Protein
biologics are an important class of drugs, but the necessity
for frequent parenteral administration is a major limitation. Drug-delivery
materials offer a potential solution, but protein-material adsorption
can cause denaturation, which reduces their effectiveness. Here, we
describe a new protein delivery platform that limits direct contact
between globular protein domains and material matrix, yet from a single
subcutaneous administration can be tuned for long-term drug release.
The strategy utilizes complementary electrostatic interactions made
between a suite of designed interaction domains (IDs), installed onto
the terminus of a protein of interest, and a negatively charged self-assembled
fibrillar hydrogel. These intermolecular interactions can be easily
modulated by choice of ID to control material interaction and desorption
energies, which allows regulation of protein release kinetics to fit
desired release profiles. Molecular dynamics studies provided a molecular-level
understanding of the mechanisms that govern release and identified
optimal binding zones on the gel fibrils that facilitate strong ID–material
interactions, which are crucial for sustained release of protein.
This delivery platform can be easily loaded with cargo, is shear-thin
syringe implantable, provides improved protein stability, is capable
of a diverse range of in vitro release rates, and most importantly,
can accomplish long-term control over in vivo protein delivery.
Objective:
In this in vitro study we have used an RNA quantification technique, nanoString, and a conventional protein analysis technique (Western Blot) to assess the genetic and protein expression of B16 murine melanoma cells following a modest magnetic nanoparticle hyperthermia (mNPH) dose equivalent to 30 minutes @ 43°C (CEM43 30) and/or a clinically relevant 8 Gy radiation dose.
Methods:
Melanoma cells with mNPs(2.5 μg Fe/106 cells) were pelleted and exposed to an alternating magnetic field (AMF) to generate the targeted thermal dose. Thermal dose was accurately monitored by a fiber optic probe and automatically maintained at CEM43 30. All cells were harvested 24 hours after treatment.
Results:
The mNPH dose demonstrated notable elevations in the thermotolerance/immunogenic HSP70 gene and a number of chemoattractant and toll-like receptor gene pathways. The 8 Gy dose also upregulated a number of important immune and cytotoxic genetic and protein pathways. However, the mNPH/radiation combination was the most effective stimulator of a wide variety of immune and cytotoxic genes including HSP70, cancer regulating chemokines CXCL10, CXCL11, the T-cell trafficking chemokine CXCR3, innate immune activators TLR3, TLR4, the MDM2 and mTOR negative regulator of p53, the pro-apoptotic protein PUMA, and the cell death receptor Fas. Importantly a number of the genetic changes were accurately validated by protein expression changes, i.e., HSP70, p-mTOR, p-MDM2.
Conclusion:
These results not only show that low dose mNPH and radiation independently increase the expression of important immune and cytotoxic genes but that the effect is greatly enhanced when they are used in combination.
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