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. 2013 Jun;30(6):1597-607.
doi: 10.1007/s11095-013-0998-3. Epub 2013 Feb 6.

Microfluidic synthesis of PEG- and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers

Renee R Hood  1 , Chenren Shao, Donna M Omiatek, Wyatt N Vreeland, Don L DeVoe
Affiliations

Microfluidic synthesis of PEG- and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers

Renee R Hood et al. Pharm Res. 2013 Jun.

Abstract

Purpose: A microfluidic hydrodynamic flow focusing technique enabling the formation of small and nearly monodisperse liposomes is investigated for continuous-flow synthesis of poly(ethylene glycol) (PEG)-modified and PEG-folate-functionalized liposomes for targeted drug delivery.

Methods: Controlled laminar flow in thermoplastic microfluidic devices facilitated liposome self-assembly from initial lipid compositions including lipid/cholesterol mixtures containing PEG-lipid and folate-PEG-lipid conjugates. Relationships among flow conditions, lipid composition, and liposome size were evaluated; their impact on PEG and folate incorporation were determined through a combination of UV-vis absorbance measurements and characterization of liposome zeta potential.

Results: PEG and folate were successfully incorporated into microfluidic-synthesized liposomes over the full range of liposome sizes studied. Efficiency of PEG-lipid incorporation was inversely correlated with liposome diameter. Folate-lipid was effectively integrated into liposomes at various flow conditions.

Conclusions: Liposomes incorporating relatively large PEG-modified and folate-PEG-modified lipids were successfully synthesized using the microfluidic flow focusing platform, providing a simple, low cost, rapid method for preparing functionalized liposomes. Relationships between preparation conditions and PEG or folate-PEG functionalization have been elucidated, providing insight into the process and defining paths for optimization of the microfluidic method toward the formation of functionalized liposomes for pharmaceutical applications.

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Figures

Figure 1
Figure 1
(a) Photograph of the thermoplastic liposome synthesis chip, and (b) numerical simulation of hydrodynamic flow focusing in the microfluidic device, illustrating the diminishing mole fraction of ethanol along the center of channel as the alcohol (red) and aqueous buffer (blue) streams interact at a flow rate ratio of 70 within a 190 μm wide microchannel.
Figure 2
Figure 2
Size distributions for liposomes composed of 0%, 5%, and 10% PEG-PE at each FRR. With increased flow focusing (higher FRR values), the diameters of the liposomes decrease in size. This trend is seen across all populations of liposomes, as well as the decrease in average size of liposome across the different lipid compositions at each FRR.
Figure 3
Figure 3
Average geometric diameters of liposomes synthesized from lipid solutions with 0%, 5%, and 10% PEG-PE. Error bars are derived from the differential distribution of each liposome population, taken as the full width at half maximum divided by the modal diameter as seen in Figure 2.
Figure 4
Figure 4
Zeta potentials of 0%, 5%, and 10% PEG-PE liposomes. The absolute value of zeta potential decreased with increasing PEG-PE content at each given size, indicating the shielding effect of DCP, an anionic surfactant included in the liposomes, by the PEG molecules on the exterior of the liposomes.
Figure 5
Figure 5
Absorption measurements at λ=520 nm performed in line with AF4 for 0%, 5%, and 10% PEG-PE liposomes. Each datum reflects the integrated absorbance intensity over a 3 min elution period centered on the liposome elution peak. Liposomes with 5% and 10% PEG-PE show significantly enhanced absorbance due to the presence of PEG on the liposomes.
Figure 6
Figure 6
Cryo-TEM images of liposomes formed using a lipid solution containing (a) 0% and (b) 10% PEG-lipid. Imaging results confirm the formation of unilamellar vesicles absent micelles or aggregates in both cases. Small ice crystals or artifacts that commonly form during vitrification, sample transfer, or elevation of specimen temperature due to the electron beam during cryo-TEM imaging seen in several images [28,29] do not reflect the presence of lipid aggregates.
Figure 7
Figure 7
UV-vis absorption data in line with AF4 from folate (10% PEG-PE + 2% folate-PEG-PE) liposome samples, normalized to control (0% PEG-PE + 0% folate-PEG-PE) liposomes. Experimental values of liposome folate content are overall in agreement with theoretical values.
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