1. Introduction
Lipidic-based nanoparticles are clinically and markedly established nanosystems for the delivery of drug molecules, supplements and cosmetic substances, having shown great benefit in the formulation, administration, and delivery of poorly water-soluble bioactive ingredients. Since they are lyotropic liquid crystalline systems, their structure and morphology depend on the concentration of the composing lipids and phospholipids, as well as on their molecular geometry, which is related to their chemical structure [
1,
2,
3]. Their conformation might be lamellar (i.e., a bilayer/membrane), cubic, hexagonal etc., allowing for incorporation of lipophilic, hydrophilic, or even amphiphilic bioactive ingredients. In the case of oral administration, by predissolving these molecules in lipidic vehicles, we overcome the dissolution process, which is usually the rate-limiting step for their absorption in the blood and as a result, for their final effectiveness, especially in the case of lipophilic agents. Lipidic nanoparticles are innovative excipients, presenting variety in their structure and properties in vitro and in vivo, including digestibility and absorption. The nature of their components profoundly affects the final biopharmaceutical and pharmacokinetic profiles of the delivered bioactive ingredients [
4].
Among lipidic nanoparticles, liposomes are one of the most promising classes for biomedical applications. They are present in the clinic for years and are still studied, modified, and functionalized, to develop advanced delivery systems for therapeutic molecules. Liposomes are closed pseudospherical structures, consisting of concentric lipid bilayers, which are mainly built by phospholipids and entrap aqueous media. They are inherently thermodynamically unstable in a colloidal dispersion and for this reason, they are usually stabilized through the addition of cholesterol and other types of biomaterials, such as polymers [
1,
2,
3,
5,
6]. Apart from structural units, these molecules contribute to the final functionality of liposomes, through physicochemical, thermodynamic and biophysical principles. Of great importance for the final liposome properties, stability and functionality is the self-assembly process, which is a thermodynamically driven spontaneous process that is determined by the geometric characteristics and the critical packing parameter of the mixed molecules [
7,
8].
Liposomes and lipidic vehicles are novel industrial products for pharmaceutical, cosmeceutical and nutraceutical formulations [
9,
10,
11,
12,
13,
14]. The various methods for the production of lipidic vehicles and liposomes include solvent injection, detergent dialysis, freeze-thaw, reverse-phase evaporation, sonication, homogenization and the dehydration-rehydration method, of which one typical technique is the Bangham method (thin-film hydration method). Most of these methods lead to the production of multilamellar vesicles (MLVs), which need to be further processed through size-reduction techniques, so that small unilamellar vesicles (SUVs) are developed, which are utilized for various applications. Another method, microfluidics, is the method of choice for scale-up applications of liposomes and lipid-based systems, with several modifications having been developed. The advantages offered by the microfluidics technology can benefit the production of liposomes and include accurate handling of nanoliter volumes, precise control of the interface position, diffusion-dominated axial mixing and continuous operation for low production volumes [
15,
16].
According to Patil and Jadhav, 2014, “Conventional techniques for liposome preparation and size reduction’ remain popular as these are simple to implement and do not require sophisticated equipment. However, issues related to scale-up for industrial production and scale-down for point-of-care applications have motivated improvements to conventional processes and have also led to the development of novel routes to liposome formation.” [
16]. At the same time, liposomal products are becoming more and more complex, in the attempt of functionalization through surface modification, which leads to integration of more formulation steps, increases the production cost, and renders their evaluation more challenging. For successful scale-up, the prerequisites are few manufacturing steps and the absence of harmful organic solvents. In addition, liposomal development, as part of pharmaceutics, is much dependent on quality assurance and cost. Regarding quality assurance, liposomes, and drug delivery nanosystems in general are affected by production scalability, reproducibility, availability of equipment, expertise, stability of the incorporated bioactive molecule and long-term stability [
17]. Moreover, it has been suggested that liposomal drug development can benefit from continuous manufacturing, a processing concept where raw materials constantly flow into a process and product constantly flow out that has been applied to produce biologics [
18].
The present investigation is an example of the application of a simple method to produce of lipidic vehicles and liposomes, which is a modified heating method (MHM) (
Figure 1) [
19,
20]. The method comprises stirring a mixture of an amphiphilic lipid and a charged lipid or a fluidity regulator in a liquid medium comprising water and a liquid polyol, heating the mixture in two steps, wherein the temperature of the mixture in the second step is higher than the temperature in the first step and allowing the mixture to cool down to room temperature.
4. Discussion
The role of glycerine concentration in the final properties of the vehicles is evident, where the utilization of higher amounts of the molecule led to HSPC:SA particles of better physicochemical properties, i.e., smaller size, and polydispersity. The liquid polyol drives/enhances the hydration process of the amphiphilic lipid(s), facilitating their self-assembly in smaller vehicles and managing the thermodynamic content of the lipidic vehicles. In addition, glycerine offers several advantages in lipidic vehicle and liposomal formulations. It is a biocompatible, bioacceptable and nontoxic isotonising and dispersant agent for the nanoparticles, enhancing their properties and stability. In addition, it serves as a cryoprotectant during freezing and thawing processes, while its removal from the final product is not necessary [
23]. In previous studies, lipidic formulations with EPC and SA, without glycerine were developed by dissolution of the lipids in chloroform, evaporation of the solvent and afterwards, by further processing the final hydrated formulations, in order to achieve the desired physicochemical properties (
Table 4). The present approach excludes toxic solvents and size reduction methods that similar studies have utilized in the past, providing a nontoxic, simple, and scalable method for lipidic vehicle preparation. Chloroform and sonication or extrusion through polycarbonate filters are very common elements in liposomal preparation and their avoidance ensures safety and saves time, effort, and costs [
24,
25,
26].
The combination of lipids is also very important for the final physicochemical properties, since HSPC:SA and EPC:CHOL:SA behaved differently, with the CHOL amount inside the system also affecting this behavior. Generally, the process depends on the fluidity/mobility of liquid crystalline materials above their phase transition temperature (
Tm), as well as their rigidity below that point. For HSPC and EPC,
Tm is ~53 °C and ~−5 °C respectively, which is associated with different self-assembly behavior and membrane stability [
27,
28]. HSPC is more rigid and stable, while EPC is more fluid and unstable and requires a membrane stabilizer, such as CHOL. As a result, CHOL, as a fluidity regulator, led to physicochemical properties according to its concentration. Finally, the observed positive zeta potential of the prepared lipidic vehicles is attributed to the positive charge of SA (pK
a~10.65) in the hydration medium, i.e., purified H
2O with different concentrations of glycerine, where pH is slightly acidic and the amino group of SA is protonated [
29].
The different colloidal stability between HSPC and EPC nanosystems is probably associated with the fluid nature of the EPC, compared with HSPC, combined with the multiple thermodynamic transitions it underwent during the measurements (from storage temperature of 4 °C to room temperature and back). As a results, the EPC:CHOL:SA 9:1.8:0.25 nanoparticles rearranged during day 15 and agglomerated due to increased phospholipid fluidity into larger particles, which then disassociated and gradually returned to normal. On the other hand, the EPC:CHOL:SA 9:0.5:0.25 system was unstable, owed to the low concentration of CHOL. The observed colloidal stability of HSPC:SA systems is attributed to the positive zeta potential of these vehicles, which comes from SA and according to the Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory, it leads to electrostatic repulsion between the particles inside the suspension [
30,
31].
All extruded formulations were above 200 nm before the extrusion process, HSPC:SA being 245.6 nm, EPC:SA 353.5 nm and DPPC:SA 250.2 nm. Interestingly, after only the first pass through the membrane filters, their size was decreased to 135–155 nm, while polydispersity improved as well, especially for EPC (reduction by about 0.350). Then, the following cycles had less effect on the lipidic vehicle properties, mainly homogenizing the HSPC system and reducing the size of the ones with EPC and DPPC. The efficiency of the size reduction method in the pilot scale is very important for future industrial applications [
32,
33].
The lyophilized systems were concerned to be stable during the freezing, lyophilization and reconstitution processes. In cases where the lipidic vehicles are subjected to lyophilization, the liquid polyol, e.g., glycerine, plays a key role in the physical stability and cryoprotection of lipidic vehicles during lyophilization and reconstitution [
34]. Specifically, 20%
v/v of the molecule led to almost absolute conservation of the physicochemical properties, while 15%
v/v led to only a slight increase in the particle size and polydispersity, indicating the importance of glycerine concentration levels for this process. An additional advantage of utilizing glycerine as a cryoprotectant is the avoidance of carbohydrates, which are traditionally used for the purpose of lyophilization. Stability of lipidic nanoformulations is imperative for their clinical application and quality, including efficacy and safety. For this reason, these products reach the clinic in their lyophilized form, rendering of fundamental importance the study of the effect of the lyophilization and reconstitution process on their properties, which will in turn affect their biological stability and effect [
34].
Curcumin is an extremely lipophilic compound that is characterized by poor bioavailability and rapid metabolism and, as a result, liposomes and lipid nanoparticles are extensively studied as vehicles for the protection and delivery of the molecule [
35,
36,
37,
38]. Concerning curcumin incorporation, the herein observed phenomena indicate the interaction of the molecule with the phospholipid bilayer and the possible displacement of SA molecules from the hydrophobic segment of the membrane, leading to alteration of the vehicle physicochemical properties. We also must note the relatively higher concentration of utilized curcumin inside the developed formulations (9:0.25:0.8), compared with past studies. In a previous study, the molecule was incorporated inside EPC liposomes at a 14:1 lipid:drug molar ratio (84% ± 15% incorporation efficiency), leading to a slight decrease in the particle size, accompanied by a slight increase in their polydispersity (
Table 5) [
39]. In the present study, a comparable case is that of HSPC:SA with glycerin at 20%
v/v. We observed that, even though the curcumin concentration was higher (around 25% more curcumin), the present MHM works well in producing drug-loaded lipidic vehicles, also considering the important advantages that this method offers, compared with previously established ones.
Concerning the process parameters of the MHM, of crucial importance for the final formulation quality are concentrations, temperature, heating and cooling rate, stirring speed and duration. In
Figure 4, we summarize the process parameters that affect the final vesicular properties, i.e., their size and polydispersity. Out of these, we have shown herein that of crucial importance are the lipidic vehicle composition, the concentration of glycerine utilized, the duration of heating the formulation at high temperature and the potential utilization of a size reduction method, such as extrusion. Concerning the method steps, heating the mixture at 60 °C, which is above the
Tm of the utilized phospholipids, serves to solubilize the ingredients in the aqueous medium, which are then heated at higher temperature, i.e., 90 °C, in order to form assemblies of small size while in the liquid crystalline phase [
23,
40]. The duration of heating at high temperature is important and determines the final physicochemical properties of the lipidic vehicles (
Table 1). The cooling step is mandatory to be able to measure these properties. The method consistency in producing specific size and polydispersity lipidic vehicles is evident from the measurement standard deviations in
Table 1, as well as from the repeatable size distributions in
Figure S1.
The present approach offers important advantages in the production of lipidic vehicles and liposomes, compared with other established methods (
Table 6) [
16]. First, it is nontoxic, by not utilizing chlorinated or other volatile organic solvents. Furthermore, it does not require the use of size reduction methods, such as sonication or centrifugation and leads to particles of adequate size and polydispersity for biomedical applications [
41]. In addition, it does not require the use of reduced pressure, it is simple and easy to scale up. The process utilizes the hydration from the aqueous medium molecules and cosolvent molecules, and mechanical shock resulting from the stirring process and heating shock coming from the temperature increase. This leads to the formation of lipidic vehicles and liposomes with desirable characteristics. Through the MHM, it is also possible to incorporate bioactive ingredients inside the lipidic vehicles, such as drugs or active pharmaceutical ingredients (APIs), nutraceuticals, cosmeceuticals etc., by adding them inside the initial liquid mixture, with respect to their thermal stability, hydrophilicity/lipophilicity and other parameters that might affect their incorporation into the lipidic vehicles. It is also possible to subject the final formulations to size reduction methods, such as extrusion and lyophilization methods, to optimize them for various applications.
5. Conclusions
The present study describes a simple method, suitable for developing various lipidic vehicles, e.g., liposomes, in the absence of chlorinated and other volatile organic solvents. Prior art processes to produce lipidic nanocarriers have a number of disadvantages, including the utilization of toxic solvents that should be avoided. Furthermore, many prior art processes are complex and/or require considerable amount of resources, time and effort, while the size and homogeneity of the carriers achieved by most of them is not satisfactory and, for this reason, additional steps for size reduction and homogenization are needed. The present investigation provides a process to produce lipidic vehicles which successfully addresses the aforementioned disadvantages. The process can be easily scaled up and utilized in the industry, with or without the use of size reduction techniques, due to simple method parameters, accurate and repeatable results, time efficiency, low cost, absence of chlorinated and volatile organic solvents and safety.
The role of glycerine and its concentration in efficiently producing nanoparticles has been demonstrated, while the composition of the lipidic system is also important. Concerning colloidal stability, HSPC:SA and EPC:CHOL:SA formulations with 20% v/v glycerine were stable in due time, however, freeze-drying was carried out, in order to produce stable lyophilized products. The latter were found to maintain their physicochemical properties after reconstitution to the initial volume. The extrusion process applied was also found to be effective in reducing the particle size and homogenizing primarily after the first pass of the vehicles through the polycarbonate filters. Finally, the molecule of curcumin was formulated with lipidic vehicles prepared through the MHM. The proposed method is efficient in producing lipidic nanoparticulate vehicles, which may incorporate bioactive molecules, may be utilized directly, without the use of downsizing techniques and may also be lyophilized to produce stable and consistent products.