-thawing method

Fig. 2.

Characteristics of the fabricated small gelatin nanoparticles A, B Low-magnification (25,000×) and high-magnification (50,000×) SEM images of the GNPs fabricated using conventional and freezing-thawing method. C, D Size distributions of the GNPs fabricated using conventional and freezing-thawing method

Fig. 3.

Chemical analysis of the fabricated small gelatin nanoparticles by Fourier-transform infrared spectroscopy (FT-IR)

It has been reported that the size of nanoparticles is an important factor in determining their cytotoxicity [16]. Generally, nanoparticles below 100 nm in size are known to be the least cytotoxic, whereas the very small nanoparticles (~ 1 nm) are very toxic to cells [14]. To investigate the cytotoxicity of the fabricated GNPs, MG-63 and tenocytes were used as model osteoblasts and fibroblasts, respectively, for WST-1 assay [15]. Both cell groups showed higher cell viability than that in the control group (0% of GNPs) at all concentrations of the nanoparticles (Fig. 4). Interestingly, the viability of the osteoblasts increased with increasing GNP concentrations. These results indicated that the fabricated GNPs were nontoxic to cells.

Fig. 4.

Cytotoxicity of the fabricated small gelatin nanoparticles at different concentrations (0%, 0.05%, 0.1%, 0.5%, 1%, and 2%) for 24 h. Viability of osteoblasts (MG-63 cells) and viability of fibroblasts (tenocytes). *p values < 0.05

In general, the size of nanoparticles is known to be one of the most important factors in determining their drug release rate in vitro and in vivo. To confirm the drug release profiles of the fabricated GNPs, in vitro drug release experiments were conducted. Figure 5 shows the model drug (i.e., trypan blue) release profiles at the concentrations of 0.05%, 0.1%, and 0.2%. The results showed that the initial burst of release of the model drug from the GNPs occurred in the first 3 days at all drug concentrations, with 40%, 30%, and 20% of the loaded drug being released at 0.05%, 0.1%, and 0.2% trypan blue concentrations, respectively. This could be explained by the drug being adsorbed onto the external surface of the GNPs [16]. Moreover, because of the weak mechanical properties of gelatin, more drugs would have been released earlier [17]. The results showed not only the initial burst of release but also that the release curves were similar for all three drug concentrations. However, the drug release rate was higher with a higher amount of drugs loaded. With regard to the mechanism of drug release by the GNPs, it is known that this may be controlled by diffusion, in accordance with the equation developed by Higuchi for understanding the release of drugs by solid matrices [18] (Fig. 6).

Fig. 5.

In vitro trypan blue release from small gelatin nanoparticles treated with different trypan blue concentrations (0.05%, 0.1%, and 0.2%, v/v)

Fig. 6.

Comparison of the trypan blue release profiles obtained between the mathematical model and the actual experiments (in vitro release from small gelatin nanoparticles treated with trypan blue concentrations of 0.05%, 0.1%, 0.2% v/v)

Mathematical modeling on the drug release profile of new nanoparticles is a very important factor for biomedical applications. The understanding of the drug release profile of nanoparticles and the release tendency through mathematical modeling enable the use of an appropriate amount of drug and release control using nanoparticles. Therefore, we proposed a simple mathematical model using the conventional diffusion equation. The diffusion equation (Eq. 1) was solved using the constant diffusion coefficient obtained through the initial first 3 days of drug release, as described above [19, 20]. Additionally, the results were calibrated by considering the first 3 days of burst release. Figure 4B shows the comparisons between the release model results at the three different drug concentrations and the experimental results.

Discussion

As the particles showed various characteristics according to their sizes, our fabricated GNPs were distinguishable from conventional GNPs and gelatin micro particles (GMPs). First, cell viability was on average over 20% higher in the presence of our GNPs than in that of GNPs over 100 nm and GMPs [21]. This is consistent with the result of a previous study where nanoparticles of less than 100 nm had minimal cytotoxicity [14]. Second, the drug release rate of the small GNPs appeared to be faster than that of conventional GNPs and GMPs. Specifically, the release rate of our small GNPs was 20% higher than that of GMPs [21, 22]. Many studies have shown that smaller particles release drugs faster because of the increased surface area [23]. However, the drug release rate of particles is determined not only by the size but also the structure of the polymer, rate of degradation, concentration of the drug, and degree of hydrophilicity. Therefore, it is important to consider the complexity of these various factors when attempting to understand the drug release mechanisms of nanoparticles.

Modeling is a mathematical representation of all phenomena, and it is important to systematically design new devices for modeling-controlled release profiles. In particular, because drug release from nanoparticles has unique properties owing to their small sizes, it is important to understand the release mechanisms and to predict the drug release profiles. Although a number of mathematical models describing the drug release profiles of micro- and nanoparticles have been reported, there are no models for predicting the drug release profiles of GNPs. From this point of view, for the prediction of the drug release profile of small GNPs developed through the novel freezing-thawing method, we present a simple mathematical model based on Ficks second raw, and this mathematical model is almost consistent with the drug release profile of the actual small GNPs. These results show the understanding of drug release profiles from small GNPs and the potential for biomedical application of small GNPs using simple mathematical modeling.

Here, we propose the possible application of small GNPs. The prepared small GNPs through the freezing-thawing method could be applied to various biomedical fields. For example, similar to other nanoparticles, the small GNPs could be used in a wide range of fields related to nanomedicine, that is, from in vitro and in vivo diagnostics to nanotherapeutics, vaccine delivery, and regenerative medicine. Although future works are still necessary to investigate the advantages of the small GNPs, we believe that they may overcome the limitations of conventional GNPs in drug delivery; for example, they may not accumulate in organs such as the liver and spleen or penetrate biological barriers in specific tissues. In other words, the small GNPs may enable a more localized drug delivery that cannot be achieved by the conventional GNPs because of their large size effects.

In summary, we developed a new freezing-thawing method for manufacturing small and stable GNPs of less than 100 nm. Not only were these small GNPs demonstrated to be noncytotoxic, but their increasing concentration also induced an increase in the viability of fibroblasts. Moreover, the drug release rate from the small GNPs was faster than that from conventional GNPs owing to their small size, and the drug release profiles differed according to the concentration of the model drug. The results of the simple mathematical model (based on Ficks second law) for the drug release profiles of the small GNPs were consistent with the actual experimental results. We conclude that the fabricated small GNPs could be used as efficient drug delivery and tissue engineering platforms in various biomedical applications based on the gelatin hydrogel.

Declarations

Conflicts of interest

The authors declare no conflicts of interest.

Ethical statement

There are no animal experiments carried out for this article.