The potential tissue repair properties of stem cells have prompted their investigation for use in multiple clinical applications. The most recognisable is the use of stem cell transplants for regenerative medicine. For such clinical applications, not only is it important to identify the position of transplanted cells within tissue but it is also vital to monitor the state of the cell. In particular for stem cell transplants, identifying whether delivered stem cells have differentiated into the desired mature cells would be desirable. This technology is missing.Non-invasive imaging techniques are already playing a critical role in the development of clinical applications of cell transplants. Herein a method of single cell detection is proposed, where contrast evolves via the enzymatically triggered release of magnetic nanocrystals from cellulose triacetate encapsulated iron oxide nanoparticles.

Encapsulation of Fe₃O₄ within cellulose-triacetate.
Fe₃O₄ nanocrystals were synthesized using the thermal decomposition of iron oleate¹. Nanocrystals were encapsulated at 25% w/v within cellulose triacetate (Sigma-Aldrich,USA) by means of emulsification² or nanoprecipitation³. In brief 10mgmL^-1 25% w/v cellulose triacetate was dissolved in DCM and added 1:4 dropwise into 1% PVA solution, tip sonicated and added to dH₂O. Nanoprecipitation encapsulation was achieved by dissolution of 10mgmL^-1 cellulose triacetate in acetone and precipitated into 1:4 dH₂O.

Measurement of particle enzymatic degradation.
Nanoparticle formation and diameter was measured by SEM using an AURIGA® CrossBeam Dual Column SEM-FIB Workstation (Carl Zeiss,Germany). Images were taken before and after cellulase incubation. Cellulase activity was determined by conversion of cellulose into glucose using a cellulase assay (Worthington-Biochemical,USA). 0.1mgmL^-1 of nanoparticles were incubated with 1% v/v cellulase at 37^oC. Released glucose was determined in a hexokinase/glucose-6-phosphate dehydrogenase system and measured at 340 nm.

Measurement of relaxivity, r₂*.
Nanoparticles were incubated for 24 hours, 37^OC with or without 0.1mgmL^-1 cellulase (Worthington-Biochemical,USA) then suspended in 1% (w/v) agarose at 0.5 and 0.1 mgmL^-1 of iron. T₂* measurements were made using a 7T Bruker BioSpec 70/30 USR with Paravision 6.0, using a large volume transmit/receive coil to acquire multi-gradient echo sequence at resolution of 400x400 um with slice thickness 1 mm. T₂* was measured 3 times in each tube then r₂* relaxivity was calculated for each particle type.

Cellular uptake.
NP uptake was investigated using coumarin 6 loaded NP's. In brief, mMSC's were incubated for 3 hours with 0.1mgmL^-1 cellulose triacetate NP's. Fixed, permeabilized cells were subsequently visualized using phalloidin (cytoskeleton) and DAPI (nuclei) staining. Images were taken on

Data analysis and statistics.
A one-way ANOVA was undertake to assess the statistical significance of differences between mean values of cellulase activity and again between r₂* values before and after cellulase addition. A P value of <0.05 was considered significant.

SEM images showed the successful fabrication of 300-400nm nanoparticles (Figure 1a), which underwent degradation in the presence of 0.1mgmL^-1 cellulase (Figure 1b). The amount of cellulase activity (units/mg) was measured over 24 hour (Figure 1c) which convincingly showed that cellulase degraded cellulose triacetate nanoparticles. Statistical analysis also showed that the activity of cellulase was similar upon cellulose triacetate NP’s and cellulose NP’s.After particle incubation with cellulase, a significant increase in relaxivity was seen (figure 2). Those NP’s fabricated by nanoprecipitation (figure 2b) demonstrated a 3 fold increase in r₂*, successfully indicating that enzymatically degrading the cellulose biopolymer coat results in a change in contrast (Figure 2c). Incubation of cellulose triacetate NP's with mMSC's showed evidence of cellular uptake (figure 3).

Cellulose triacetate nanoparticles can undergo triggered enzymatic degradation by cellulase, in a similar way to cellulose only nanoparticles. Successful removal of the cellulose triacetate by enzymatic degradation results in the modulation of relaxivity, r₂* and evolution of contrast as encapsulated Fe₃O₄ is released.These nanoparticles were demonstrated as capable for endocytosis by mMSC's making them suitable for cell labelling.

Cellulase degradable cellulose triacetate NP’s offer promise as an in-situ cellular imaging agent. The small NP diameter makes such particles ideal for cellular endocytosis. The long term vision of this work is to create transgenic stem cells which express catabolic enzymes under the control of a promoter activated during stem cell differentiation. Following expression, these enzymes will be shuttled to the endosome/lysosome. Co-localization of the enzyme and nanoparticles within the endosome/lysosome during differentiation will then allow for the evolution of contrast.