Protein Replacement Therapy with Cationic Lipid Assisted Intracellular Delivery
therapeutics have unique advantages and challenges that set this therapy apart from
others. The delivery of proteins is regarded as a safe and direct treatment approach
with advantages such as high tolerances, low off target effects, and potent efficacy.
However, there are significant challenges in delivering protein past the cell membrane
where a substantial portion of ther... read moreapeutic proteins require delivery. Therefore, there
remains a need for a vehicle capable of delivering protein intracellularly while
maintaining its integrity, structure, and function. Thus, the work presented herein
focused on expanding the cationic lipidoid delivery platform to encompass a broad range
of protein applications requiring intracellular delivery. An effective lipidoid for
delivery of recombinant PTEN protein was identified, characterized, and therapeutically
evaluated for intracellular replacement of PTEN in PTEN-null cancer cells. The lipidoid
structure EC16-80 demonstrated the most effective intracellular delivery compared to a
range of lipidoid structures and displayed an encapsulation efficiency of 80% complexed
PTEN. The interaction forces that contribute to the nanocomplexation were determined to
be both electrostatic and hydrophobic in nature, based on dissociation studies and
thermotropic measurements, respectively. The biological effects of delivering PTEN with
EC16-80 resulted in dose dependent cellular apoptosis and were confirmed to be the
result of PTEN antagonizing the AKT pathway, as displayed through western blot.
Furthermore, the delivery of PTEN protein predominantly affected PTEN deficient cells
compared to cells with wild-type PTEN. Together, these results highlight EC16-80/PTEN
protein delivery as a potential new therapeutic strategy selectively against PTEN
deficient cancers. Additionally, the effect of protein charge modification on PTEN was
found to result in more favorable particle characteristics (decrease in hydrodynamic
radius and surface charge and increase in % complexed PTEN) and delivery efficacy
(decrease in IC50). Lastly, the lipidoid EC16-80 also displayed intracellular delivery
with both modified RNase-A protein to inhibit lentivirus infection and with modified
β-Glucocerebrosidase as protein replacement therapy. Together, this work
establishes a new treatment strategy for PTEN-deficient cancers by directly delivering
PTEN protein using lipidoid complexes. Furthermore, the ability of EC16-80 to deliver
various protein structures in multiple disease models highlights the diverse utility in
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Qiaobing Xu.
Committee: David Kaplan, Hyunmin Yi, and Eric Park.
Keyword: Biomedical engineering.read less