ABSTRACT
The career development activities and studies delineated in this K01 application are designed to equip Dr.
Theocharidis, the Principal Investigator (PI), with expertise in biomaterials and multi-omics in order to become
an independent investigator. Non-healing diabetic foot ulcers (DFUs) affect millions of Americans and lead to
devastating consequences. Existing treatments are inefficient in promoting wound closure. The ongoing steep
surge in diabetic population necessitates new strategies to accelerate healing of diabetic wounds. Unlike acute
uncomplicated wounds, the linear progression from one phase of wound healing to the next is impaired in DFU,
which is characterized by a chronic low-grade inflammation. This could be the primary reason that growth factor
treatments that act during the proliferative phase have been unsuccessful. Single-cell RNA-sequencing (sc-
RNAseq) offers extensive insights into cell function and disease pathophysiology by allowing the mapping of the
transcriptomic landscape of individual cells in heterogeneous tissues like DFUs. A recently completed study led
by the PI focused on differences between DFU patients who healed their ulcers and those who failed to heal
them and investigated molecular changes via sc-RNAseq of surgically removed DFUs. Comparative analysis
unveiled genes and pathways significantly associated with successful wound repair.
Delivery of messenger RNA (mRNA) into recipient cells has the potential to enable functional protein
expression with tremendous therapeutic implications. Ongoing work by the PI has revealed that alginate hydrogel
dressings that facilitate lipid nanoparticle (LNPs)-mediated delivery of healing associated modified CHI3L1
mRNA and IL-2 mRNA to the injury site, markedly improve wound healing in diabetic mice. Based on these
findings, I hypothesize that (i) topical wound treatment with advanced biomaterials capable of tailorable delivery
of mRNAs, corresponding to genes shown to promote DFU healing can lead to novel treatments. Further, I
hypothesize that (ii) altering the LNPs’ surface by conjugating antibodies targeting specific cell type receptors
can streamline their application and maximize their pro-reparative impact.
These hypotheses will be explored across three aims. The first aim will focus on fabricating alginate hydrogels
for effective topical delivery of mRNA loaded LNPs. During the second aim, I will employ multi-omics approaches
to establish LNP cell internalization and intracellular mRNA translation processes, together with particle
biodistribution, as well as to identify mechanisms of action. In the third aim, LNPs will be endowed with cell
specificity by conjugation of antibodies on their surface to maximize the platform’s efficiency and also evaluated
in a mouse model of increased clinical relevance. Successful completion of this multidisciplinary proposal will
result in highly novel data that will substantially expand our knowledge on DFU pathophysiology and advance
the fields of biomaterials, RNA therapeutics and cutaneous wound repair. This can lead to the development of
innovative much-needed interventions for the management of DFU.