Formulation of Monodisperse Non-Ionic Surfactant Vesicles (NISV) by Microfluidic Mixing Compared to Other Formulation Methods
Introduction
Non-ionic surfactant vesicles (NISV), or “niosomes,” are synthetic bilayer vesicles typically formed by the self-assembly of non-ionic surfactants, cholesterol, and the addition of a charged species. NISV exhibit more advantages over liposomes, in terms of cost and stability, and constituent surfactants have a wider range of chemistries that can be selected to provide greater potential for innovation related to vesicle composition. NISV have been used to deliver hydrophilic drugs that are encapsulated in the interior aqueous compartment or adsorbed on the bilayer surface, as well as hydrophobic drugs that are localised within the lipid bilayer of the NISV.1 Various methods have been used in vesicle preparation, providing different characteristics. We have investigated the physical characteristics of NISV prepared by three different manufacturing methods: thin-film hydration (TFH), heating method, and microfluidic mixing. The main factors affecting the control of NISV production by microfluidics were also given careful consideration.
Experimental Methods
NISV formulations composed of monopalmitin glycerol (MPG), cholesterol (Chol), and dicetyl phosphate (DCP) in a molar ratio of 5:4:1 of MPG/Chol/DCP were prepared by the three methods as previously described.2 NISV suspensions prepared by the TFH and heating methods were manually extruded using an Avanti miniextruder containing a 100 nm pore diameter polycarbonate membrane at 50°C to reduce the particle size and distribution. Physical characteristics such as particle size, polydispersity index (PDI), and zeta potential (ZP) were measured by dynamic light scattering. Morphological examination of the NISV was performed by atomic force microscopy (AFM).
Results and Discussion
Dynamic light scattering revealed that the particle size of the extruded NISV prepared by the TFH method and heating method were small and monodisperse, whereas the non-extruded particles were large and polydisperse (Table 1). However, particles prepared by microfluidic mixing were small with a narrow particle distribution. Moreover, because all the particles prepared by the three methods used the same lipid compositions, the ZP values for the extruded particles prepared by the TFH and the heating methods and by microfluidics were the same with no significant difference (P > 0.05) (Table 1). Microfluidics produces small, monodisperse particles in minutes, whereas the other methods took hours to obtain equivalent results. Traditionally, the production of small particles using the TFH and heating methods requires the use of a post-manufacturing size-reduction step to produce particles of the required size and to reduce the PDI. This has restricted the use of these methods to bench scale because a much longer industrial-scale process is required to produce a consistently sized end product. However, microfluidic mixing allows larger scale production of controlled particle sizes with homogenous distribution in a single step without the need for post-manufacturing size reduction.
Regarding the stability of the NISV, the TFH and heating methods (post extrusion) and microfluidic mixing produced stable particles with respect to size with no significant change when stored at four different temperatures over two months (Fig. 1). This suggests that
the method of preparation had no effects on particle stability. Although temperature can have an energy input into the system and can sometimes lead to changes in the crystalline structure of the lipids and potentially cause changes in the ZP that might affect the stability of the particles,3 in this study all three methods exhibited excellent stability over the range of temperatures (4, 25, 37, and 50°C) with no significant increase in the average particle size, PDI, and ZP (P > 0.05) when stored for two months even at the higher storage temperatures. These data indicate that microfluidics not only enables rapid, robust, and scalable production of NISV but also supports the stable formation of these vesicles, which is necessary for applications requiring prolonged shelf life such as pharmaceutical drug delivery.
Morphological observations of AFM images confirmed the formation of spherical particles of NISV regardless of the method of preparation (Fig. 2). These results confirmed that the particles prepared by microfluidics in a single step are similar to the extruded particles prepared by the more traditional TFH and heating methods.
For the formation of lipid-based particles through microfluidic mixing, the total flow rate (TFR) and the flow rate ratio (FRR) of aqueous to solvent
Figure 1. Size stability of NISV prepared by the thin-film Figure 2. AFM images of NISV prepared three methods: (A) thin-film hydration
hydration (TFH) method, heating method, and microfluidic method post extrusion, (B) heating method post extrusion, and (C) microfluidic mixing.
mixing and stored over 60 days at 4, 25, 37, and 50°C. © Elsevier, reproduced by permission.2
The data represent the mean ± SD (n = 3). © Elsevier,
reproduced by permission.2
streams were anticipated to be crucial factors in particle preparation.4,5 Figure 3 shows the changes of the particle size by changing the FRR from 1:1 to 5:1
Figure 3. Size changes of NISV prepared at different total flow rate and flow rate ratio of the aqueous and lipid phase. The data represent the mean ± SD (n = 3). © Elsevier, reproduced by permission.2(aqueous/lipid phases) and the TFR from 0.5 to 12 mL/min.
As the aqueous/ethanol FRR increased from 1:1 to 5:1, a significant (P < 0.05) reduction in NISV size was observed and found to be TFR dependent. At a TFR < 3 mL/min, the difference between the particle size prepared at FRR of 3:1 and 5:1 was not significant (P > 0.05). However, at higher TFR (>3 mL/min), the difference between these two FRRs was significant (P < 0.05). The FRR strongly affected the final solvent concentration. At lower FRR (1:1), the final solvent concentration increased, thus boosting the production of larger particles owing to particle fusion and lipid exchange, whereas at higher FRR (5:1), the chance of producing large particles was reduced as a result of decreased solvent concentration. Moreover, the TFR was shown to have a significant (P < 0.05) effect on particle size, in which the increase in the TFR from 0.5 to 9 mL/ min resulted in an overall reduction in particle size at all the FRR. However, further increase in the TFR above 9 mL/min was not associated with a significant decrease in particle size at all the FRR. The effect of the TFR on particle size is still debatable. Although some researchers have reported that TFR does not have a significant effect,6 others have reported the contrary.7 In our previous work, we have demonstrated that the aqueous medium used also has a significant effect on NISV characteristics when prepared by microfluidics.8 So microfluidic mixing allows the production of NISV with a tuned particle size by varying the TFR, FRR, and aqueous medium.
Conclusions
In this work, the characteristics of NISV prepared by microfluidics were compared with those prepared by the conventional TFH and heating methods. Microfluidic mixing enabled preparation of small, monodisperse particles in a single step, without the need of a size-reduction step as in the case of the other methods. The method of preparation did not have significant effects on particle stability. Using microfluidic mixing, a homogenous NISV suspension was prepared with high reproducibility. FRR and TFR between the two phases of the microfluidic mixing are factors that have significant effects on particle characteristics, which can be optimised to produce NISV with a defined size, which is important in developing an effective drug delivery system. This work demonstrates the promise of microfluidic mixing in NISV preparation to facilitate the development and optimisation of these dispersions for nanomedicine applications at both bench and industrial scales.
Acknowledgements
The authors acknowledge the Jordanian Ministry of Higher Education and Scientific Research and Yarmouk University in Jordan for funding this work. Also, the authors thank Dr. Michele Zagnoni from the Department of Electronic and Electrical Engineering for help with the miniextruder.
References
1. Kumar, GP, Rajeshwarrao, P. Nonionic surfactant vesicular systems for effective drug delivery—An overview. Acta Pharm. Sin. B 1(4): 208-219 (2011).
2. Obeid, MA, Gebril, AM, Tate, RJ, Mullen, AB, Ferro, VA. Comparison of the physical characteristics of monodisperse non-ionic surfactant vesicles (NISV) prepared using different manufacturing methods. Int. J. Pharm. 521(1-2): 54-60 (2017).
3. Heurtault, B, Saulnier, P, Pech, B, Proust, JE, Benoit, JP. Physico-chemical stability of colloidal lipid particles. Biomaterials 24: 4283-4300 (2003).
4. Carugo, D, Bottaro, E, Owen, J, Stride, E, Nastruzzi, C. Liposome production by microfluidics: Potential and limiting factors. Sci. Rep. 6: 25876 (2016).
5. Bottaro, E, Nastruzzi, C. “Off-the-shelf ” microfluidic devices for the production of liposomes for drug delivery. Mater. Sci. Eng. C 64: 29-33 (2016).
6. Jahn, A, Vreeland, WN, DeVoe, DL, Locascio, LE, Gaitan, M. Microfluidic directed formation of liposomes of controlled size. Langmuir 23: 6289-6293 (2007).
7. Yu, B, Lee, RJ, Lee, LJ. Microfluidic methods for production of liposomes. Methods Enzymol. 465: 129-141 (2009).
8. Obeid, MA, Khadra, I, Mullen, AB, Tate, RJ, Ferro, VA. The effects of hydration media on the characteristics of non-ionic surfactant vesicles (NISV) prepared by microfluidics. Int. J. Pharm. 516(1): 52-60 (2017).
