PrincetonUniversity

Nanomedicine: from high tech to global health

Robert K. Prud’homme, Brian Johnson,Walid Saad, Ying Liu, Marian Gindy, Stephanie Budijono, Margarita Herrera-Alonso,Varun Kumar,Suzanne D’Addio, Robby Pagels,

Chet Markwalter, Brian WilsonDept. Chemical Engineering

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Support: NSF, NIH, Gates Foundation, BASF, Evonik, Merck, J&J, GSK, Celator, Optimeos; Nanomedicine for BBB-crossing in CNS oncologic pathologies MAECI PROJECT 2019-2021

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Next Generation Nanoparticles (NPs)

Tumor vasculature

Nanoparticles

11 µm

“…over the next few years some of the complex theranostic strategies published rampantly in chemistry journals will fall out of contention…For me, something that’s too difficult to make or too complex to sustain in large-scale production is not what we are interested in.”

J.Janijic C&EN Sept 26,2011.

“…over the next few years some of the complex theranostic strategies published rampantly in chemistry journals will fall out of contention…For me, something that’s too difficult to make or too complex to sustain in large-scale production is not what we are interested in.”

J.Janijic C&EN Sept 26,2011.

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Block copolymer directed rapid precipitation

Nanoparticle formation by Flash NanoPrecipitation

Non solvent

Copolymer Solute Solvent

DeadMicelle

PROTECTEDNANO

PARTICLE

tmix

Micellizationtagg

tngNucleationand Growth

Johnson, Prud’homme AICHE J (2003), Liu, Prud’homme, Fox, Chem Engr. Sci. (2008)

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• Control super -saturation by changing solvent quality or solute concentration.

• Higher super saturation leads to smaller particles

Control of particle size

0 100 200 300 400 500 60050

100

150

200

250

300 1:1 H2O:THF 2:1 H2O:THF 3:1 H2O:THF 5:1 H2O:THF

Parti

cle D

iam

eter

(nm

)

Average Re

1. Mixing intensity

2. Super-saturation

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NP Formation: PS to PLA NPsWant to shift from model polystyrene (PS) to biocompatible polylactic acid (PLA). How does size depend on species in core?

• What is in the core doesn’t matter

• The ratio of core to BCP matters, Ccore /CBCP

• The total mass matters

PS-b-PEG

PS-b-PEG

PLA-b-PEG

PLA-b-PEG

Pagels Nano Letters 18 1139-1144 (2018)

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NP Formation: PS to PLA NPsWant to shift from model polystyrene (PS) to biocompatible polylactic acid (PLA). How does size depend on species in core?

• What is in the core doesn’t matter

• The ratio of core to BCP matters, Ccore /CBCP

• The total mass mattersPagels Nano Letters 18 1139-1144 (2018)

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Drugs ConsideredClofazimine

Ref: Design and Solidification of Fast-Releasing Clofazimine Nanoparticles for Treatment of Cryptosporidiosis”. Molecular Pharmaceutics. 14(10) 3480-3488 (2017); “Rapid Recovery of Clofazimine-loaded Nanoparticles with Long-term Storage Stability as Anti-Cryptosporidium Therapy”. ACS Applied Nano Materials. 1(5) 2184-2194 (2018)

OZ439

Ref: “Encapsulation of OZ439 into Nanoparticles for Supersaturated Drug Release in Oral Malaria Therapy”. ACS infectious diseases. 4(6) 970-979 (2018); “Spray drying OZ439 nanoparticles to form stable, water-dispersible powders for oral malaria therapy”. Journal of Translational Medicine (2019), just accepted

Lumefantrine

Ref: “Amorphous Nanoparticles by Self-assembly: Processing for Controlled Release of Hydrophobic Molecules”. Soft Matter. 15 2400-2410 (2019)

Delamanid

Ref:https://app.box.com/s/lvghy956h7pmzvdd1nlj9zzeotabijke

Ecumicin

Ref:https://app.box.com/s/5lggnm4tv84kcb0cry0tscby9oszzqzi

TBA 354

Ref:https://app.box.com/s/57cromgxhg8uyqiz4zegtafwqbclg3nk

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Clofazimine – Zein/casein

Direct spray dryingredisperses to NP

Mol Pharm (2017)

40X higher dissolution in GF

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Long-term Zein Clofazimine NPs

Day FaSSIF FeSSIF

0 5.4 ug/mL 5.4 ug/mL

1 4.5 ug/mL 4.8 ug/mL

3 4.7 ug/mL 5.9 ug/mL

7 6.2 ug/mL 5.6 ug/mL

14 4.6 ug/mL 5.1 ug/mL

• Open vial: 75 % RH, 50 C

• Have gone to 28 da +

• FeSSIF: influence was notsignificant

0

20

40

60

80

100

120

140

0 50 100 150 200 250 300 350 400

Rele

ase

(%)

Time (min)

FaSSIF Day 0

Day 1

Day 3

Day 7

Day 14

0

20

40

60

80

100

120

0 50 100 150 200 250 300 350 400

Rele

ase

(%)

Time (min)

FeSSIF

Fasted Intestinal Fluid

Feasted Intestinal Fluid

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siRNA for gene silencing

•siRNA complex cleaves mRNA and stops protein expression

•Problem is delivery

+

buffersiRNA

ethanolcationic lipid

structural lipid

+

+++

++ +

+

+

•Successful prep of 100 nm PEG protected siRNA

•Stable and transfect efficiently

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1 mL batch

1.5 L/min 5 L/min

CIJConfined Impinging Jet

MIVMMulti Inlet Vortex Mixer

Micro-MIVMµ−Multi Inlet Vortex Mixer

Markwalter, et al JOVE 143 (2019), J Pharm Sci 107 (2018); Feng, J Trans Med (2019)

Gates: 3700 tons/yr

Four Scales of CIJ & MIVM

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Equivalent NP formation vs Reynolds No.

Similar NPs produce from batch (1 ml scale) to 5 L/min

Feng, et al. J. Translational Medicine (2020)

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Active targeting

Small molecule:(mannose, folate, LHRH)

• Conjugate targeting group to block copolymer• Do hard chemistry and characterization on

conjugate• Quantitatively assemble targeted NP with

control on % targeting groups• Conjugate chemistry: click chemistry, maleimide,

carbodiimide, carbamate

Gindy, Biomaterials (2008)Akbulut Adv Fn Mat (2009)

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Variable domain antibody targeting

ELN

–N

anop

artic

les 2

013

-000

48

Blank Cells

W26A-NPs (300 ug/mL)

•14K fibrinogen peptide scaffold (Centryin)

•Pre functionalize BCP•Assemble NP

Control (PEG NP)

5% ligand targeted

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Inverse Flash NanoPrecipitation (iFNP)

Precipitation process for the encapsulation of hydrophilic therapeutics

iFNP uses a non-polar antisolvent to produce NPs with a water-

soluble core:

iFNP has been demonstrated on a variety of water-soluble molecules:

All ≥ 50% loading Other encapsulated molecules include: polysaccharides, insulin,

ovalbumin, peroxidase, Trp cage , mRNA

Pagels & Prud’homme, J. Cont. Release (2015)Markwalter, et al. AAPS (2020)

20

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Unique Technology Capability: RNA Encapsulation

mRNA has been encapsulated (50% loading) into inverted nanoparticles by iFNP

Cationic lipids are not required for encapsulation of RNA –different from currently available technologies

Model RNA used: RNA from Torula utilis, Mr 5,000-8,000 Da (15-25 nucleotides)

0

2

4

6

8

10

12

14

16

1 10 100 1000

Inte

nsity

Diameter, nm

50 wt% RNA

PK1 avg: 40 nmPDI: 0.24

0

0.5

1

1.5

2

2.5

3

230 250 270 290 310 330 350

Abso

rban

ce, A

.U.

Wavelength, nm

Solvent blankRNA controlNPs

95% encapsulation efficiency

RNA control – no

polymer

RNA encapsulated

by iFNP

RNA precipitate (left) and nanoparticles (right) in

CHCl3

Particle size distribution Encapsulated RNA is protected from extraction into an

aqueous phase

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Unique Technology Capability Co-Encapsulation

RNA (33 wt% loading) and a model protein (33 wt% loading)

0

5

10

15

20

10 100 1000 10000

Inte

nsity

Diameter, nm

RNA + HRPNPs

0.0

0.5

1.0

1.5

2.0

2.5

3.0

225275325375425Wavelength, nm

RNA +HRPRNA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

225275325375425

Abso

raba

nce

Wavelength, nm

Brine phase

Organic NP phase

Nanoparticle Size Distribution Nanoparticle Encapsulation Efficiency

RNA

HRPHorseradish peroxidase,

44 kDa

PLA-b-PAsp

Unencapsulated Encapsulated

Nanoparticle Composition, wt%

Encapsulation Efficiency

RNA HRP Polymer RNA HRPRNA NPs. 50% -- 50% 92% --HRP NPs. -- 50% 50% -- 99%

RNA + HRP NPs.33.3% 33.3% 33.3% 93% 99%

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Cu-64 PET/CT imaging allows tracking of biodistribution in vivo

23

Neutral PEG coating leads to tumor accumulation

3 hours post injection 24 hours post injection

Negative coating promotes immune cell interactions

24 hours post injection

For Discussion with Spark Therapeutics Only Please Do Not Distribute

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Summary (prudhomm@Princeton.edu)

Flash NanoPrecipitation (FNP) Polymer-directed, rapid precipitation process Hydrophobic drugs, and drugs made hydrophobic by ion pairing Scaleable: mg to 1000 kg Enables targeting incorporation and imaging (PET, fluorescence, MRI)

Inverse Flash NanoPrecipitation (iFNP) Polymer-directed, two step rapid precipitation process Hydrophilic peptides, proteins, mRNA, DNA High loading (20-50%), high encapsulation efficiency (80-99%) Makes nanoparticles, or microparticles for depot delivery (2 mo release)