Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

Summary

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Abstract

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (> 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 °C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

Introduction

LTSL formulation is a clinically relevant liposomal product that has been developed to deliver the chemotherapeutic drug doxorubicin (DOX) and allows efficient burst drug release at clinically attainable mild hyperthermia (T ≈ 41 °C)¹. The LTSL formulation consists of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the lysolipid 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (MSPC; M stands for "mono") and PEGylated lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀). Upon reaching the phase transition temperature (T_m<....

Protocol

1. Equipment setup

1. Assemble the syringe pumps and SHM as follows.
   1. Connect the "To Computer" port of the secondary syringe pump (Pump 02, for aqueous solution) to the "To Network" port of the master syringe pump (Pump 01, for ethanol lipid solution) using Pump to Pump network cable (Figure 1, yellow).
   2. Connect the "To Computer" port of the master pump to the "RS232 Serial" port of the computer using PC to Pump .......

Discussion

The presented protocol describes the preparation of low temperature-sensitive liposomes (LTSLs) using a staggered herringbone micromixer (SHM). The LTSL10 formulation enables temperature-triggered burst release of doxorubicin within 5 minutes at a clinically attainable hyperthermic temperature of 42 °C. Indocyanine green (ICG) can also be co-loaded for photothermal heating triggered the release of DOX. The method relies on: (i) self-assembly of phospholipids into liposomes under a homogenized solvent environment pro.......

Materials

Name	Company	Catalog Number	Comments
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)	Lipoid	PC 16:0/16:0 (DPPC)
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000)	Lipoid	PE 18:0/18:0-PEG 2000 (MPEG 2000-DSPE)
1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC)	Avanti Polar Lipid	855775P-500MG	Distributed by Sigma-Adrich; also known as Lyso 16:0 PC (Not to be confused with 14:0/18:0 PC, which is also termed MSPC)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375-100G
Adapters, Female Luer Lock to 1/4"-28UNF	IDEX Health & Science	P-624	Requires 2 units. For the inlets
Adapters, Union Assembly, 1/4"-28UNF	IDEX Health & Science	P-630	Requires 2 units. (One unit included 2 nuts and 2 ferrules)
Ammonium Sulfate ((NH4)2SO4)	Sigma-Aldrich	31119-1KG-M
Bijou vial	VWR	216-0980	7 mL, clear, polystyrene vial
Centrifugal Filter Unit	Sigma-Aldrich	UFC801008	10 kDa MWCO, Amicon Ultra-4 Centrifugal Filter Unit
Centrifuge	ThermoFisher Scientific	Heraeus Megafuge 8R	With HIGHConic III Fixed Angle Rotor
Cuvette	Fisher Scientific	11602609	Disposable polystyrene cuvette, low volume, for DLS measurement
Dialysis Kit - Pur-A-Lyzer Maxi	Sigma-Aldrich	PURX12015-1KT	12-14 kDa MWCO
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	34943-1L-M
DLS Instrument	Malvern Panalytical	Zetasizer Nano ZS90
Doxorubicin Hydrochloride (DOX)	Apollo Scientific	BID0120
DSC Instrument	TA Instruments	TA Q200 DSC
DSC Tzero Hermetic Lids	TA Instruments	901684.901	For DSC measurement
DSC Tzero Pans	TA Instruments	901683.901	For DSC measurement
DSC Tzero Sample Press Kit	TA Instruments	901600.901	For DSC measurement
Ethanol	VWR	20821.330	Absolute, ≥99.8%
FC-808 Fibre Coupled Laser System	CNI Optoelectronics Tech	FC-808-8W-181315	FOC-01-B Fiber Collimator included.
Ferrule, 1/4"-28UNF to 1/16" OD	IDEX Health & Science	P-200	For the outlet
Fibre Optic Temperature Probe	Osensa	PRB-G40
Glass Staggered Herringbone Micromixer (SHM)	Darwin Microfluidics	Herringbone Mixer - Glass Chip
Heating Tape	Omega	DHT052020LD	Can be replaced by other syringe heater such as "HTC" or "SRT series" for slower heating. Manual wiring to a 3-pin plug required for 240V models
Indocyanine Green	Adooq	A10473-100	Distributed by Bioquote Limited (U.K.)
Luer-lock Syringe, 5 mL	VWR	613-2043	Hanke Sass Wolf SOFT-JECT 3-piece syringes, O.D. 12.45 mm
Microplate Reader	BMG Labtech	FLUOstar Omega	Installed with 485 nm (exictation) and 590 nm (emission) filters
Microplate, 96-well, Black, Flat-bottom	ThermoFisher Scientific	611F96BK	For fluorescence measurement in microplate reader
Microplate, 96-well, Clear, Flat-bottom	Grenier	655101	For absorbance measurement microplate reader
Nut, 1/4"-28UNF to 1/16" OD	IDEX Health & Science	P-245	For the outlet
PC to Pump Network Cable for Aladdin, 7ft	World Precision Instruments	NE-PC7	Optional: Syringe pumps can be operated manually
Pump control software - SyringePumpPro Software License for 2	World Precision Instruments	SYRINGE-PUMP-PRO-02	Optional: Syringe pumps can be operated manually
Pump to Pump Network Cable for Aladdin, 7 ft	World Precision Instruments	NE-NET7	Optional: Syringe pumps can be operated manually
Size exclusion chromatography (SEC) column	GE Life Science	17085101	Sephadex G-25 resin in PD-10 Desalting Columns
Sodium chloride (NaCl)	Sigma-Aldrich	31434-1KG-M
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881-500G
Syringe Pumps & Cable (DUAL-PUMP-NE-1000)	World Precision Instruments	ALADDIN2-220/AL1000-220
Thermostat Temperature Controller	Inkbird	ITC-308	Can be replaced by other syringe heater kit/thermostat
Triton X-100	Sigma-Aldrich	X100-100ML
Tubing, ETFE (1/16" OD)	IDEX Health & Science	1516
USB To RS-232 Converter	World Precision Instruments	CBL-USB-232	Optional: For computer without RS-232 port
Water Bath	Grant Instruments Ltd.	JB Nova 12