Use of surface plasmon resonance for probing cell-matrix interactions

Transcription

1 Use of surface plasmon resonance for probing cell-matrix interactions Reichert Technologies Webinar September 20, 2016 Michael Hill Debanjan Sarkar Lab: Laboratory of Biomaterials and Regenerative Therapeutics State University of New York at Buffalo 1

2 Outline of the presentation Introduction and Motivation Part I: Characterization of surface energy of immobilized proteins using liquid contact angle studies Part II: Test of SPR for non-specific adhesion applications using endothelial cells Part III: Test of SPR for specific adhesion applications using white blood cells Summary and conclusions 2

3 Surface forces regulating cell adhesion o Van der Waals Forces: Long range; decay as 1/R 6 o H-bonding and double 200 Å layer: Strong but fall off more quickly with distance - Non-specific longer range forces are important during cell adhesion 10 Å 3 Å - Protein-coats present their own surface field. Specific interactions overcome these shielding effects. 3

4 Specific vs. non-specific biological adhesion Specific forces: due to a pattern of short-ranged hydrogen bonding or electrostatic forces (<1nm under physiological conditions) -Can be defined by their ability to transmit across albumin blankets Arginylglycylaspartic acid (RGD) peptides: bind specifically with domains of integrins - specific arrangement of polar and non-polar groups responsible for such bonding Polar Apolar Receptor-Ligand Interactions Polar Albumin 4

5 Surface Plasmon Resonance (SPR): A brief introduction Evanescent wave: set up by shining a light with resonant frequency of a thin gold film -Since wave is a near field-effect, molecules interacting with surface are detected by changing surface refractive index (micro refractive index units (µriu)) Range of signal: few 100 nm from the surface 5

6 SPR studies: experimental setup Reichert SR7500DC: modular tubing and flow chamber -wide possible range of flow rates SPR gold chip has carboxyl/peg terminated SAM -integrated with a flow chamber to enable cell attachment under shear Controlled shear and real-time signal is major advantage to cell adhesion studies A = EDC-NHS B = Protein C = Ethanol amine 6

7 SPR versus previous cell adhesion metrics Passive Attach Manual Count Cell Attachment Under Defined Shear Atomic Force Microscopy Cost Low intermediate High Intermediate Complexity Low intermediate High Intermediate Challenge Adhesion SPR No Yes Yes Yes Resolution Low Intermediate High High Natural No Yes No Yes Real Time No No Yes Yes Speed High High Low high 7

8 Summary of Experiments Part I: Surface energy of immobilized protein surface is estimated for non-specific cell adhesion application Part II: SPR is used to test adhesive interaction of endothelial cells to protein surfaces of differential surface energy Part III: Specific interaction between Immobilized P-selectin and white blood cells (HL-60) across a blanket of albumin. Study confirms use of SPR for measurements across multiple protein interlayers 8

9 Introduction In vivo Endothelial Cells (ECs): come into contact with two classes of extracellular matrix proteins: Basement membrane (collagen IV/Laminin dominated) Stromal tissue (collagen I/III dominated) ECs are typically separated from stromal tissue by basement membrane When injury occurs, the cells contact and invade the stromal tissues, and this induces angiogenesis 9

10 Methods -Collagen I (stromal protein), Matrigel (basement membrane), and serum (control protein) were immobilized -Contact angle measurements using various diagnostic liquids were performed for biosurface energy estimation Low surface energy High surface energy 10

11 Surface energy theories and biological adhesion I. Critical Surface Tension (CST) No serum serum Biomaterials CST (dynes/cm) (Weiss and Blumenson 1967) 11

12 Surface energy theories and biological adhesion II. Kaelble s Method γ p = polar γ d = dispersive - Multiple liquids used, similar to CST - Equation is solved numerous times for each liquid pair - Arithmetic average is taken - Values outside standard deviation rejected 12

13 Surface energy theories and biological adhesion III. Van Oss- Good-Chaudhury Theory (vogct) γ + = Lewis acid γ - = Lewis base - 2 polar and 1 apolar liquid are measured - 3 simultaneous linear equations are solved.. O.. O.. O.. O.. O.. O.. O.. O.. O.. O.. O.. O.. O.. O 13

14 SPR studies: Surface energy measurement of proteins on SPR chip Collagen I: greater γ p and γ + than other proteins (greater surface energy) Matrigel: lowest γ p with lower γ + (less surface energy) 14

15 SPR studies. cell attachment studies - Differential cell adhesion to immobilized proteins can be correlated to protein surface energy - Proteins with higher surface energy have greater tendency to bind cells Flow rate reduced Cells 15

16 20x 10x SPR studies. Visualization of cell monolayers at 24h on three substrates Collagen I Matrigel Serum 200 µm 200 µm 16

17 Discussion Surface energy measurements may be a predictor of optimal conditions for cell capture and adhesion. This can be used to optimize cell binding conditions on the sensor chip. Protocols were established for the formation of confluent live endothelial cell monolayers. Cell binding was most efficient when extra-cellular matrix proteins were immobilized at physiological ph. 17

19 SPR studies: Higher surface energies correlate with greater hyper-osmolar shock response Collagen I Matrigel Serum 100 mm hyperosmolar mannitol Isotonic HEPES 19

20 20x 10x SPR studies. Greater strength of binding on collagen I substrates Collagen I Matrigel Serum 200 µm 200 µm 20

21 Discussion Collagen I: Higher γ p and γ + correlate with increased cell adhesion strength. This is the condition for ECs on stromal tissue as they undergo angiogensis. Collagen I: Immobilized arrays of Lewis acid and Lewis base groups Matrigel : Lower γ p and γ + correlates with reduced EC interaction. This occurs when cells form monolayers on basement membranes Matrigel : Cross-linked structure causes shielding of Lewis acid/base. Young s modulus of adhesion calculated via SPR Matrigel ~0.5MPa & Collagen ~2MPa All results correlate well with AFM + = Lewis acid - = Lewis base 21

23 Introduction White blood cells: recruited to specific tissue sites upon injury or inflammation Endothelial cells: line the blood vessels, express selectins in injured tissues -White blood cells first tether onto selectins such as P-selectin on the endothelium via glycoprotein ligands including PSGL-1 (Pselectin glycoprotein ligand-1). 23

24 Non-specific adhesion of HL-60 A B C D A = EDC-NHS B = IgG C = Ethanol amine D = P-selectin Non-specific adhesion: overpowers the effects of specific adhesion when the long-range forces are not blocked by a blanket 24 of albumin

25 Isolation of specific effects of HL-60 recruitment BSA blocking: ensures specific interactions are probed MAb KPL-1 (anti-psgl-1 mab) : confirms specificity - SPR response across multiple protein interlayers 25

26 Discussion Specific adhesion of HL-60: SPR confirms the specificity of cell adhesion across multiple protein interlayers - Distance of SPR signal is limited to few 100 nanometers - Cells are large ( s of µm) colloidal objects - SPR can be used to closely monitor the nature by which cells spread on ligand bearing substrates in a narrow distance scale. Something that cannot be done using standard microscopy. 26

27 Conclusions SPR: Used to probe cell-matrix interactions in the context of both specific (BSA blocked) and non-specific cell adhesion systems The two types of adhesion often co-exist in particular biological contexts SPR is useful to dissect intermolecular force characteristics of cell adhesion in different model systems, especially at short length and time scales Hyperosmolar shock studies with SPR: can quantify strength of cell interactions with proteins Cheaper, simpler, easier compared to past methods Similar quantitative results in comparison to Atomic Force Microscopy 27

28 Future Work Visualization of cells: It is useful to visualize cells during SPR studies. This can enable: - Correlation of SPR signal to kinetics of cell spreading Regeneration of the sensor chip: Removing cells while keeping immobilized protein intact is not possible: - High shear may cause cohesive rupture of cells - Digestive enzymes may alter protein film Methods to release the entire chemistry on SPR sensor chip would be helpful. - Model development: SPR is typically used to measure monovalent binding interactions. Estimation of kinetic on-off data for multivalent cellular interactions requires more research. 28

29 Q & A 29

30 Appendix 30

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32 P-Selectin versus Control 19Fc Surfaces 19Fc : Control IgG fragment - Slope of P-Selectin signal increases > 3-fold compared to 19Fc control substrate - Cells continue to spread during dissociation phase - Further increase in signal upon introduction of more HL-60 cells. - Cells do not bind the control 19Fc substrate efficiently 32

33 EDC-NHS Methods 80 µg/ml protein 1 M ethanolamine Chip removed and cells cultured overnight Cells spread for 3 hours cells/ml (Endothelial cells) 4 33

34 EDC-NHS Methods 80 µg/ml protein 1 M ethanolamine mm hyperosmolar mannitol cycles Cells spread for 3 hours cells/ml (Endothelial cells) 4 34

35 EDC-NHS Methods 80 µg/ml IgG antibody immobilized 1 M ethanolamine cells/ml (cell binding facilitated by PSGL-1) 6 2% BSA 5 5 µg/ml P-selectin 4 35

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37 Human Embryonic Kidney CELL-PROTEIN BINDING 37

38 HEK Cells Capture 38

39 200 nm Fibrinogen Injection Surface Heterogeneity Model k a1 = 2.40 e 3 M -1 s -1 k d1 = 8.51 e -3 s -1 K D1 = 3.54 mm k a2 = 9.74 e 3 M -1 s -1 k d2 = 2.57 e -4 s -1 K D2 = 26.4 nm 39