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Particle Size Analyses of Polydisperse Liposome Formulations with Multispectral Nanoparticle Tracking Analysis Pushpendra Singh October 16, 2019 The views expressed are those of the authors and should not be construed to represent the positions

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The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army or the DoD.

Particle Size Analyses of Polydisperse Liposome Formulations with Multispectral Nanoparticle Tracking Analysis Pushpendra Singh

October 16, 2019

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2

Outline

  • Liposomes and their application in medicine
  • Liposome manufacturing process
  • Significance of particle size distribution for liposomes
  • Challenges in particle size characterization
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3 Sercombe et al., Front. Pharmacol. 2015, 6: 286

  • Heavily being used in pharmaceutical industry as drug delivery system and vaccine

Liposomes in Medicine

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Liposomes in Medicine

4

  • Specialized

delivery vehicles that mimic biological membranes

  • Shield a drug from degradation and increase its lifespan in

circulation

  • Possess the ability of encapsulation of hydrophilic drug as

well as capability of solubilizing lipophilic drug Modulate pharmacokinetics and biodistribution of drug to minimize side effects

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http://www.horiba.com

Liposome

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  • Drying of Lipids
  • Lipid Hydration
  • Downsizing of Lipid Vesicles

Liposome Manufacturing Process

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7 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs

Liposome Manufacturing Process

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8 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs

Liposome Manufacturing Process

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9 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs

Liposome Manufacturing Process

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10 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs

Liposome Manufacturing Process

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11 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs Downsizing

Liposome Manufacturing Process

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12 Sharma and Verma , Int. J. Green Pharm. 2017, 6: 286 Lipids in organic solvent Rotary Evaporator Aqueous Solution Dried Lipid Film Agitation MLVs LUVs/SUVs Downsizing Filtration

Liposome Manufacturing Process

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  • Sonication
  • Extrusion
  • Microfluidization

Downsizing Methods

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Microfluidization

http://www.horiba.com

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Hydrated multilamellar vesicles Small size liposomes

Liposome Size Distribution

http://www.horiba.com

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Liposomes in Vaccines

16

  • Adjuvant: Enhance and direct the adaptative

immune response to vaccine antigens

  • Our lab created Army Liposome Formulation (ALF)

that contains various immunostimulants (e.g., Lipid A, QS21 and Aluminum salt)

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Adapted from Pasquale et al., Vaccine 2015, 3: 320-343

Development of vaccine design

17

Live Pathogen Attenuated Pathogen Recombinant Immunogen

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Licensed Vaccines

2017

Shingrix

18 Adapted from Pasquale et al., Vaccine 2015, 3: 320-343

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Experimental vaccine:

  • Malaria
  • Ricin
  • Ebola
  • Anthrax
  • HIV
  • Dengue
  • Cancer

Human Studies Vaccines: 14 Phase I & 2 Phase II

  • Malaria
  • HIV
  • Cancer
  • Meningococcus

ALF in Vaccines

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  • Dimyristoyl phosphatidylcholine (DMPC)
  • Dimyristoyl phosphatidylglycerol (DMPG)
  • Cholesterol
  • Monophosphoryl lipid A (MPLA)

Core composition of ALF

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ALF: Liposomes with MPLA ALFQ: ALF + QS21 ALFA: ALF + aluminum salt ALFQA: ALFQ + aluminum salt

Types of ALF

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Rationale

To establish a stand-alone method for particle size characterization

  • f

polydisperse liposomal adjuvants

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Why care about particle size?

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Why care about particle size?

Moyer et al., J Clin. Invest. 2016,126: 799-808

Biodistribution and Kinetics

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Moyer et al., J Clin. Invest. 2016,126: 799-808

Biodistribution and Kinetics

Why care about particle size?

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Moyer et al., J Clin. Invest. 2016,126: 799-808

Processivity Biodistribution and Kinetics

Why care about particle size?

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ALF: size vs. immune response

Endpoint Titer

Beck et al., J. Control Release 2018, 275: 12-19

Monodisperse Polydisperse

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Challenges of particle sizing methods

Dynamic Light Scattering (DLS) Laser Diffraction (LD) Nanoparticle Tracking Analysis (NTA)

Autocorrelation of scattered intensities to deduce average particle size

  • Range 30 -10000 nm
  • does not work on polydisperse sample

Narrow and wide angle scattering to deduce average particle size

  • Range 10 nm - 5 mm
  • does not work on polydisperse sample

Tracking of each particle undergoing Brownian (random) motion to determine particle size

  • Small size range 30 - 1000 nm
  • works on polydisperse sample
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ViewSizer

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Manzo and Garcia-Parajo Rep. Prog. Phys. 2015, 78: 124601 Ernst and Kohler Phys. Chem. Chem. Phys., 2013,15: 845-849

How does it work?

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Particle size distribution by ViewSizer

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Alone Mix

Singh et al., Int. J. Pharm. 2019, 566: 680-686

Size analysis of standards

DLS LD NTA MANTA

PSL beads ….. 70 nm

  • ---- 200 nm

.-.-. 600 nm 1300 nm

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Alone Mix

PSL beads ….. 70 nm

  • ---- 200 nm

.-.-. 600 nm 1300 nm

DLS LD NTA MANTA

Size analysis of standards

Singh et al., Int. J. Pharm. 2019, 566: 680-686

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Alone Mix

PSL beads ….. 70 nm

  • ---- 200 nm

.-.-. 600 nm 1300 nm

DLS LD NTA MANTA

Size analysis of standards

Singh et al., Int. J. Pharm. 2019, 566: 680-686

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Alone Mix

PSL beads ….. 70 nm

  • ---- 200 nm

.-.-. 600 nm 1300 nm

DLS LD NTA MANTA

Size analysis of standards

Singh et al., Int. J. Pharm. 2019, 566: 680-686

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Liposomes

  • Lwet:

Uniform (monodisperse) small sized liposomes manufactured by microfluidization

  • Llyo: Heterogenous (polydisperse) sized

liposomes nano to micron size

36

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Particle size distribution by ViewSizer

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Linear range of sensitivity

LYO

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Snapshots

Lwet Monodisperse Llyo Polydisperse

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Liposome size distribution in Lwet

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Liposome size distribution in Llyo

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DLS LD NTA MANTA

  • ----- Lwet

 Llyo

Size analysis of liposomes

Singh et al., Int. J. Pharm. 2019, 566: 680-686

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DLS LD NTA MANTA

  • ----- Lwet

 Llyo

Singh et al., Int. J. Pharm. 2019, 566: 680-686

Size analysis of liposomes

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Summary

  • Multispectral

Nanoparticle Tracking Analysis can measure particle size 50 - 2000 nm in polydisperse liposomes

  • Lyophilized liposome formulation contain

>90% particles in the range of 500 - 1500 nm

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Acknowledgements

LAAR Gary Matyas Zoltan Beck WRAIR Nelson Michael Robert Gramzinski LTC(P) Julie Ake HJF Sandhya Vasan Merlin Robb HORIBA Jeffrey Bodycomb

Kuba Tatarkiewicz

ANATOM Bill Travers Sean Travers

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Publication

Singh, P., Matyas, G.R., Beck, Z. (2019) Particle Size Analyses

  • f

Polydisperse Liposome Formulations with a Novel Advanced Nanoparticle Tracking Technology.

  • Int. J. Pharm. 566: 680-686.

doi: 10.1016/j.ijpharm.2019.06.013.

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Thank You

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www.mantainc.com

Particle size distribution by ViewSizer

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Particle size distribution by ViewSizer

www.mantainc.com