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NANOMEDICINE

Update: 2017-01-15 15:03      View:

1.     Characterization of Doxorubicin-Carrying Liposomes

 

Introduction

In nanomedicine development, nanoparticles are used as carriers to deliver payloads such as therapeutic agents; thus, the simultaneous detection of both nanoparticles and their cargos is desirable. Doxil (doxorubicin-carrying liposomes) is the first FDA-approved nanomedicine (1995), and DLS and cryo-TEM are the two most commonly used methods for size analysis. While DLS is not appropriate for heterogeneous samples, the three-dimensional (3D) reconstruction of cryo-TEM usually takes 2-3 days. Doxoves, a research-grade product of PEGylated liposomal doxorubicin whose physical characteristics and pharmacokinetics are comparable to those of Doxil, is analyzed as a model system. Monodisperse silica nanoparticles are used as the standard to calibrate the size measurement of the Doxoves nanoparticles based on their SS burst areas. A substantial amount of variation in both size and doxorubicin content can be observed among individual particles.

 

Instrument configuration

The Flow NanoAnalyzer is equipped with a 532 nm CW laser, and the detection channels are side scatter and orange fluorescence (PE).

 

Results


 
Figure 1. Characterization of doxorubicin-encapsulating liposomes.

 

Discussion

1.      Flow NanoAnalyzer is sufficiently sensitive to detect both the scattered light and the intrinsic fluorescence of the doxorubicin emitted from each individual liposome.
2.      Employing monodisperse silica nanoparticles with known sizes to build a standard calibration curve, the SS burst area of Doxoves is converted to particle size.
 
 

ACS Nano, 2014, 8,10998.



 

2.     Measurement of Particle Count and Fraction of siRNA Loading for Gene Delivery Systems

 

Introduction

Nanoparticle-mediated gene therapy has received considerable attention over the past two decades. Lipid nanoparticles (LNPs) encapsulating siRNA are currently the most extensively clinically validated means of enabling RNA interference. However, it is challenging to determine the precise particle count and the fraction of loaded particles. Although, in principle, particles can be counted in cryo-TEM images, the nonuniformity of LNP distribution in vitrified samples on electron microscopy grids makes it difficult to obtain a precise particle count. Moreover, the comparable size, shape, and electron density of empty and siRNA-loaded LNPs render cryo-TEM less effective in determining the fraction of siRNA loading. Flow NanoAnalyzer is used for quantitative, multi-parameter characterization of siRNA loaded LNPs. Upon fluorescent staining with SYTO 82, the fraction of siRNA loading is determined.

 

Instrument configuration

The Flow NanoAnalyzer is equipped with a 532 nm CW laser, and the detection channels are side scatter and orange fluorescence (PE).

 

Results


Figure 1. Characterization of siRNA nanomedicine after SYTO 82 staining.

 

Figure 2. Concentration measurement of unstained siRNA-loaded LNPs by internal standard method.

 

Discussion

1.      Flow NanoAnalyzer is able to detect the SS signal from single nanomedicine, and upon SYTO 82 staining, empty and siRNA-loaded LNP samples are discriminated, and the fraction of siRNA loading is calculated.
2.      Using fluorescent silica nanoparticles of a known concentration as an internal standard, the particle concentration of siRNA-loaded LNPs is measured. Based on the concentration and molecular weight (~13,500 Da) of the encapsulated siRNA, the average number of siRNA load is calculated.

ACS Nano, 2014, 8,10998.