Development of AFM Platform for Directly Measuring the Nanoparticle-Nanoparticle and Nanoparticle-Cell Interactions

Transcription

1 Development of AFM Platform for Directly Measuring the Nanoparticle-Nanoparticle and Nanoparticle-Cell Interactions Georgios Pyrgiotakis, Christoph Blattmann, Sotiris Pratsinis, Philip Demokritou Harvard School of Public Health

2 Nano-bio interactions The use of engineered nanomaterials (ENMs) in commercial products, pharmaceutical applications and industrial processes continuously increases. Environmental and occupational exposures are considered by experts to be inevitable. Key mechanism of the nanoparticle fate is the nanoparticle cell interactions. The carefully controlling these interactions can enhance or mitigate the particle uptake by cells and exploited to target specific cells.

3 Nanoparticle - Protein interaction Macroscopic: in-vivo, in-vitro toxicity cell lines, animals etc Microscopic: Protein binding, particle aggregation etc Molecular: Protein confirmation on particles Biological assays Inhalation studies Instillation studies Proteomics Particle sizing Particle uptake Computer simulations Spectroscopy Connection Gap Indirect method: DLS: Aggregation, hydrodynamic diameter, PDI Proteomics: Information of what proteins are bided but not on thickness Missing a tool that will directly measure and quantify these interactions

4 Research Goal Systematic study of nanoparticle nanoparticle and nanoparticle cell interactions and how they relate to Nanoparticle Properties NP Properties Corona Thickness Interactions The developed approach has to be: Nanoparticle independent Able to be used with a wide variety of cells Able to be used in various physiological liquids Adding an meaningful layer of information 4

5 Atomic Force Microscope (AFM) Tip Sample Piezoelectric for stage movement The tip is bending up and down based on the interaction forces The deflected laser is changing location on the photo-detector If the tip is well characterized the interaction force can be calculated 5

6 Study Design Tasks Develop the appropriate AFM platform and methodology Measure the NP NP interaction force and relation to inherent particle properties: Material Size Measure the NP Cell interactions and correlate to material properties and experimental conditions Case study: Fe 2 O 3 and CeO 2 NPs of controlled primary particle size

7 Approach Nanoparticle Nanoparticle Interactions Nanoparticle Cell Interactions Modify the AFM tips by attaching Engineered Nanoparticles. Prepare substrates with the same nanoparticles attached on them. Measure the force between the NPs in various media. Use the same AFM tip as before. Grow the cells on appropriate substrate. Measure the interaction force between the NPs and the cells at the appropriate media.

8 Particle Synthesis and AFM tip functionalization PLATFORM DEVELOPMENT

9 Platform development & protocol Synthesize NP and NP films by VENGES Attach NPs to AFM tips Image NP films and AFM tips (SEM) Determine cantilever spring constant (AFM) Conduct force measurements in desired medium re-image AFM tips to ensure NP adherence (SEM) 9

10 47.9 nm 27.2 nm 5.4 nm Nanoparticle characterization XRD characterization TEM Imaging CeO 2 Fe 2 O nm 24.5 nm 90.6nm

11 Examples of substrates imaged by SEM and AFM SiO 2 Fe 2 O 3 CeO 2

12 Functionalized AFM tips Samples of the various tis after the preparation and before usage

13 Functionalized AFM Tips: Stability over Time Freshly prepared tip Fe2O3 L (90.6nm) 200 Measurements in air 200 Measurements in water 200 Measurements in RPMI 200 Measurements in RPMI+10% FBS 13

14 Pyrgiotakis et al., Langmuir, 2013 NANOPARTICLE NANOPARTICLE INTERACTIONS

15 Forces measured with AFM

16 Adhesion Forces The attraction of the NPs increases with increasing the NP size. The attraction of the NPs is independent Of the NP primary particle size. 16

17 Repulsive Layer Thickness (RLT) The attraction of the NPs increases with increasing the NP size. The attraction of the NPs is independent Of the NP primary particle size. 17

18 Repulsive Layer Thickness The values are in the same range for both materials. Iron oxide shows a strong dependence with the primary particle size. The data correlate well the measured hydrodynamic diameter 18

19 Work in Progress NANOPARTICLE CELL INTERACTIONS

20 Particle Cell Interactions Different tips with more sensitive cantilevers: The tip has to yield before the cells Controlled the force: No puncture to the cell membrane. Experimental design: 1. A549 Cells and RPMI+10%FBS 2. Nanoparticle size: Large vs. Small 3. Particle Cell contact time: 30 s vs 180 s 20

21 Particle Cell Interactions In order to analyze the data we need to develop a set of parameters to compare the data: 1) User parameters: Particle size, Particle Cell contact time (30 s vs 180 s) 2) Multiple adhesive forces measure: Step Detachment forces (multiple particles detaching) Final Detachment force 21

22 Large 90.6 nm Small 10.1 nm Number of Force Steps: Fe 2 O 3 Example 30 Seconds 180 Seconds

23 Force Quantification 30 Seconds Interaction time 180 Seconds Interaction time The overall adhesion forces have almost doubled between the 30 and 180 seconds The more Step Detachment forces maintained the same levels indicating similar types of adhesion The same trends in the interactions maintained eg: Larger iron oxide particles have lower Final Detachment force Smaller particles more Step Detachment forces

24 Conclusions The Atomic Force Microscope is a promising platform that has the potential to address the need to measure the NP NP and NP cell interactions. Does not depend on the particle or media optical properties It directly measures the interaction We developed an method to functionalize the AFM tips/substrates which can be used can for a variety of industry relevant particles, cells and physiological media. Our preliminary results shows that the NP NP and NP Cell interactions can depend both on the material and the primary particle size.

25 Acknowledgements Center for Nanotechnology and Nanotoxicology at the Harvard School of Public Health, the NIEHS center (ES ), Harvard Career Incubator Fund NSF grant no European Research Council under the European Union s Seventh Framework Program (FP7/ , ERC grant agreement no ) Sandra Pirela Center for Nanotechnology and Nanotoxicology Harvard School of Public Health Margaret Thomson Manager of the Transgenic laboratory Children s Hospital and Developmental Diabetes Research Center

26 QUESTIONS? THANK YOU!