Custom siRNA/ASO-LNP | RNA Medicine Core

Custom siRNA-LNP/ASO-LNP Synthesis

siRNA-LNP Synthesis involves the formulation of custom lipid nanoparticles (LNPs) for the delivery of small interfering RNA (siRNA) and antisense oligonucleotide (ASO) molecules. We employ specialized equipment such as the Lipid Nanoparticle Synthesis Pack from Elveflow to produce LNPs with high encapsulation efficiency and low toxicity. The facility also employs cutting-edge instruments for siRNA-LNP analysis and characterization. These include, but are not limited to: 1) Analytical UPLC with PDA, ELS, and MS detectors, ideal for LNP formulation quality and quantitative analysis. 2)Dynamic Light Scattering (DLS) systems, powerful instruments for particle size and zeta potential measurement. 3)Quantitative PCR (qPCR), which is used for a variety of genomic research applications.

siRNA-LNP/ASO-LNPs QC

Lipid Nanoparticle Synthesis Pack

The lipid nanoparticle Pack offered by Elveflow is designed for generation of lipid nanoparticles (LNP) using microfluidics techniques. Thanks to the pressure-driven flow control system, a wide range of flow rates (TFR) – from µL/min to 10s of mL/min – can accommodate both low (µL) and large (L) volume production. This versatile platform can be easily scaled up to meet your requirements from the screening to the production stage.

Specifications and Features:

Reduced mixing time
Increased homogeneity
High monodispersity (polydispersity index (PDI) lower than 0.2;
Continuous production and high throughput
Nanoparticle production integration and automatisation
Wide range of monodisperse nanoparticle sizes possible due to precise flow rate control
Excellent repeatability from one run to another
Possibility of working with both small (μL) and large (L) volumes using the same system.

ACQUITY® UPLC System with PDA eλ, ELS Detectors, or MS Dectector

Ultra performance liquid chromatography (UPLC) is a significant advancement over the conventional high performance liquid chromatography (HPLC) that achieves dramatic increases in resolution, speed and sensitivity in liquid chromatography. The UPLC technology allows users to work at higher efficiencies with a much wider range of linear velocities, flow rates, and backpressures.

Specifications and Features:

The PDA eλ detector:
- Wavelength range: 190 to 800 nm
- PDA signal linearity range: up to 2.0 AU
- Simultaneous 2D and 3D operation
- Able to quantitate trace impurities

The ELS detector:
- 60° scatter angle detection to minimize stray light and optics polarization for long-term optimal sensitivity
- Nanogram-level detection limits
- Improved detection for semi-volatile compounds
- Able to detect lipids

Optimized MS/MS detection
MS ionization options:
- Electrospray and multi-mode ionization
- Atmospheric chemical ionization
- Dual-mode atmospheric pressure photo-ionization / atmospheric chemical ionization interface;

The facility is equipped with two UPLC systems from Waters. The ACQUITY® UPLC system with PDA eλ and TQD detectors allows users to characterize their formulations with 2D/3D spectroscopy light absorbance as well as their molecular weight. The PDA eλ detector allows users to detect and quantify lower concentrations of sample analytes and compare spectra across a broad wavelength range, while the TQD detector features the highest levels of tandem quadrupole MS selectivity, robustness, speed, and accuracy. The second ACQUITY® UPLC system with PDA eλ, and evaporative light scattering (ELS) and detectors allows users to characterize their formulations with 2D/3D spectroscopy light absorbance, improved lipid detection. The ELS detector complements the PDA eλ detector by providing enhanced sensitivity for the detection of semi-volatile components and low peak dispersion such as lipid-based nanomedicine.

Zetasizer Pro-Red

The Zetasizer Pro-Red is a popular dynamic light scattering (DLS) system from Malvern. It is an easy-to-use system and is commonly used to measure the size (hydrodynamic diameter), polydispersity, molecular weight and zeta potential of the sample.

Measurement modes:

Size measurement
Zeta potential measurement
Molecular weight measurement
Kinetics and trend measurements

Specifications and Features:

Size range: 0.3nm to 10 um
Zeta potential range: -150 to 150 mV
Molecular weight range: 1000 to 2 x 107 Daltons
Laser wavelength: He-Ne 633 nm
Sample volume: 12 to 500 μL for size, 1mL for zeta potential
Temperature range: 2 to 90 °C

DelsaMax™ Pro Particle Characterization Instrument

One of the fastest nanoparticle analyzers, the DelsaMax™ Pro from Beckman Coulter allows for the simultaneous analysis of both particle size and zeta potential for sample volumes as small as 45 μl, in less than one second. With an unprecedented 32 detectors working simultaneously, the instrument provides much reduced run time and increased measurement accuracy.

Specifications and Features:

Size range: 0.4 to 10,000 nm, hydrodynamic diameter
Molar mass range: 5×107 g/mol (Da)
Minimum sample volume: 45 μL (170 μL for zeta potential)
Minimum measurement time: 1 second
Ion strength range: 0 to 50 mS/cm
Mobility range: no practical limit

Evaluation of gene expression or gene knock down level Real-Time PCR (qPCR)

Real-time polymerase chain reaction (real-time PCR), or quantitative PCR (qPCR), is a versatile technique that is used to amplify and simultaneously detect and quantify target nucleic acid sequences.

The StepOnePlus™ real-time PCR system from Applied Biosystems is a 96-well real-time PCR instrument perfect for both first-time and experienced users. With intuitive data analysis and instrument control software, this system is designed to deliver precise, quantitative real-time PCR results for a variety of genomic research applications.

For messenger RNA detection, reverse transcription is required prior to qPCR. The Veriti 96-Well Thermal Cycler from Applied Biosystems is a versatile instrument that provides such feature. In addition, this thermal cycler allows for fast RT-PCR for users who are unable to perform qPCR on their samples.