Perfluorocarbon nanodroplets for oxygen delivery for bone repair

Abstract

Around 2-10% of bone fractures result in delayed or non-union fractures.
These typically require surgery, which can be painful and carry a risk of infection.
There is, therefore, a critical need for minimally invasive treatment methods.
Many bone diseases linked to unsuccessful fracture healing, like osteoporosis and
osteonecrosis, are associated with hypoxia. Perfluorocarbons are inert and have
very high oxygen solubilities. They can be stabilised in aqueous solutions to form
nanodroplets. These perfluorocarbon nanodroplets have previously been shown
to successfully increase the oxygen tension in tumours. This study tested the hypothesis
that perfluorocarbon nanodroplets relieve the effects of hypoxia on cells
of osteoblastic and osteoclastic lineage in a manner that aids bone repair.
Perfluoropentane (PFP) nanodroplets with a 1,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC) and polyoxyethylene (40) strearate (PEG(40)s) shell were
fabricated using sonication. Temperature, PFP concentration, sonicator tip height,
total sonication time, sonication amplitude and post-processing techniques were
varied to evaluate their effect on nanodroplet characteristics through nanoparticle
tracking analysis. The optimised formulation was investigated for cytotoxicity on
cells of both osteoblastic (MC3T3E1, Saos-2 and BMSC) and osteoclastic (PBMC)
lineages using Alamar blue, Picogreen and cell counting. Alkaline phosphatase
enzyme and calcification served as osteogenic markers in BMSCs exposed to
the nanodroplets in normoxia and hypoxia, while osteoclastic activity was
measured by manually assessing osteoclast number and size in peripheral blood
mononuclear cells (PBMCs). Hypoxic stress was measured by quantifying HIF-1
protein expression in Saos-2s by western blotting and VEGF secretion by ELISA.
Nanodroplets exhibit low cytotoxicity on osteoblastic cells but significantly reduce
osteoclast numbers. Treatment with 0.1% v/v nanodroplets led to a marked
increase in calcification. Osteoclast number and size were evaluated manually after
long-term nanodroplet treatment. A decrease in osteoclast number and size
when treated long-term occurred in both normoxia and hypoxia, while short-term
treatment reduced osteoclast numbers in normoxia but increased them in hypoxia.
Microscope examination showed a preferential association of nanodroplets with
phagocytic cells over other cell types.
This work demonstrated that precise control over sonication parameters enables
better control over nanodroplet size and repeatability. The resulting nanodroplets
were capable of significantly increasing mineralisation and decreasing
hypoxic stress in osteoblastic cells, as well as relieving hypoxia-induced apoptosis
in osteoclastic cells. Overall, nanodroplets present a promising new oxygen delivery
method for relieving hypoxic stress in bone cells.

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Identifiers

ORCID for Nick Evans: orcid.org/0000-0002-3255-4388

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