Background:

It has been shown that uniform nanodiscs and nanovesicles can

self-assemble in phospholipid mixtures composed of long- and

short- chain lipids. Previously, we were able to manufacture

large quantities of such nanoparticles (NPs), which entrapped

the hydrophobic fluorescence dye (e.g., Nile Red), and stabilize

the NP structures by either charged or polyethylene glycol

conjugated (PEGylated) lipid. The original proposal is to apply

these NPs is for drug delivery carriers to target cancer cells. In

addition to this application, here we demonstrate another

potential application for biosensor using these NPs.

• The preparation protocols of uniform lipid-

based NPs are simple, robust, environmentally

friendly and scalable.

• The cellular uptake of nanodiscs is than that of

nanovesicles by CCRF-CEM , presumably due

to more internalization pathways are available

for discs than vesicles.

• The NPs can be used for instrument-free, high-

sensitivity and low-cost pathogen sensors.

Summary

Graduate Students:

Ming Li (IMS, UCONN), Andrew Hu (Mech.

Eng.), Yan Xia, Ying Liu (Chem. Biomol.

Eng.) and Wafa Aresh (Biomed. Eng.)

Scalable Manufacture of Multi-Functional Lipid-Based Nanoparticles

(Grant #: NSF-CMMI 1131587) Mu-Ping Nieh1, Tai-Hsi Fan1, Yong Wang2

University of Connecticut1, Pennsylvania State University2

Spontaneous Structural Diagram

Materials & Strategy

Zwitterionic long-chain lipids:

dimyristoyl (di-C14) phosphatidylcholine (DMPC) or

dipalmitoyl (di-C16) phosphatidylcholine (DPPC)

Zwitterionic short-chain lipid:

dihexanoyl (di-C6) phosphatidylcholine (DHPC)

PEGylated lipid:

Polyethylene glycerol (2000) - distearoyl (di-

C18) phosphatidylethanolamine (PEG-DSPE)

Charged long-chain lipids:

dimyristoyl (di-C14) phosphatidylglycerol (DMPG) or

dipalmitoyl (di-C16) phosphatidylglycerol (DPPG)

Fluorescence

Dye

(hydrophobic

molecules)

Increasing T

Nanodisc-to-Nanovesicle Transition

Decreasing T

Nanodiscs Spherical vesicles Oblate vesicles

Cellular Uptake CCRF –CEM (T-lymphoblastic leukemia)

Fluorescence Confocal Microscopy

• The cell uptake of nanodiscs is 3 ~ 5 times of that of nanovesicles.

Fluorescence 0 mm

0 mm

3 mm

3 mm

5 mm

5 mm

7 mm

7 mm

Fluorescence Flow Cytometry

Cel

l co

un

ts

Fluorescence Int.

Cellular

Uptake

Mechanism

Inhibitors Inhibited Mechanism

Chlorampramazine Clathrin

Filipin III Caveolae

Cytochalasin D Macropinocytosis

Amiloride Macropinocytosis

Wortamannin Macropinocytosis

Nocodazole Microtubules

0

20

40

60

80

100

120

Med

ian

flo

ure

cen

ce u

pta

ke

(% o

f co

ntr

ol)

Discs Vesicles

Clathrin

Caveolae

Macrpinocytosis Microtubules

Vesicles only take Clathrin- and

Caveolae-mediated pathways, while

nanodiscs take all four pathways .

(1) Drug Delivery Carrier

(2) Biosensing

Induced NP Aggregation by

polymer linker

Individual nanodiscs

50 nm

“strung” nanodiscs

100 nm

“strung” vesicles

Addition of the Polymer Linkers

high charge

density

low charge

density

108 CFU/mL: 104 102 0

Instrument-free Pathogen

Sensing

No

coverage }