Modulating the Therapeutic Microenvironment Using Nanostructured - - PowerPoint PPT Presentation

Tej ejal A l A. Des esai, i, Ph PhD Ernest st L L Pr Prien Professo essor and C nd Chair Direc ector, UCSF E F Engineer eering and nd Appl pplied ed S Scien ences I es Initiative

• Dept. of Bioen

engineer eering a and nd T Ther herape peutic S Scien ences es

Modulating the Therapeutic Microenvironment Using Nanostructured Materials

Advanced biomaterials for therapeutic delivery

Picture credit: Zhang, Drug Discovery Today

Material Size Surface Shape

CANCER DIABETES HEART DISEASE PSORIASIS ARTHRITIS …

• Direct biophysical stimulation
• Biomolecule carrier/presenter

Micro and Nanostructures Cell –Material Interactions Therapeutic Systems

How can material structure modulate biologic function for therapeutic purposes?

Desired Routes of Drug Delivery

Challenges to epithelial drug delivery

5

Revisiting the small intestine

100 10-2 10-4 10-6 10-8 Length (m)

Small intestine Villi Enterocytes Microvilli

Fox C et al., Journal of Controlled Release, 2015

Drawbacks of paracellular permeation enhancers

• Chemical permeation agents

induce opening of tight junctions

• Examples: Surfactants,

chelators, and toxins

• Toxicity to cells
• Non-reversible tight junction
• pening

Permeation enhancer (red) opens tight junctions for increased drug permeation Non-reversible tight junction damage Membrane damage and cell toxicity

Opening Epithelial Barriers

Sun et al. Physiological Reviews, 2017.

Topographical cues can enhance permeation

• f drug between tight junctions

Nanostructured Film Drug Molecule Cells

Diameter: 200 nm Height: 300 nm Diameter: 800 nm Height: 16 µm High Drug Permeation Low Drug Permeation

Kam et al., Nanoletters, 2014; Stewart et al., Exp. Cell Res, 2017

Sun, et al. Physiological Reviews, 2017 Walsh, et al. Nano Lett, 2015

Nanostructured microneedles enhance transdermal drug delivery

200 nm diameter

Fox et al., JCR 2015 Fox et al. ACS Nano 2016

Nanostructured planar particles for enhanced oral delivery

Nanostructures can be tuned to facilitate permeation

LD100 LD200 LD500 HD100 HD200 HD500

The process is reversible and involves remodeling of tight junctions

b) a) c)

d

Remove 10 µm 10 µm 10 µm 20 µm 20 µm 20 µm

ZO-1 (tight junction protein), Caco-2 nuclei, F-Actin

Paracellular or transcellular? Mechanism?

FITC-IgG present at apical cell-cell borders

TJ scaffolding protein ZO-1 shows altered morphologies upon NS film treatment

Total level keep the same  reversible

Z-stack-1 Z-stack-2 Z-stack-3 Z-projection

CRISPR-based gene editing to visualize tight junctions

Endogenous mCherry-ZO1 ZO-1 ICC

Clone isolation

Inserted allele Original allele Inserted allele Original allele

After In Vitro barrier model screening

mCherry ZO-1

Fluorescent protein fusion of TJP-ZO-1

ZO-1 proteins fused with mCherry-reporter at N-terminus through CRISPR

NS 10-40 mins NS 45-75 mins

Time-Lapse Video of ZO-1 during Nanostructure Treatment (MAX Z-projection, 2 locations 1s=5mins) TEER>>1800

Dynamic changes in TJs with nanostructure contact

Large aggregates of ZO-1protein are formed on apical side when treated with NS film

Active interaction between aggregates and border ZO-1

NS-film treated cells have faster recovery from FRAP

t = 0s t = 100s t = 200s t = 300s Photobleaching in frame

NS-treated Non-treated

Junctional protein Claudin-4 colocalizes with ZO-1 aggregates

Non-treated Flat NS endogenous mCherry-ZO-1 + AAV exogenous YFP-Cldn3, Z projection of ~1um

Video

Using physical cues to alter tight junction permeability: implications for delivery

Harnessing nanotopographical cues for therapy

Smooth Muscle Cells Endothelia l Cells Epithelial Cells Fibroblasts

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