Fluorescence labeled PEI-based gene delivery systems for near infrared imaging in nude mice

Gene therapy is a research area where nucleic acids are transferred
into cells to treat neoplastic, metabolic and hereditary diseases.
Delivery of genetic material into living organisms can be achieved
with viral or non-viral vectors. Viral gene carriers are very
efficient but present some major disadvantages due to their
pathogenicity and immunogenicity. Nonviral carriers are based on
synthetic molecules binding and condensing nucleic acids into
small, virus-like particles. The aim of this thesis was to study
the biodistribution and tumor targeting properties of non-viral
gene vectors based on polyethylenimine (PEI) after systemic
injection into mice. The gene vectors were labeled with fluorescent
dyes emitting in the near infrared (NIR), which allowed studying
their bio-distribution in living animal over time. Owing to its
amine groups PEI has a high positive charge density that enables
electrostatic interactions with negatively charged nucleic acids
and their efficient compaction into nucleic acid-PEI complexes,
called polyplexes. The net positive surface charge of these
polyplexes permits interactions with negatively charged cell
surface molecules, thus leading to their internalization into the
cell. To avoid unspecific interactions with blood components and
nontarget tissues after intravenous application, polyplexes were
shielded with the hydrophilic molecule polyethylene glycol (PEG).
PEI-based gene carrier systems were tested on two subcutaneously
implanted tumor types: Human Hepatocellular Carcinoma (HUH7) and
Murine Neuroblastoma (N2a). HUH7 cells express epidermal growth
factor (EGF) receptors, while N2a cells express transferrin (Tf)
receptors on their surfaces. To enable targeting of the polyplexes
to the tumor cells, polyplexes were generated containing the
ligands EGF and Tf for targeting of HUH7 cells and N2a cells
respectively. The targeted polyplexes were then intravenously
injected into immunodeficient, athymic nu/nu mice in which HUH7 or
N2a tumor cells had been previously set under their skin. To
monitor the biodistribution of polyplexes throughout the mouse
organism and to evaluate their gene delivery capability into the
neoplastic cells, polyplexes were labeled with fluorescent dyes
(Alexa 750, NIR 797) or near infrared emitting quantum dots (QD),
whose fluorescent expression signal was detected and analyzed with
a device for imaging in vivo. All fluorescent molecules and quantum
dots were biocompatible and non-toxic. They emitted light in the
near infrared area of the spectrum, thus avoiding overlapping
phenomena with autofluorescent biomolecules or absorption of light
by hemoglobin. With all dyes used for polyplex labeling a
fluorescent signal could be observed in organs like liver and lung
being clearly distinguishable from background fluorescence. Among
the fluorescent molecules tested, quantum dots were identified
being the most suitable method for in vivo studies, showing the
highest signal/noise ratios. PEG-shielding led to best tumor
targeting efficiency when administering EGF or Tf-targeted
polypelxes in mice bearing HUH7 and N2a tumors respectively. A
clear fluorescent signal specific for tumor tissue was detected;
the imaging software used allowed quantitative analysis of this
signal. For this reason this system is now available for further
experimental applications.