Magnetic Heating Stimulated Cargo Release with Dose Control using Multifunctional MR and Thermosensitive Liposome

Abstract

Rationale: Magnetic resonance imaging (MRI) is one of the most widely used diagnostic tools in the clinic. In this setting, real-time monitoring of therapy and tumor site would give the clinicians a handle to observe therapeutic response and to quantify drug amount to optimize the treatment. In this work, we developed a liposome-based cargo (cancer drugs) delivery strategy that could simultaneously monitor the real-time alternating magnetic field-induced cargo release from the change in MRI relaxation parameter R₁ and the location and condition of liposome from the change in R₂. The tumor site can then be monitored during the cargo release because liposomes would passively target the tumor site through the enhanced permeability and retention (EPR) effect. Physical insights from the experimental results and corresponding Monte Carlo spin dynamics simulations were also discussed. Methods: Superparamagnetic iron oxide (SPIO) nanoparticles, diethylenetriaminepentaacetic acid gadolinium(III) (Gd(III)-DTPA), and a model cancer drug (fluorescein) were co-loaded in PEGylated thermosensitive liposomes. The liposomes were characterized by transmission electron cryo-microscopy (cryoTEM), dynamic light scattering (DLS), and inductively coupled plasma optical emission spectrometry (ICP-OES). Alternating magnetic field (AMF) was used to create controlled mild hyperthermia (39-42°C) and facilitate controlled cargo (fluorescein) release from the thermosensitive liposomes. MRI relaxation parameters, R₁ and R₂, were measured at room temperature. The temporal variation in R₁ was used to obtain the temporal profile of cargo release. Due to their similar sizes, both the gadolinium and cargo (model cancer drug fluorescein) would come out of the liposomes together as a result of heating. The temporal variation in R₂ was used to monitor SPIO nanoparticles to enhance the tumor contrast. Monte Carlo spin dynamics simulations were performed by solving the Bloch equations and modeling SPIO nanoparticles as magnetized impenetrable spheres. Results: TEM images and DLS measurements showed the diameter of the liposome nanoparticle ~ 200 nm. AMF heating showed effective release of the model drug. It was found that R₁ increased linearly by about 70% and then saturated as the cargo release process was completed, while R₂ remained approximately constant with an initial 7%-drop and then recovered. The linear increase in R₁ is consistent with the expected linear cargo release with time upon AMF heating. Monte Carlo spin dynamics simulations suggest that the initial temporal fluctuation of R₂ is due to the plausible changes of SPIO aggregation and the slow non-recoverable degradation of liposomal membrane that increases water permeability with time by the heating process. The simulations show an order of magnitude increase in R₂ at higher water permeability. Conclusion: We have performed MR parameter study of the release of a cargo (model cancer drug, fluorescein) by magnetic heating from thermosensitive multifunctional liposomes loaded with dual contrast agents. The size of the liposome nanoparticles loaded with model cancer drug (fluorescein), gadolinium chelate, and SPIO nanoparticles was appropriate for a variety of cancer therapies. A careful and detailed analysis with theoretical explanation and simulation was carried out to investigate the correlation between MRI relaxation parameters, R₁ and R₂, and different cargo release fractions. We have quantified the cargo release using R₁, which shows a linear relation between each other. This result provides a strong basis for the dosage control of drug delivered. On the other hand, the fairly stable R₂ with almost constant value suggests that it could be used to monitor the position and condition of the liposomal site, as SPIO nanoparticles mostly remained in the aqueous core of the liposome. Because our synthesized SPIO-encapsulated liposomes could be targeted to tumor site passively by the EPR effect, or actively through magnetofection, this study provides a solid ground for developing MR cancer theranostics in combination of this nanostructure and AMF heating strategy. Furthermore, our simulation results predict a sharp increase in R₂ during the AMF heating, which opens up the exciting possibility of high-resolution, high-contrast real-time imaging of the liposomal site during the drug release process, provided AMF heating could be incorporated into an MRI setup. Our use of the clinically approved materials, along with confirmation by theoretical simulations, make this technique a promising candidate for translational MR cancer theranostics.

Keywords: AMF-controlled drug release; alternating magnetic field (AMF); magnetic hyperthermia; magnetic resonance theranostics; thermosensitive multifunctional liposome.

Figure 1

A schematic diagram of model drug/cargo (fluorescein) and contrast agents (magnetic nanoparticles and Gd(III)-DTPA) released from thermosensitive PEGylated liposomes under mild hyperthemia with alternating magnetic field (AMF). It illustrates the liposomes membrane permeability change and release of Gd(III)-DTPA and fluorescein upon AMF-induced heating.

Figure 2

Characterization of liposomes. (A) Dynamic light scattering (DLS) of synthesized liposomes. It demonstrates the distribution of nanoparticles with an average diameter 231 nm and polydispersity 0.134. (B) CryoTEM images show the homogeneous distribution and morphology of liposomes in different magnifications. They show an average diameter around 200 nm and successful formation of bilayered spherical liposomes. The darker regions mark the presence of SPIO inside the liposome. A representative red circle is drawn to show such an SPIO concentrated region. The magnified liposome in blue box of the right panel shows the SPIO encapsulation inside the aqueous core of liposomes. Larger aggregates of SPIO appeared as darker spots.

Figure 3

Cumulative cargo (fluorescein) release profile. (A) Cumulative cargo (fluorescein) release profile is shown as a function of the cumulative AMF heating time. Dotted red curve demonstrates the percentage of cargo release at different time. The characteristic pattern indicates the initial slow rate of release, then an increased rate of release between 30 to 45 minutes, and finally leveling off, indicating the complete release. Dotted black curve shows the spontaneous release of cargo from liposomes without AMF treatment and flat line depicts no leakage is observed otherwise. (B) Cumulative cargo (fluorescein) release profile is shown as a function of the maximum bulk temperature (before cooling to room temperature). Fluorescence measurements were performed at room temperature. It demonstrates that the maximum change in the bulk temperature during the process from no cargo release to complete release is about 3°C. The dotted lines through the data points are included in each case as a guide to the eye (see text for details).

Figure 4

Correlation between changes in MR relaxation rates and cargo (fluorescein) release. (A) Red curve indicates the percentage change in R₁ and black curve indicates the percentage change in R₂. It demonstrates R₁ linearly increases with cargo release, and shows maxima after 69.8% increase, when cargo release was complete. R₂ is held nearly constant; however, it shows an initial 7%-drop (from the second to the third point, corresponding to the path of no cargo release to about 6% cargo release) and then a gradual increase to the initial value. Please see the text for the plausible explanation. The dotted lines through the data points are included in each case as a guide to the eye. (B) Fitted linear plot for percentage change in longitudinal relaxation rate (R₁) with percentage cargo (fluorescein) release. The fitted linear equation: % Change in R₁ = 0.686 × (% Change in cargo release) with squared correlation coefficient R² = 0.997.

Figure 5

MR relaxation rates were measured at different dilutions before and after complete AMF heating. (A) R₁ versus Gd concentration before and after complete AMF heating. It shows the slope of the fitted linear plot before (black) r₁= 7.84 s^-1 mM^-1 (Gd) and after complete AMF heating (red) r₁ = 13.38 s^-1 mM^-1 (Gd). (B) R₂ versus Fe concentration before and after complete AMF heating. It shows the slope of the fitted linear plot before (black) r₂= 108.01 s^-1 mM^-1 (Fe) and after complete AMF heating (red) r₂=108.93 s^-1 mM^-1 (Fe). The uncertainties on R₁ and R₂ are < 1%, whereas the uncertainties on the concentration are ~ 5%.

Figure 6

The effects of liposome membrane permeability and SPIO aggregation inside liposome on CPMG R₂ relaxation rates. (A) CPMG relaxation rates R₂ are plotted as a function of the liposome membrane permeability at three different aggregation states with D_pp (inter-SPIO nanoparticle distance) / r_p (single SPIO radius) = 2, 6, and 10, respectively. The radius of a single SPIO, r_p = 5 nm. (B) CPMG relaxation rates R₂ are plotted as a function of the degree of aggregation, Dpp/r_p, at different liposome membrane permeability conditions. The dotted lines through the points are included in each case as a guide to the eye.