Effect of Liposomal Encapsulation on the Chemical Exchange Properties of Diamagnetic CEST Agents

Exogenous chemical exchange saturation transfer (CEST) contrast agents such as glucose or 2-deoxy-d-glucose (2-DG) have shown high sensitivities and significant potential for monitoring glucose uptake in tumors with MRI. Here, we show that liposome encapsulation of such agents can be exploited to enhance the CEST signal by reducing the overall apparent exchange rate. We have developed a concise analytical model to describe the liposomal contrast dependence on several parameters such as pH, temperature, irradiation amplitude, and intraliposomal water content. This is the first study in which a model has been constructed to measure the exchange properties of diamagnetic CEST agents encapsulated inside liposomes. Experimentally measured exchange rates of glucose and 2-DG in the liposomal system were found to be reduced due to the intermembrane exchange between the intra- and extraliposomal compartments because of restrictions in water transfer imposed by the lipid membrane. These new theoretical and experimental findings will benefit applications of diamagnetic liposomes to image biological processes. In addition, combining this analytical model with measurements of the CEST signal enhancement using liposomes as a model membrane system is an important new general technique for studying membrane permeability.


I. Derivation of a six-site exchange model
For the derivation of a six-site model we consider the chemical exchange of the intraliposomal water magnetization with the extra-liposomal water magnetization. The intraliposomal magnetization is described by a five-site chemical exchange while the chemical exchange of the intra-liposomal water magnetization from a single hydroxyl group of glucose or 2-DG with the extra-liposomal water can be consider as a two-site system because it is described by a single exchange rate known as the intermembraneexchange rate.
A two-site exchange model is described by a set of differential equations known as Bloch-McConnell equations (S1-S6): From our simulations, we found that by setting 0 = 0 = 0 the resulting z-Magnetization will not change from its theoretical value. Taking this into account S1-S6 can be solved as follows: From S1 we derive: (S13) Then combing equations (S13) and (S10) we have = ( , ) and using equation (S10) and (S11) = ( , ) which will lead to = ( , ) (S14). (S14): Finally using (S12) and (S14): The longitudinal water magnetization for a five-site exchange model was derived from previous work 1 and can be written as follows: where DC is the duty cycle defined as DC= ( + ). ⁄ For shaped RF pulses 1 is described by the average 1 ̅̅̅̅̅ defined as follows: where Β is the fractional concentration of a single hydroxyl group from glucose or 2-DG and ΒA its chemical exchange rate with water. To expand this into a five site-exchange system we simply add another 3 terms (for the rest of the hydroxyl protons in glucose or to Equation (S17). Finally, (S17) is substituted to (S16) for calculating the exchange rates of hydroxyl groups in glucose or 2-DG. S5 PdI values measured by DLS must be converted into standard deviation (σ) values.

II. Calculation of encapsulation efficiency
When the particle size distribution can be fitted to a Gaussian distribution, the relationship between PdI and σ and the average hydrodynamic radius (r) can be described by the following equation:

III. Determining monosaccharide concentrations of liposomal samples
Overall and exterior glucose and 2-DG concentrations for liposome formulations were obtained using the Glucose GO Assay Kit ® supplied by Sigma-Aldrich. The kit is an enzymatic, colorimetric assay intended to measure glucose concentration utilising the enzyme, glucose oxidase. The assay reagent contains glucose oxidase (500 units Overall sugar concentrations for liposomal samples were measured after addition of Triton X-100 which was used to disrupt the liposome bilayer and cause uniform dispersion of the encapsulated contents throughout the total sample volume. Overall concentration test solutions consisted of DI water (490 µL), 3% Triton X-100 (5 µL) and liposomal sample (5 µL).
The assay reagent conditions were found to cause monosaccharide leakage from liposomes so in order to measure exterior sugar concentrations liposome samples were centrifuged at 4000 rpm for 5 minutes. When subjected to centrifugation liposomes formed a pellet and did not release encapsulated monosaccharide allowing 5 µL of the     *this liposome sample had been centrifuged and some exterior solution pipetted off to increase lipid and monosaccharide concentration. This was carried out to aid detection of small quantities of leakage in the early stages of the experiment.

VI. Release over time experiments
Glucose (L1) and 2-DG (L2 and L3) liposomes were incubated at 37 °C using a BIOER mixing block with slow agitation at 350 rpm. Before the start of an experiment the initial exterior monosaccharide concentration was confirmed to be negligible (< 1 mM) using the Glucose GO Assay ® and an aliquot of exterior solution was kept aside to obtain a 0 min data point in the assay conducted at the end of the experiment. Overall monosaccharide test solutions were obtained as usual (5 µL liposomes, 5 µL 3% Triton).
Once heating at 37 °C was commenced, aliquots (40 µL) were taken from the incubated liposome sample at regular time points, decanted into a 0.35 mL Eppendorf, dipped in an ice bath to immediately stop leakage and then stored in the fridge until the end of the experiment. Once all time points aliquots had been collected, the aliquots were centrifuged at 10,400 rpm and 4 °C for 1 h, and 5 µL of supernatant was pipetted off to be used in the Glucose GO Assay ® to determine exterior monosaccharide concentration.
Determining exterior concentrations for all time points in a single assay was found to be more accurate than conducting several assays throughout the experiment. Following completion of the assay, the A 540 of test solutions were measured in triplicate and readings obtained for the original exterior monosaccharide and overall monosaccharide S14 concentrations (measured in the same assay) were used to convert each time point A 540 reading into a percentage leakage value ( Figure S2). Figure S2. Release of glucose 2-DG from liposome formulations L1-3 (Table S20) Figure S4. Simulated Z-spectra obtained from a six-site chemical exchange with Rcest = 10, B 1 = 1.5 μT.   Figure S6. Simulated Z-spectra obtained from a six-site chemical exchange for various Rcest at B 1 = 1.5 μT and 5.06 μT. 5-site Z-spectra are displayed for comparison.