Microfluidic preparation of polymer nanospheres
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In this work, solid polymer nanospheres with their surface tailored for drug adhesion were prepared using a V-shaped microfluidic junction. The biocompatible polymer solutions were infused using two channels of the microfluidic junction which was also simultaneously fed with a volatile liquid, perfluorohexane using the other channel. The mechanism by which the nanospheres are generated is explained using high speed camera imaging. The polymer concentration (5–50 wt%) and flow rates of the feeds (50–300 µl min−1) were important parameters in controlling the nanosphere diameter. The diameter of the polymer nanospheres was found to be in the range of 80–920 nm with a polydispersity index of 11–19 %. The interior structure and surfaces of the nanospheres prepared were studied using advanced microscopy and showed the presence of fine pores and cracks on surface which can be used as drug entrapment locations.
KeywordsPolymethylsilsesquioxane Perfluorohexane Microfluidics Surface morphology Nanopheres Nanocarriers Nanobiotechnology
A major challenge faced during the preparation of solid polymer nanospheres for advanced drug delivery is the ability to generate reproducible near-monodisperse polymer nanospheres having the desired matrix structure and surface morphology (Bhatt and Shah 2012; Sackmann et al. 2014; Zhang et al. 2012). Solid polymeric nanospheres have received considerable attention due to their potential applications. These include therapeutic agents, such as proteins, genes and drugs (Bourges et al. 2003; Capretto et al. 2012; de Jalón et al. 2001; Hall et al. 2007; Mundargi et al. 2008), disease detection and therapy (Byrne et al. 2008), multimodal contrast enhancement (Kim et al. 2010; Pisani et al. 2008; Schneider et al. 1992; Xu et al. 2009), cell/enzyme experiments, targeted therapeutic applications (Fernandez-Fernandez et al. 2011; Gao et al. 2008; Xu et al. 2011), chemical reagents (Meier 2000; Yu et al. 2011) and controlled delivery (Zhang et al. 2012). In order to conceive polymer nanospheres with a desired structure, numerous techniques including emulsion polymerization, suspension polymerization (Jahn et al. 2008; Liu et al. 2010; Shestopalov et al. 2004; Song et al. 2006), spray drying (Vehring 2008), phase separation (Chan et al. 2005; Chang et al. 2010), electrohydrodynamic techniques (Eltayeb et al. 2013b; Jayasinghe et al. 2004; Nangrejo et al. 2008), self-assembly (Chan et al. 2005; Cui et al. 2011; Shestopalov et al. 2004) as well as microfluidics (Sun et al. 2013) have been developed over the past few decades.
A popular method is microfluidics widely used in the preparation of polymer nanospheres due to the fact that microfluidic technologies offer compelling advantages, including cost-effective preparation and easy and effective control of fluid flow over the other methods (Seiffert 2011; Stride et al. 2008). Several microfluidic methods with different device geometries, including T-junctions, flow focusing devices and co-flow or cross-flow capillaries for generating continuous droplets and subsequently polymer nanospheres, have been described in the literature (Dendukuri and Doyle 2009; Köhler et al. 2011; Liu and Qin 2013; Song et al. 2010; Wang 2013; Xu et al. 2012). In particular, droplet-based microfluidic methods have been widely used to prepare discrete and independently controllable droplets leading to polymer nanospheres with various geometries and polydispersity (Kamio et al. 2008; Serra and Chang 2008; Song et al. 2010).
Polymethylsilsesquioxane (PMSQ) has been used as a model micro/nanosphere material due to its interesting chemical, physical, drug release and biocompatibility properties (Quintanar-Guerrero et al. 1998; Xu et al. 2005). Studies conducted by Ye et al. (2010) using a microfluidic technique have shown that solid PMSQ microspheres 28 µm in diameter have been produced via monodisperse droplet generation. In addition, Chang et al. (2010) used the process of co-axial electrohydrodynamic atomization to prepare submicrometer capsules using PMSQ and a volatile liquid, perfluorohexane (PFH).
Solvent volatility has an influence on the preparation of polymer nanospheres with an enhanced surface roughness (Arshady 1991). In order to enhance the desired matrix structure and surface morphology, a volatile liquid, PFH has been used as an excipient in microfluidic techniques due to its very limited solubility and miscibility with organic solvents and most compounds, and very low toxicity which is preferred in the encapsulation of hydrophilic and lipophilic drugs (Kucuk et al. 2014; Mana et al. 2007). Kucuk et al. (2014) reported that having a tailored rough surface on the polymer nanospheres resulted in increased drug accessibility to the release medium and thus correlated with a higher initial burst release. It is clear that the aforementioned properties and applications confirm that PFH is a suitable excipient in terms of drug delivery requirements to generate polymeric nanospheres.
In this work backed by high speed camera footage, we used a V-shaped microfluidic junction device to generate near-monodisperse polymer nanospheres from droplets and investigated how system parameters (flow rates of PMSQ and PFH) and solution properties influenced the sphere size and surface roughness in a one-step process.
Materials and methods
PMSQ powder, average molecular weight 7,465 g mol−1, was purchased from Wacker Chemie AG, GmbH, Burghausen, Germany. Liquid PFH was provided by F2 Chemicals Ltd., Lea, UK (purity grade, 99.7–100 %; density, 1,710 kg m−1). Ethanol was procured from the Sigma-Aldrich (Poole, UK; purity grade, 99.7–100 %; density, 790 kg m−1).
5, 10, 20, 30, 40 and 50 wt% PMSQ was dissolved in ethanol in a sealed vial for 1,800 s at ambient temperature (23 ± 2 °C), using a magnetic stirrer.
Characterisation of solutions
The standard data sheet of F2 Chemicals Ltd. provided the physical properties of PFH. The polymer solutions were characterised to measure surface tension, viscosity and density using calibrated equipment. A VISCOEASY-L rotational viscometer (Schott GERÄTE GMBH, Germany) and an Ostwald U-tube viscometer were used to measure the viscosity. A tensiometer K9 (Kruss GmbH, Germany, standard Wilhelmy plate method) was used to determine the surface tension. A standard 25-ml density bottle was used to measure the density. All experiments were conducted at the ambient temperature (23 ± 2 °C), and ethanol was utilized as a cleaning and standardising agent prior to characterisation experiments.
Preparation of nanospheres
Optimization studies were conducted to obtain monodisperse nanospheres by varying the polymer (PMSQ) concentration (5–50 wt%), the flow rate of the PMSQ solutions and of the PFH (in the range 50–300 µl min−1). The flow processes were observed using a Phantom V7 high speed camera (provided by Engineering and Physical Science Research Council of the UK).
Characterisation of nanospheres
Droplets were observed using a Nikon Eclipse ME-600 optical microscope (Nikon Co, Tokyo, Japan) as soon as they were generated. Samples of collected spheres were left to dry for 48 h at the ambient temperature (23 ± 2 °C) in a desiccator. They were then sputter coated for 200 s to apply a thin layer of gold to prepare them for SEM imaging (5 kV). A JEOL JSM 6301 F SEM was used to characterise the size and morphology of the produced nanospheres. 200 nanospheres were studied using image analysis software (ImageJ 1.47n, Wayne Rasband National Institute of Health, USA).
Transmission electron microscopy (TEM, JEOL JEM 1010) was used to characterise the internal structure of the nanospheres. For TEM, the collected nanospheres were suspended in distilled water and placed on a copper grid (provided by Agar Scientific Ltd., Stansted, UK).
Atomic force microscopy (AFM) was used to investigate the surface of the produced nanospheres. The images were obtained by scanning the resulting spheres kept on a mica surface in air under ambient conditions using an AFM (Bruker Multimode 8.0, Santa Barbara, CA, USA; Veeco Nanoscope analysis software Version V 6.14r1) operated using the tapping mode. Dried samples were scanned by Bruker silicon nitride tips with a force constant of 0.12 N m−1 at 1 Hz with a resolution of 512 × 512 pixels for all images. To avoid structural changes of the sample, the tip loading force was minimized.
Results and discussion
Mechanism of nanosphere formation
Physical properties of PFH and various PMSQ solutions used in this work (mean ± standard deviation, n = 5)
Density (kg m−3)
Viscosity (mPa s)
Surface tension (mN m−1)
PMSQ 5 wt%
PMSQ 10 wt%
PMSQ 20 wt%
PMSQ 30 wt%
PMSQ 40 wt%
PMSQ 50 wt%
The generated encapsulated droplets stream down through the outlet capillary and were gathered in insoluble media at the channel exit (see supplementary information S2 provided). Upon making contact with an aqueous environment, (distilled water in collecting vial) sphere generation from these droplets becomes evident. Under an optical microscope at a post-collection time of approximately 100 s, the resultant droplets were approximately 120 µm in diameter (Fig. 1c). A cluster of spheres is seen on the droplet surface (Fig. 1c). Upon impact with the water in the collector, the droplet breaks up much like an explosion to release the PFH solvent, while the PMSQ coating forms nanospheres. The high density of spheres on the surface is brought about by the spontaneous bursting of the droplet surface. Evaporation of the PFH continues and the nanospheres shrink and adopt a rough surface (see supplementary information S3 provided). This stage leads to solidification. Eventually, PMSQ polymer nanospheres with diameter in the range of 80–920 nm were obtained.
Influence of polymer concentration
Effect of flow rate
Solid polymer nanospheres have been conceived using a V-shaped microfluidic junction device. The device used in this work offers a simple method to prepare nanospheres from polymeric droplets. It also enables optimization of nanosphere size. The sphere diameters obtained ranged from 80 to 920 nm, (polydispersity index: 11–19 %) and at the lowest PFH flow rate of 50 µl min−1, nanospheres of 120 nm diameter were generated. The solution properties (polymer concentration) and the process parameters, such as PMSQ solution and PFH flow rates, have a significant effect on the sphere diameter and characteristics, such as surface roughness, which is desirable for some therapeutic applications such as drug delivery. In current work, we are using other biodegradable polymer systems to make this processing and forming method even more generic and versatile. We are also working towards optimizing the process parameters in order to further control the polydispersity of the nanospheres and to prepare different types of nanospheres having internal porosity.
The authors are thankful to the Islamic Development Bank Merit Scholarship Programme for funding the PhD programme of Israfil Kucuk. The authors also thank the Engineering and Physical Science Research Council of the UK for providing the high speed camera and Mr Adrian Walker is especially thanked for his assistance. They gratefully thank Kevin Reeves for assistance with scanning electron microscopes in the Archaeology Department at UCL. They would also like to thank Jonathan Moffat and Bahijja Raimi-Abraham from the School of Pharmacy at UCL for the use of their atomic force microscope. Professor Paolo Colombo (University of Padova, Italy) is thanked for his helpful advice regarding the experimental work.
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