Natalie Garrett

The clinical development of therapeutic peptides has been restricted to peptides for non-CNS diseases and parenteral dosage forms due to the poor permeation of peptides across the gastrointestinal mucosa and the blood-brain barrier. Quaternary ammonium palmitoyl glycol chitosan (GCPQ) nanoparticles facilitate the brain delivery of orally administered peptides such as leucine(5)-enkephalin, and here we examine the mechanism of GCPQ facilitated oral peptide absorption and brain delivery. By analyzing the oral biodistribution of radiolabeled GCPQ nanoparticles, the oral biodistribution of the model peptide leucine(5)-enkephalin and coherent anti-Stokes Raman scattering microscopy tissue images after an oral dose of deuterated GCPQ nanoparticles, we have established a number of facts. Although 85-90% of orally administered GCPQ nanoparticles are not absorbed from the gastrointestinal tract, a peak level of 2-3% of the oral GCPQ dose is detected in the blood 30 min after dosing, and these GCPQ particles appear to transport the peptides to the blood. Additionally, although peptide loaded nanoparticles from low (6 kDa) and high (50 kDa) molecular weight GCPQ are taken up by enterocytes, polymer particles with a polymer molecular weight greater than 6 kDa are required to facilitate peptide delivery to the brain after oral administration. By examining our current and previous data, we conclude that GCPQ particles facilitate oral peptide absorption by protecting the peptide from gastrointestinal degradation, adhering to the mucus to increase the drug gut residence time and transporting GCPQ associated peptide across the enterocytes and to the systemic circulation, enabling the GCPQ stabilized peptide to be transported to the brain. Orally administered GCPQ particles are also circulated from the gastrointestinal tract to the liver and onward to the gall bladder, presumably for final transport back to the gastrointestinal tract.

Advances in pharmaceutical nanotechnology have yielded ever increasingly sophisticated nanoparticles for medicine delivery. When administered via oral, intravenous, ocular and transcutaneous delivery routes, these nanoparticles can elicit enhanced drug performance. In spite of this, little is known about the mechanistic processes underlying interactions between nanoparticles and tissues, or how these correlate with improved pharmaceutical effects. These mechanisms must be fully understood before nanomedicines can be rationally engineered to optimise their performance. Methods to directly visualise these particulates within tissue samples have traditionally involved imaging modalities requiring covalent labelling of fluorescent or radioisotope contrast agents. We present CARS, second harmonic generation and two photon fluorescence microscopy combined as a multi-modal label-free method for pinpointing polymeric nanoparticles within the stomach, intestine, gall bladder and liver. We demonstrate for the first time that orally administered chitosan nanoparticles follow a recirculation pathway from the GI tract via enterocytes, to the liver hepatocytes and intercellular spaces and then to the gall bladder, before being re-released into the gut together with bile.

... 5 D. Brayden, Drug Discov. Today 2003, 8, 976: ... 6 XZ Qu, VV Khutoryanskiy, A. Stewart, S. Rahman, B. Papahadjopoulos-Sternberg, C. Dufes, D. McCarthy, CG Wilson, R. Lyons, KC Carter, A. Schatzlein, IF Uchegbu, Biomacromolecules 2006, 7, 3452. ...

Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world
nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 μM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most
important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.

The delivery of therapeutic peptides and proteins to the central nervous system is the biggest challenge when developing effective neuropharmaceuticals. The central issue is that the blood brain barrier, is impermeable to most molecules. Here we demonstrate the concept of employing an amphiphilic derivative of a peptide to deliver the peptide into the brain. The key to success is that the amphiphilic peptide should by design self-assemble into nanofibres wherein the active peptide epitope is tightly wrapped around the nanofibre core. The nanofibre form appears to protect the amphiphilic peptide from degradation while in the plasma and the amphiphilic nature of the peptide promotes its transport across the blood brain barrier. Therapeutic brain levels of the amphiphilic peptide are achieved with this strategy, compared with the absence of detectable peptide in the brain and the consequent lack of a therapeutic response when the underivatised peptide is administered.

We show that naturally occurring chitinous nanostructures
found on the wings of the Graphium butterfly can
be used as substrates for surface-enhanced Raman scattering
when coated with a thin film of gold or silver.
The substrates were found to exhibit excellent biocompatibility
and sensitivity, making them ideal for protein
assaying. An assay using avidin/biotin binding showed
that the substrates could be used to quantify protein
binding directly from changes in the surface-enhanced
Raman scattering (SERS) spectra and were sensitive
over a concentration range comparable with a typical
enzyme-linked immunosorbent assays (ELISA) assay. A
biomimetic version of the wing nanostructures produced
using a highly reproducible, large-scale fabrication process,
yielded comparable enhancement factors and biocompatibility.
The excellent biocompatibility of the
wings and biomimetic substrates is unparalleled by other
lithographically produced substrates, and this could pave
the way for widespread application of ultrasensitive
SERS-based bioassays.