(657d) Scaleable Production of Long Wavelength Fluorescent Nanoparticles to Enable Targeting and Multiplexed Imaging

Authors: 
Prud'homme, R. K. - Presenter, Princeton University

Long wavelength (NIR) fluorescent dyes enable deep tissue penetration and avoidance of

tissue autofluorescence. In diagnostic applications and therapeutic drug delivery,

targeting is often required. Nanoparticles offer advantages in targeted delivery because

avidity of binding can be enhanced by presenting multiple copies of the targeting ligand

on the nanoparticle surface. The combination of long wavelength fluorescence and

targeting presents challenges in the production and scaleup of nanoparticles with

repeatable and quantifiable characteristics. We present a novel technology --Flash

NanoPrecipitation – a controlled precipitation process that produces stable nanoparticles

at high concentrations of encapsulated components using amphiphilic block copolymers

to direct self-assembly. Uniform particles with tunable sizes from 50 – 500 nm can be

prepared in an economical, scalable, and reliable manner.[2] The key to the process is the

control of time scales for micromixing, self-assembly, and nucleation and growth.

Stoichiometric encapsulation of components enables the assembly of complex

nanoparticles with tailored optical and targeting properties.

Most bio-imaging, even for long wavelength dyes, has employed aqueous dyes that are

conjugated onto the surfaces of nanoparticles. The long wavelength dyes are intrinsically

large, conjugated structures and significant surface attachment may interfere with

targeting. Instead we employ extremely hydrophobic dyes that remain sequestered in the

cores. This enables higher loadings than are achievable with surface-attached dyes. Also,

we show that several dyes which the community considers “hydrophobic” such as Nile

red or ICG, partition out of nanoparticle cores to other lipid compartments and do not act

as true reporters of the nanoparticle concentration or location. We report on two novel

classes of NIR dyes: a hexacene based dye that has been first used in photo-voltaic

applications, and a series of chlorin and bacteriochlorin dyes from Nirvana Sciences. The

later dyes are particularly interesting because the wavelengths can be tuned at ~10nm

intervals over the NIR window as shown in Fig. 1. The native form of these dyes is

hydrophobic, so they are ideally suited for encapsulation in our nanoparticle constructs.

The narrow emission spectra will allow multiplexing of these dyes so that multiple

populations of dyes with different targeting ligands can be injected simultaneously and

their location/fate in vivo can be ascertained.

We present targeting studies with these longwavelength

dyes to show ligand density can

be easily varied to determine optimum ligand

concentration on the nanoparticle surface.[3]

1. Taniguchi, M., et al., Accessing the near-infrared spectral region with stable,

synthetic, wavelength-tunable bacteriochlorins. New Journal of Chemistry, 2008.

32(6): p. 947-958.

2. Akbulut, M., et al., Generic Method of Preparing Multifunctional Fluorescent

Nanoparticles Using Flash NanoPrecipitation. Advanced Functional Materials,

2009. 19(5): p. 718-725.

3. D'Addio, S.M., et al., Optimization of cell receptor-specific targeting through

multivalent surface decoration of polymeric nanocarriers. Journal of Controlled

Release, 2013. 168(1): p. 41-49.Long wavelength (NIR) fluorescent dyes enable deep tissue penetration and avoidance of

tissue autofluorescence. In diagnostic applications and therapeutic drug delivery,

targeting is often required. Nanoparticles offer advantages in targeted delivery because

avidity of binding can be enhanced by presenting multiple copies of the targeting ligand

on the nanoparticle surface. The combination of long wavelength fluorescence and

targeting presents challenges in the production and scaleup of nanoparticles with

repeatable and quantifiable characteristics. We present a novel technology --Flash

NanoPrecipitation – a controlled precipitation process that produces stable nanoparticles

at high concentrations of encapsulated components using amphiphilic block copolymers

to direct self-assembly. Uniform particles with tunable sizes from 50 – 500 nm can be

prepared in an economical, scalable, and reliable manner.[2] The key to the process is the

control of time scales for micromixing, self-assembly, and nucleation and growth.

Stoichiometric encapsulation of components enables the assembly of complex

nanoparticles with tailored optical and targeting properties.

Most bio-imaging, even for long wavelength dyes, has employed aqueous dyes that are

conjugated onto the surfaces of nanoparticles. The long wavelength dyes are intrinsically

large, conjugated structures and significant surface attachment may interfere with

targeting. Instead we employ extremely hydrophobic dyes that remain sequestered in the

cores. This enables higher loadings than are achievable with surface-attached dyes. Also,

we show that several dyes which the community considers “hydrophobic” such as Nile

red or ICG, partition out of nanoparticle cores to other lipid compartments and do not act

as true reporters of the nanoparticle concentration or location. We report on two novel

classes of NIR dyes: a hexacene based dye that has been first used in photo-voltaic

applications, and a series of chlorin and bacteriochlorin dyes from Nirvana Sciences. The

later dyes are particularly interesting because the wavelengths can be tuned at ~10nm

intervals over the NIR window as shown in Fig. 1. The native form of these dyes is

hydrophobic, so they are ideally suited for encapsulation in our nanoparticle constructs.

The narrow emission spectra will allow multiplexing of these dyes so that multiple

populations of dyes with different targeting ligands can be injected simultaneously and

their location/fate in vivo can be ascertained.

We present targeting studies with these longwavelength

dyes to show ligand density can

be easily varied to determine optimum ligand

concentration on the nanoparticle surface.[3]

1. Taniguchi, M., et al., Accessing the near-infrared spectral region with stable,

synthetic, wavelength-tunable bacteriochlorins. New Journal of Chemistry, 2008.

32(6): p. 947-958.

2. Akbulut, M., et al., Generic Method of Preparing Multifunctional Fluorescent

Nanoparticles Using Flash NanoPrecipitation. Advanced Functional Materials,

2009. 19(5): p. 718-725.

3. D'Addio, S.M., et al., Optimization of cell receptor-specific targeting through

multivalent surface decoration of polymeric nanocarriers. Journal of Controlled

Release, 2013. 168(1): p. 41-49.

Figure