Cellular Entry of Gold Nanoparticles Ahmad Sohrabi Kashani Research - - PowerPoint PPT Presentation

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Cellular Entry of Gold Nanoparticles Ahmad Sohrabi Kashani Research Assistant at Bio-Optical Microsystem Lab. Mechanical, Industrial and Aerospace Engineering Department December 2017 Outline Introduction Nanoparticles applications Gold

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Cellular Entry of Gold Nanoparticles

Ahmad Sohrabi Kashani Research Assistant at Bio-Optical Microsystem Lab.

Mechanical, Industrial and Aerospace Engineering Department December 2017

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Outline

 Introduction Nanoparticles applications Gold Nanoparticles Surface Plasmon Resonance Nanoparticle-based drug delivery system Cellular entry of nanoparticles  Comparative study on cellular entry of two different types of gold nanoparticles Preparation of nanoparticles Imaging techniques Results  Possible effects of nanoparticle absorbance on biophysical properties Importance of biophysical properties Various methods for biophysical characterization Classical methods, MEMS-based methods, Microfluidic Methods Suspended- microfluidic for biophysical characterization  Summary

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http://bgr.com/2014/05/05/ nanogold-paint-smartphones-biotech/

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1- Cellular Entry of Nanoparticles

Behzadi et al, Chem Sco Rev, 2017 3

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Nanoparticles types/applications

Environment biomedical Industrial Food agriculture Cancer Therapy Imaging Drug delivery Engineering Application

Cheo et al, Chem Sco Rev, 2011 http://nanogloss.com/nanoparticles

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Nanoparticles

  • Nanoparticles are small pieces of substances

between 1 to 100 nanometers having various applications

  • Nanoparticles are classified based on their

properties

Joshua A et al. 2015, The Journal of Physical of Chemistry C

Size

Optical Properties Morphology Surface charge http://tremblinguterus.blogspot.ca/ Physiochemical properties 5

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How can you see Nanoparticles?

  • We cannot see nanoparticles with regular

microscopes  Scanning Electron Microscopy (SEM)  Atomic Force Microscopy (AFM)  Transmission Electron Microscopy (TEM)

silver nanoparticles http://www.nanoscop y.net/

Gold nanoparticles Imperial college London 6 Behzadi et al, Chem Sco Rev, 2017

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Nanogold particles

  • Gold Particles (AuNPs) have great potentials for biomedical applications
  • AuNPs are tunable in term of :

Shape Size Surface chemistry Surface chemistry Aggregation State Aggregation State

Bio-sensing Diagnosis Therapeutic Drug Delivery 7

Optical

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Gold Nanoparticles Properties: Surface Plasmon Resonance

  • Optical Properties of metallic nanoparticles: Gold and Silver
  • (The wavelength of the light are larger than the size of particles)
  • LSPR: Localized Surface Plasmon Resonance

λ

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Gold Nanoparticles Properties

  • Colloidal Gold (Suspension of submicron particles of golds in fluid)

Increasing Size Red -------- Blue

http://nanocomposix.com 9

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Influence of gold nanoparticles properties on LSPR

Size Sharper to broader Shape

Senyuk et al, Nano Letter,(2012) http://www.cytodiagnostics.com 10

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Influence of gold nanoparticles properties on LSPR

  • Aggregation

Kumar et al, Pharmacy and Pharmaceutical Science, 2014 11

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Nanoparticles for drug-delivery

Nanoparticles can be absorbed, convolutely attached, or encapsulated into particles

Limitation of conventional methods

  • Lack of selectivity toward cancerous cells
  • Systemic toxicity
  • Low therapeutic index
  • Low circulation half-life
  • Tendency to aggregate

https://www.cancer.gov/sites/ocnr/cancer-nanotechnology/treatment

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Advantages of NPs for drug delivery

Increasing treatment efficiency Increasing treatment efficiency Increasing specific location Controlled contribution Reducing dose Decreasing toxic side effects Improving patient compliance

Nanoparticle-based Drug Delivery System

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Targeting Approaches

1- Active Targeting (Pre conjugated with antibodies, small molecules and peptides) 2- Passive Targeting (EPR)

Ajnai et al, Journal of Experimental and Clinical Medicine (2014)

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Limitation of nanoparticle drug delivery system

Two determining factors should be taken into account before using nanoparticles as Drug Delivery system 1- Biocompatibility

  • Not toxic (Exposure time, dose)

Methods: viability of cell Function changes

  • Accepted by body without rejection
  • Inert or stable

2- Internalization ability (subcellular location)

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Cellular uptake of Nanoparticles

Cell-specific targeting: Attaching drugs to specially designed carries (Drug can be absorbed, covalently attached or encapsulated into nanoparticles) Entry Mechanisms:

  • L. Chou et al, Chemical Society Reviews (2012)

Endocytosis

Receptor-mediated Macropinocytosis

Phagocytosis

Important factors

NP’s related factors

Biological parameters Experimental Factor

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Clathrin- mediated Caveolae- dependent

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2- Comparative Study on cellular entry of Synthesized and Ayuverdic gold particles

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  • Scientific Report, Nature (2017)
  • Plasmonic, Springer (2017)
  • Nanoscience and Nanotechnology, ASP, (2017)
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Synthesized gold nanoparticles and Ayuverdic particles

Swarna Bhasma Powder

(http://www.planetayurveda.com)

Colloidal Gold

(https://dir.indiamart.com)

 Incinerated Gold nanoparticles (IAuPs) Traditional Indian approach:  Citrate-capped spherical nanoparticles (AuNPs)

By the reduction of chlorauric acid with sodium citrate

Coarse Powder

  • f Gold

Is Hammered into ribbon Is Mixed with herbal extracts Is incinerated at high temperature HAuCl4

Heating Adding sodium citrate Stirring Changing color

Au Particles Heating Sodium Citrate Stirring

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Characterization of particles

Scanning Electron Microscopy (SEM) IAuPs Average Size: 4500 nm (Dynamic Light Scattering) Crystal size: 60 nm Non-uniform AuNPs Average Size: 32 nm uniform

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Elemental composition of AuNPs and IAuPs

AuNPs IAuPs Au 56.88 % 89.6 ppm Mg 1.8 % 0.273 ppm Na

  • 20.9 ppm

EDS-SEM for IAuPS EDS-SEM for IAuPS

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Characterization of gold nanoparticles to cells

To test the toxicity and subcellular location Two experiment test were performed 1- under different exposure time 2- under different doses Two types of cell lines were chosen 1- HeLa (Cancerous cells) 2- HFF1- (Healthy cells) Test: Localization, entry and impacts

  • n human cells

Imaging Techniques:  Light Microscopy  SEM  Hyperspectral Imaging

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http://smashinglife.co.uk/cancer-cells-look/

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Nanoparticle in cells (Light Microscopy)

Leica DMI 6000 B inverted epifluorescence microscope 22

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Concentration and incubation time effects

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AuNPs and IAuPs in Cells (SEM)

Low con. AuNPs in Hela High con. AuNPs in HeLa Control w/o AuNPs

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Nanoparticle in cells (Live Imaging)

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Hyperspectral Microscopy CytoViva

Technology for characterization of nanomaterial in cells Combination of:

  • Hyperspectral imaging system
  • Optical Microscope

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https://cytoviva.com

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Using Hyperspectral Microscopy for AuNPs in cells

  • Presence of AuNPs
  • Location of AuNPs

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https://cytoviva.com

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Enhanced dark field imaging

Control AuNPs

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Hyperspectral imaging of AuNPs and IAuPs in cells

600~625 nm 600~700 nm

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Intracellular Localized Surface Plasmonic Sensing for Subcellular Diagnosis

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Breaking IAuPs to smaller particles

Sonication Mixer

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Broken and unbroken particles in cells

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Hyperspectral imaging of broken and unbroken IAuPs in cells

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Entry mechanism of IAuPs

Mechanicsms Before blocking After blocking (changes) Macropinocytosis 9.2 % 4.7% (-4.5% ) Clarotin-mediated 11.1 % 4.9% (-5.2% ) Both Macropincocytosis and Clarotin- mediated 9.2 % 4% (-5.2 %) Calveolin-mediated ~12 % ~ 12%

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Nanoparticles in Nucleus

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3- Impacts of nanoparticles

Robyn et al, (2014) 36

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Biophysical properties of cells  Biophysical properties of cells?

BIOMECHANICAL, bioelectrical, biochemical

 Important biophysical biomechanical properties

Size, Viscoelastic properties Mass Friction Density

Function Biological Function Mechanics

Biophysical of cells

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Components and Mechanics of Cells (Eukaryotic cell)

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Mechanics of Eukaryotic Cells

Actin filament Microtubules Intermediate Filament Membrane Cytosol Nucleus

Cytoskeleton and Cellular Structure Less contribution to mechanics of cells

Decreasing Contribution Microtubules Intermediate Filament Actin Filament Rodriguez et la, Applied Mechanics Review (2013) Suresh, Acta Biomaterialia (2007)

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Why studying Bio-Mechanical Properties of cells is important?

Bio-mechanical properties of cells during disease undergo changes Importance of Mechanical Properties

A powerful and label-free approach for diagnosis cancer at early stage Evaluate the efficiency and effectiveness of medicine or nanoparticle-based drug delivery systems

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Biochemical factors Structural changes induced in cells Changes in cell deformability Altered cell function Altered cell motility Cancer cell metastasis

Suresh, Acta Biomaterialia (2007)

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Different Methods for Deformability Characterization of Single Cells

Methods:  Classical Methods,  MEMS-based methods,  Microfluidic-based methods Classical methods: Main Advantage: Main Limitation:

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High –precision

Bao et al, Journal of Royal Society Interface (2014)

Local Deformation Whole Deformation

Low-throughput

Main Classical Methods

Choice Criteria for cell mechanics:  Size,  Elasticity  Precision  Speed

Moeendarbary et al, WIREs Systems Biology and Medicine, (2014)

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Deformability characterization: MEMS-based systems

Limitations:

Expensive External devices Non-Transparent

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Microfluidic-based systems

 Advantages High-throughput Easy fabrication  Limitation Low precision

A) Constriction-induced deformation B) Multiple Constriction channel C) Micro- aspiration D) Hydrodynamic-induced deformation

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Nanoparticle uptake effects on biomechanical of cells

  • NPs can provide desirable effects on cells

But! The intercellular effects of NPs in cells is unknown.

cell Particles Effects on stiffness mesenchymal stem cells Silica Increased Escherichia coli Hematite NPs Increased iron oxide NPs endothelial Increased Selenium NPs MCF-7 Decreased

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Summary

 Advantages of nanoparticles have made them a good candidate for medical application  Gold nanoparticles can be used for cancer diagnosis as well as cancer therapy  Nanoparticle-based drug delivery system can provide advantages comparing to conventional methods  Cellular entry and toxicity are two determining factors in choosing particles for drug- delivery systems  Nanoparticles can enter cells through different mechanisms  Nanoparticles absorption can alter biophysical properties of cells  Resolution and throughput are two important factor for bio-mechanical characterization

  • f cells

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Thank you 

45 Behzadi et al, Chem Sco Rev, 2017