Objective

Previous research in our laboratory has demonstrated the potential to encapsulate small molecule drugs into a human ferritin nanocage (HFn) mutant using an ordered assembly process. HFn is created very efficiently using our Biopart Assembly Standard for Idempotent Cloning (BASIC); a synthetic biology DNA assembly standard which enables the creation of fusion proteins by combining protein domains (GFP, human ferritin [HFn], IgG binding motif; targeting motifs) with fusion linkers. Drugs coordinate to 5 nm gold nanoparticles, and these then act as nucleation centres for the formation of ferritin nanocages. This leads to the encapsulation of drug-gold conjugates within a biocompatible nanocage. The key objective for this work is to develop HFn as a drug delivery vector capable of tissue- and cell-selective delivery of small molecule drugs. In the present study, a nanocage containing doxorubicin (dox) was formed by stoichiometric addition of HFn monomer to 5 nm gold nanoparticles loaded with dox. Drug encapsulation was measured by purifying the drug loaded nanocages using Size Exclusion Chromatography by HPLC and quantitating protein and drug by HPLC Q-TOF mass spectrometry after tryptic digest. We monitored the cytotoxic effects of HFn-dox delivery using a flow cytometry assay for cell viability. HFn-dox functionalised with a protein A domain facilitated uptake via an anti-EGFR antibody into HT-29 colonic adenocarcinoma cells. Cells treated with HFn-dox + anti-EGFR antibody presented a loss in viability due to cytotoxic effects of dox released from internalised HFn-dox. These data demonstrate the utility of functionalised HFn as viable drug delivery vectors in phenotypic cell screens and they could provide the basis of important biological tools for clinical drug delivery.

Methods

In the present study, a nanocage containing doxorubicin (dox) was formed by stoichiometric addition of HFn monomer to 5 nm gold nanoparticles loaded with dox. Drug encapsulation was measured by purifying the drug loaded nanocages using Size Exclusion Chromatography by HPLC and quantitating protein and drug by HPLC Q-TOF mass spectrometry after tryptic digest. We monitored the cytotoxic effects of HFn-dox delivery using a flow cytometry assay for cell viability.

Results

HFn-dox functionalised with a protein A domain facilitated uptake via an anti-EGFR antibody into HT-29 colonic adenocarcinoma cells. Cells treated with HFn-dox + anti-EGFR antibody presented a loss in viability due to cytotoxic effects of dox released from internalised HFn-dox.

Conclusion

These data demonstrate the utility of functionalised HFn as viable drug delivery vectors in phenotypic cell screens and they could provide the basis of important biological tools for clinical drug delivery.