MRI Monitoring of Tumor-Selective Anticancer Drug Delivery with

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Article

MRI monitoring of tumor-selective anticancer drug delivery with stable thermosensitive liposomes triggered by high-intensity focused ultrasound Hyun Ryoung Kim, Dong Gil You, Sang-Jun Park, Kyu-Sil Choi, Wooram Um, Jae-Hun Kim, Jae Hyung Park, and Young-sun Kim Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00013 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 27, 2016

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Molecular Pharmaceutics

MRI monitoring of tumor-selective anticancer drug

delivery

with

stable

thermosensitive

liposomes triggered by high-intensity focused ultrasound Hyun Ryoung Kim*,†, Dong Gil You#, Sang-Jun Park†, Kyu-Sil Choi§, Wooram Um#, Jae-Hun Kim‡, Jae Hyung Park#,∥, Young-sun Kim*, ‡ †

Bio Therapeutics Laboratory, Samsung Advanced Institute of Technology (SAIT), Samsung

Electronics Co., Ltd., #130, Samsung-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do, 443-803, South Korea §

Laboratory

Animal

Research

Center,

Samsung

Biomedical

Research

Institute,

Sungkyunkwan University School of Medicine, #50, Irwon-dong, Gangnam-gu, Seoul 138225, South Korea #

School

of

Chemical

Engineering,

College

of

Engineering,

Sungkyunkwan

University, Suwon 440-746, South Korea ‡

Department of Radiology and Center for imaging Science, Samsung Medical Center,

Sungkyunkwan University School of Medicine, #50, Irwon-dong, Gangnam-gu, Seoul 138225, South Korea ∥Department

of

Health

Sciences

and

Technology,

University, Suwon 440-746, South Korea 1

ACS Paragon Plus Environment

SAIHST,

Sungkyunkwan


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KEYWORDS: thermosensitive liposome, MRI, heat-triggered drug release, elastin-like polypeptide, drug release monitoring, intratumoral accumulation

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ABSTRACT Monitoring of drug release from a heat-activated liposome carrier provides an opportunity for real-time control of drug delivery and allows prediction of the therapeutic effect. We have developed short-chain elastin-like polypeptide-incorporating thermosensitive liposomes (STL). Here, we report the development of STL encapsulating gadobenate dimeglumine (GdBOPTA), a MRI contrast agent, and doxorubicin (Dox) (Gd-Dox-STL). The Dox release profile from Gd-Dox-STL was comparable to Gd-Dox-LTSL, however, the serum stability of Gd-Dox-STL was much higher than Gd-Dox-LTSL. MRI studies showed that the difference in T1 relaxation time between 37°C and 42°C for Gd-Dox-STL was larger than the difference for Gd-Dox-LTSL. Although relaxivity for both liposomes at 42°C were similar, the relaxivity of Gd-Dox-STL at 37°C was 2.5-fold lower than Gd-Dox-LTSL. This was likely due to Gd-BOPTA leakage from the LTSL because of low stability at 37°C. Pharmacokinetic studies showed plasma half-lives of 4.85 h and 1.95 h for Gd-Dox-STL and Gd-Dox-LTSL respectively, consistent with in vitro stability data. In vivo MRI experiments demonstrated corelease of Dox and Gd-BOPTA from STL under mild hyperthermia induced by high-intensity focused ultrasound (HIFU), which suggests STL is a promising tumor selective formulation when coupled with MR-guided HIFU.

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INTRODUCTION

The ideal chemotherapeutic regimen would be highly effective therapy by concentrating drugs at the tumor site, and minimizing exposure to healthy tissues thereby decreasing adverse effects. To increase therapeutically effective drug delivery to the tumor and minimize damage to normal tissue, target-specific drug delivery is essential. Nanosized colloidal carriers coated with a hydrophilic polymer, e.g., polyethylene glycol (PEG), show prolonged survival in the circulation, owing to a stealth effect that makes the particles “invisible” to macrophages by reduced opsonization on the surface.1-3 Because of leaky vasculature4 and poor lymphatic drainage5 in the solid tumor microenvironment, PEGylated nanocarriers can selectively extravasate from blood vessels through the permeable endothelium and accumulate at the tumor site (enhanced permeability and retention effect).6-7 Liposomeencapsulated doxorubicin (Dox) (Doxil®) is a representative nanomedicine that has prolonged survival in the circulation. Compared to free Dox, this liposome formulation successfully reduced acute cardiotoxicity. However, the therapeutic efficacy of Doxil® was not substantially improved compared to free Dox, in spite of its enhanced tumor targeting.8-9 The reason may be that drug release from the nanocarrier was not sufficient even though liposomes accumulated in the tumor. Thus, a triggering mechanism for drug release should be incorporated into the nanomedicine to improve bioavailability of drug. One of the most advanced triggering systems is a liposomal carrier activated by mild hyperthermia, i.e., lysolipid-based temperature sensitive liposome (LTSL, Thermodox®).10-15 To further improve stability and heat responsiveness, several other heat-triggered platforms have been reported such as block copolymer-incorporated liposomes,16,17 peptide-lipid 4


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hybrids,18 synthetic lipid containing liposomes,19-20 and surfactant-inserted liposomes.21 Thermodox® is currently being evaluated in several ongoing clinical trials for the treatment of breast cancer and bone metastases by using heating sources such as microwave or high intensity focused ultrasound (HIFU). These trials are continuing in spite of the failure of a Phase III study on Thermodox® in combination with radiofrequency ablation for the treatment of primary hepatocellular carcinoma. Direct in vivo drug release monitoring at site of heat application may help understand the reasons for this failure. Drug release monitoring and delivery tracking of thermosensitive liposomes may provide real-time information regarding the treatment efficiency specifically at the target site. When the thermosensitive liposome co-encapsulating MRI contrast agent and anticancer drug is delivered to the tumor site and is heated through HIFU application, it is able to release the contents simultaneously. The effectiveness of drug delivery and drug release by heating can be assessed by the detection of MRI contrast agent through MRI modality.22-25 Through this approach, the prediction of delivery efficiency after treatment is feasible along with individually tailored delivery parameters according to the individual vasculature and tumor microenvironment. LTSL loaded with a gadolinium-based MRI contrast agent and Dox (GdDox-LTSL) was investigated by several researchers.26-30 Gd-Dox-LTSL had low stability of in serum-containing media and this may be because the lysolipid might readily interact with proteins (e.g., albumin) and induce drug leakage from the liposomes.30-31 To enhance stability of liposomal membranes and increase the potential for drug monitoring, surfactant-coated liposomes and liposomes inserted with synthetic polymers were created.32-33 However, toxicity remained a problem because of non-biodegradability, which limits their clinical application. 5



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We previously reported in vitro and in vivo evaluation of a short chain elastin-like polypeptide (ELP) conjugate incorporated into thermosensitive liposomes (STL). ELP, consisting of [VPGXG]n (X≠Proline) pentapeptide repeat, was used as heat-triggered moiety in STL because of the temperature-dependent phase transition property. STL were highly stable under physiological conditions (37°C), and were capable of rapid release of encapsulated drug with mild hyperthermia (42°C).34 In the current study, we developed STL encapsulating gadobenate dimeglumine, clinically used MRI contrast agent, and anticancer drug Dox (Gd-Dox-STL). We hypothesized that Dox release at the target tumor site could be monitored by MRI when HIFU-induced mild hyperthermia was applied to the tumor after administration of Gd-Dox-STL (Fig. 1). The in vitro Dox release profile from the liposomes and the stability of liposomes under physiological conditions were measured. We compared in vitro MRI performance between Gd-Dox-STL and Gd-Dox-LTSL. In addition, we carried out pharmacokinetics and biodistribution studies in normal mice and in vivo evaluation of the theranostic potential in a mouse tumor model by combining liposomes with HIFU stimuli and MRI.

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Figure 1. A) Schematics of in vivo MRI imaging in a mouse. Thermosensitive liposomes loaded with gadobenate dimeglumine (Gd-BOPTA) and doxorubicin (Dox) and incorporating short chain elastin-like polypeptides (Gd-Dox-STL) were intravenously injected through the tail vein of mice and MRI imaging was recorded. B) Gd-BOPTA and Dox were simultaneously released from Gd-Dox-STL by heat stimuli of high intensity focused ultrasound (HIFU). C) HIFU was applied to tumor site and Gd-BOPTA and Dox were released into blood vessel close to tumor. Dox tracking can be performed by T1 enhanced MRI imaging of gadolinium contrast agent.

EXPERIMENTAL SECTION

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Materials.

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1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

glycero-3-phosphocholine

(DSPC),

(DPPC),

1,2-distearoyl-sn-

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(polyethylene glycol)-2000] (DSPE-PEG), 1-stearoyl-sn-glycero-3-phosphocholine (MSPC), and cholesterol were purchased from Avanti Polar Lipid, Inc. (Alabaster, AL, USA). Chemically synthesized elastin-like polypeptide (ELP), [VPGXG]n, and modified ELPs conjugated with lipid (C18-ELP) were provided by Peptron, Inc. (Daejeon, Korea). Doxorubicin (Dox) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Gadobenate dimeglumine

(Gd-BOPTA),

with

the

IUPAC

name,

2-[2-[2-[bis(2-oxido-2-

oxoethyl)amino]ethyl-(2-oxido-2-oxoethyl)amino]ethyl-(2-oxido-2-oxoethyl)amino]-3phenylmethoxypropanoate;

gadolinium

(3+);

hydron;

(2R,3R,4R,5S)-6-

(methylamino)hexane-1,2,3,4,5-pentol, (Multihance®) was obtained from Bracco Diagnostics (Ferentino, Italy.).

Preparation of Thermosensitive Liposomes Loaded with Gd-BOPTA and Dox. Dox was loaded into liposomes formulated with DPPC/DSPC (75/25 mol %):DSPE-PEG2000:cholesterol

= 55:2:10 (mol ratio) using an ammonium sulfate gradient method. In

summary, lipids were dissolved in chloroform and the solvent was removed under reduced pressure. The thin film of lipids was hydrated at 60°C in 250 mM ammonium sulfate containing 125 mM Gd-BOPTA by vortexing and sonication. The liposome suspension was extruded at 60°C through polycarbonate membrane successively with 400 nm, 200 nm, and 100 nm pore sizes to facilitate formation of homogeneous liposomes. The exterior buffer of the liposome suspension was exchanged with 25 mM Tris⋅HCl (pH 9.0) by size exclusion chromatography using a Sephadex (G-50) column. Dox was added to the liposome 8


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suspension at 1:0.2 ratio (w/w, DPPC:Dox), and the mixture of liposome suspension and Dox was incubated at 37°C for 1 h. Subsequently, 2 mol % of the lipid-conjugated ELP was added to the liposome and incubated at 25°C for 1 h. Finally, unloaded Dox and lipid-conjugated ELP were removed by size exclusion chromatography, using phosphate buffered saline (PBS, pH 7.4) as the eluent. For LTSL loaded with Dox and Gd-BOPTA contrast agent (Gd-DoxLTSL), LTSL was formulated with DPPC:MSPC:DSPE-PEG-2000 = 90:10:4, prepared using a previously published procedure.30 The concentration of Dox was estimated from the absorbance at 490 nm (UV-VIS Spectrometer) of the liposome dissolved in dimethyl sulfoxide (DMSO) after purification with the Sephadex (G-50) column. The concentration of phosphorus and gadolinium (Gd) were measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) using an ICPS-8100 (Shimadzu, Japan), after the destruction of liposomes with nitric acid at 180°C.

Size and Morphology of Liposomes. Liposome diameters were determined by dynamic light scattering (DLS) at 25°C using a Zetasizer Nano ZS (Malvern Instruments, UK) with a He-Ne laser at a wavelength of 633 nm and a detection angle of 90°. The Gd-Dox-STL morphology was observed using a cryogenic transmission electron microscope (cryo-TEM). Samples for cryo-TEM were prepared on porous carbon film-supported grids. Thin aqueous film blotted with filter paper was fabricated by Vitrobot (FEI) and immediately plunged into liquid ethane. The resulting grids were stored in liquid nitrogen and transferred to a cryotransfer holder (Gatan). Cryo-TEM images were obtained using a CCD camera (2k,

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Gatan) with a Tecnai F20 field emission gun electron microscope operated at 200 kV (FEI) in low dose mode.

Doxorubicin Release from Liposomes. Since the fluorescence of concentrated Dox inside liposomes is quenched and only the fluorescence of released Dox can be measured, Dox release from Gd-Dox-STL and Gd-Dox-LTSL was determined by measuring the fluorescence intensity of the suspension liquid. Each aliquot of liposome suspension was incubated in a preheated chamber at different temperatures for 5 min, or the corresponding time for time course of Dox release, then the fluorescent intensity was monitored at 615 nm with an excitation wavelength of 490 nm by a fluorescence spectrometer (PerkinElmer, Envision 2104-multilabel reader). The percentage of Dox released was calculated according to the following equation:

% release =

( −

) × 100 ( −

)

in which Ft and Fi respectively denote the fluorescent intensities of the heated and initial liposome suspension. Ff is the fluorescent intensity of the liposome solution after addition of 1% Triton X100 containing ethanol, to completely disrupt the liposomes. To compare the stability of Gd-Dox-STL and Gd-Dox-LTSL under physiological conditions, fluorescence was measured after different incubation times at 37°C in a culture medium with 10% serum, and calculated using the above equation.

In Vitro Magnetic Resonance Imaging. To obtain T1 (relaxation time) weighted-images and longitudinal relaxation rate (R1), Gd-Dox-STL and Gd-Dox-LTSL were incubated at 37°C 10


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and 42°C for 5 min in an agarose gel (1% w/v). The quantification graph of T1-weighted image was analyzed using Image J 1.48v (Wayne Rasband, National Institutes of Health, USA). The T1 relaxation times were measured by a spin-echo pulse sequence on a 4.7 T MRI system (Biospec 47/40, Bruker, Karlsruhe, Germany) for both liposomes containing different concentrations of Gd-BOPTA in a range from 0.05 to 0.5 mM. The MRI parameters used were as follows: TE/TR = 9.5/350 ms, NEX = 4, FOV = 50 × 60 mm2, matrix size = 192 × 192, slice thickness = 1 mm. The relaxivity was determined by plotting the 1/T1 as a function of the concentration of Gd.

Tumor-Bearing Animal Model. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), and our facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International, protocol No. H-A9-003). The murine squamous cell carcinoma, SCC-7 cell line was grown in 150-cm2 dishes containing 20 mL of culture medium with 1% antibiotics at 37°C in a 5.0% CO2 atmosphere and 95% relative humidity. Cells were detached from the culture dish with cell dissociation solution (Sigma-Aldrich) and collected. SCC-7 cells (1×106) suspended in 50 µL in Dulbecco's phosphate-buffered saline (Gibco). The suspension was mixed with Matrigel (BD Bioscience, Bedford, MA, USA) at a 1:1 ratio for a total cell suspension volume of 100 µL. BALB/c nude mice (5-week old male, 20 ± 3 g) (Orient Bio, Sungnam, Korea) were used for the in vivo tumor-bearing animal model. SCC-7 cell suspension was subcutaneously injected into the lateral part of the both thighs, since breathing motions there were minimal during HIFU therapy.

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Pharmacokinetics and Biodistribution Studies. To measure the pharmacokinetic properties of the liposomes, Gd-Dox-STL and Gd-Dox-LTSL (5 mg of DOX/kg) were injected into BALB/c mice (Orient, Seongnam, Korea) via the tail vein as previously reported.34 Pharmacokinetics and biodistribution experiments were carried out with unheated mice model. Blood was collected from the tail vein at various time points (30 min to 24 h) after injection. Gd quantities in the blood were analyzed by ICP-AES (Agilent 7500A, Palo Alto, CA, USA). The plasma half-lives of Gd-Dox-STL and Gd-Dox-LTSL were calculated from the regression curve obtained by four-parameter logistic curve fitting using SigmaPlot (Systat Software Inc., San Jose, CA, USA). In order to determine the biodistribution of Gd-Dox-STL and Gd-Dox-LTSL, SCC-7 cells (5×106 in 0.3 ml PBS) was subcutaneously injected into the thigh of BALB/c nude mice. The tumor was allowed to grow for a week, reaching ~10 mm in diameter. At twenty-four hours after intravenous injection of the liposomes (5 mg of DOX/kg) the mice were sacrificed and samples of muscle, liver, heart, spleen, kidney, and tumor were excised. The tissue samples were washed with PBS and weighed after being completely dried at 36°C for 48 h. Gd quantities in organs were analyzed by ICP-AES (Agilent 7500A).

HIFU System. An animal HIFU system (Therapy Imaging Probe System, Phillips Research, Briarcliff Manor, NY, USA) was used to generate pulsed HIFU. The natural focus and diameter of the annular array transducer (8 elements) were both 80 mm. The focal zone was 1.5 mm × 1.5 mm × 6.0 mm (with a center sonication frequency of 1.0 MHz, at -6 dB). Tumor-bearing mice were held on a custom-made treatment bed equipped with an acoustic absorber. The HIFU transducer and part of the mouse’s body were submerged in water for acoustic coupling. The water was degassed (dissolved oxygen level, 1-2 ppm) for 2 h before 12


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treatment using a Stand-Alone Inline Degasser (Philips Research). A thermostatically controlled heater (NTT-2200, Eyela, Tokyo, Japan) was used to maintain degassed water at 37°C. Re-oxygenation was minimized with small floating polypropylene balls (diameter, 20 mm; Cole-Parmer, Vernon Hills, IL, USA).

HIFU-Induced Hyperthermia. During all hyperthermia treatments, the mice were anesthetized by inhalation of isoflurane (Forane; Baxter, Deerfield, IL, USA). HIFU parameters were determined based on the previous studies34,35 and pilot experiments in which iterative adjustments were made based on direct temperature measurements using a thermocouple wire (diameter = 50 µm, unsheathed; Physitemp Instruments Inc., Clifton, NJ, USA) inserted directly into the tumor. HIFU was applied for 30 min to the cranial part of the tumor along a trajectory composed of nine spots (3 × 3 configuration, 1-mm interval between spots, 5 s per spot, and 40 cycles total). The following parameters were chosen: total acoustic power of 16 W, sonication frequency of 1.0 MHz, pulse repetition frequency of 5 Hz, and duty cycle of 50% (i.e., 100 ms on and 100 ms off). These parameters were determined to yield an average temperature at the focus point between 42-43°C.

In Vivo MRI Data Acquisitions for T1 Mapping. All mouse MRI images were obtained using a horizontal 7.0 T MRI System (Bruker Biospin, Billerica, MA, USA) equipped with a 20 cm gradient set capable of supplying up to 400 mT/m with a 100 µs rise-time. A birdcage coil (72 mm i.d., Bruker Biospin) was used for excitation, and an actively decoupled phased array coil was used for signal reception. The head of the mouse was carefully fixed using a bite/ear bar and two flexible cushions were used to fix the position of the thigh tumors before 13



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placement in the magnet. Axial images including whole tumor regions were acquired with a RARE with variable repetition time (RARE-VTR) sequence by using the following parameters: imaging matrix, 150 × 94 (reconstruction of 150 × 125 matrix); imaging resolution, 200 × 200 µm2; RARE factor, 2; effective TE, 10.7 ms; multiple TR, 575, 600, 800, 1500, 3000, 5000 ms; and slice thickness, 1.5 mm without any interslice gap.

MRI Data Analysis. Saturation recovery T1 mapping method was used to estimate T1 value for each voxel. T1 was calculated by fitting signal intensities into three-parameter exponential curves using the following equation: M (t) = M

0

× (1 − A × exp ( − TR/T 1)) ,

where M(t) is the signal intensity at a particular TR, M0 is the equilibrium signal, and A is a factor to account for incomplete inversion. Nonlinear least-squares fitting was implemented using the lsqnonlin function of MATLAB R2009a (The MathWorks, Natick, MA, USA). A region of interest analysis was performed in order to average regional T1 values for the cranial half of the tumors. The changes in the longitudinal relaxation rates between before and after HIFU therapy were then calculated (∆R1 = ∆(1/T1)).

In Vivo Evaluation of MRI Monitoring and HIFU-Induced Mild Hyperthermia. These experiments were performed on the 8th day after tumor cell injection, when average tumor volume was approximately 250 mm3 (7-8 mm in diameter, ± 25%). The mice were randomly allotted into the treatment group (n = 6) or the control group (n = 3). In the treatment group, 0.2 mL of Gd-Dox-STL containing 4 mM of Gd-BOPTA and 0.1 mg of Dox was injected through the tail vein. The same volume of PBS was administered in the control group. In both 14


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groups, HIFU therapy was performed only on the right side tumor immediately after drug administration. This resulted in four sub-groups based on the administration of Gd-Dox-STL or vehicle and the presence or absence of HIFU-induced hyperthermia. MRI was performed before and after HIFU therapy. Baseline MRI was conducted under anesthesia. The mice were anesthetized with isoflurane (5% for induction, 2% for the animal set-up and 1-1.5% during the MR experiment) in a mixture of O2 and N2 gases (3:7) delivered to a nose cone with spontaneous respiration throughout the experiment. HIFU therapy was performed for 30 min using the method described above. Immediately after HIFU therapy, the mice were euthanized in order to prevent washout of the drug from the tumor, and a postmortem MRI was carried out. After MR evaluation, the cranial half of each tumor specimen was collected to assess drug accumulation in the tumor. For Dox quantification, the tissues were homogenized after adding lysis buffer. The homogenized tissue was fully mixed with lysis buffer (0.1 mL), distilled water (0.1 mL), 10% Triton X (octyl-phenol-polyoxyethylen-ether, Sigma) (0.05 mL), and indophenyl acetate (Sigma-Aldrich) (0.75 mL) and kept at -40°C for 12 h. Then the sample was centrifuged for 10 min at 1200 rpm, and the supernatant (0.1 mL) was collected and placed in a 96-well plate. Dox fluorescence was evaluated using a multi-label counter (Ex 485 nm/Em 572 nm) (Wallac VICTOR2 1420 Multilabel HTS counter, PerkinElmer, Waltham, MA, USA). For Gd quantification, inductively coupled plasma mass spectrometry (ICP-MS, Nexion 300, PerkinElmer) was utilized after preparing the specimens as described above.

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Statistical Analysis. The Mann-Whitney test was used to compare the values of ∆R1 in MRI and Dox fluorescence between each tumor group in the in vivo experiment. Data were expressed as means ± standard error of mean. A p-value of < 0.05 was considered statistically significant. Data were analyzed using IBM SPSS Statistics 19.0 (International Business Machines Corp., Armonk, NY, USA).

RESULTS Preparation and Characterization of the Liposomes. Gd-BOPTA, a MRI contrast agent, was employed for monitoring of drug release from thermosensitive liposomes. In order to optimize Gd-BOPTA encapsulation into STL, we tried two different procedures, one was to load Gd-BOPTA in the hydration buffer, (initial loading; IL), and the other was to incubate Gd-BOPTA with the STL in an additional step after the extrusion (additional loading; AL) (Supplemental Fig. 1). To enhance the permeability of liposome membrane during AL, different temperatures (25, 37, 42, and 60°C) were tested. Dox and Gd-BOPTA loading were measured and 42°C was determined as the optimal temperature (data not shown). Gd-DoxLTSL was also prepared and Gd-BOPTA was loaded in the hydration buffer (initial loading) according to a previously published method.30 Phosphorus and Gd concentration in Gd-DoxSTL and Gd-Dox-LTSL are shown in Table 1. The gadolinium/phosphorus (Gd/P) concentration ratio indicates the Gd loading efficiency in the liposome.

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Table 1. Concentration of phosphorus and gadolinium of STLs prepared by different procedure, GdBOPTA loading at the hydration step (initial loading: IL) and additional step after extrusion (additional loading: AL), and LTSL.

STL

STL

(initial loading: IL)

(additional loading: AL)

Phosphorus (mM)

14.21

2.6

3.229

Gadolinium (mM)

3.5

9.2

0.445

Gd/Phosphate ratio

0.246

3.538

0.137

Dox loading (ug/ml)

315

101

42

Size (nm)

123

168.8

105.6

PDI

0.042

0.092

0.05

Sample

LTSL

The phosphate concentrations of Gd-Dox-STL prepared by IL and AL, and LTSL prepared by IL were 14.21 mM, 2.6 mM, and 3.229 mM, respectively, and the gadolinium concentrations were 3.5 mM, 9.2 mM, and 0.445 mM, respectively. The Gd/P ratios of ILprepared, AL-prepared STL and IL- prepared LTSL was 0.246, 3.538, and 0.137, respectively. To determine the optimal STL preparation procedure, drug release from STL was tested (Supplemental Fig. 2). STL prepared by AL showed a poor release profile, i.e., 80% release at 30°C. We used only the IL procedure for loading Gd-BOPTA into STL for all subsequent experiments. The Dox loading concentration prepared by IL was 315 ug/ml. As 0.5 mg/ml of Dox was added to the liposome suspension, the loading efficiency of Dox is 63%. The mean 17



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diameter of Gd-Dox-STL (prepared by IL) as measured by laser light scattering was 123 ± 12 nm (Fig. 2A) and the Cryo-TEM images confirmed the size and revealed that Gd-Dox-STL had a spherical shape that maintained the lipid bilayer structure (Fig. 2B).

B

A

Figure 2. Physicochemical characterization of thermosensitive liposomes loaded with gadobenate dimeglumine (Gd-BOPTA) and doxorubicin (Dox) and incorporating short chain elastin-like polypeptides (Gd-Dox-STL). A) Particle size distribution and B) Cryotransmission electron microscopy (TEM) image of Gd-Dox-STL.

In Vitro Dox Release and Stability of Gd-Dox-STL and Gd-Dox-LTSL. To compare the temperature sensitivity of Gd-Dox-STL and Gd-Dox-LTSL, we investigated the Dox release profile over the range of 25°C to 55°C (Fig. 3A). While the fluorescence of Dox inside liposome is self-quenched due to its high concentration, released Dox has fluorescence intensity that is proportional to the concentration of Dox in the suspension. The amount of released Dox was measured after 5 min incubation at the different temperatures. Gd-DoxSTL showed rapid drug release at 39°C and reached maximum release at 42°C. The phase 18


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transition temperature of Gd-Dox-STL and Gd-Dox-LTSL was 40.3°C and 40.5°C, respectively. Gd-Dox-LTSL had a similar pattern to Gd-Dox-STL.

A

19



B

C 100 42°C

Dox release (%)

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80

41°C 40°C

60

39°C

40 20

0 0

5

10

15

20

25

Time (min)

20


30

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Figure 3. A) In vitro doxorubicin (Dox) release profile from thermosensitive liposomes loaded with gadobenate dimeglumine (Gd-BOPTA) and Dox and incorporating short chain elastin-like polypeptides (Gd-Dox-STL) and lysolipid-containing thermosensitive liposomes loaded with Gd-BOPTA and Dox (Gd-Dox-LTSL) in phosphate buffered saline (PBS). The liposomes were incubated for 5 min at the different temperatures in the range from 25°C to 55°C, and the amount of Dox released was analyzed by fluorescence intensity of Dox using a fluorometer. Data are mean ± S.D. (n = 3). B) The time course release of Dox from Gd-DoxSTL and Gd-Dox-LTSL at 37°C in 10% serum-containing culture media. Dox releases were measured at different times using a fluorometer. Data are mean ± S.D. (n = 3). C) Time course of Dox release from Gd-Dox-STL at various temperature (39, 40, 41, and 42°C).

Liposome stability is an important factor for clinical application because a more stable nanocarrier arrives at the tumor site without substantial loss of drug in the bloodstream. The stability of Gd-Dox-STL and Gd-Dox-LTSL was assessed in 10% serum-containing culture media at physiological temperature (37°C) by measuring drug leakage as function of time. Gd-Dox-STL was highly stable. Drug loss was minimal after 30 min (1.1%) and only 5% after 2 h incubation at 37°C (Fig. 3B). STL maintained a large amount of Dox inside the liposome even after 24 h showing only 19.4% Dox release (data not shown). In contrast, GdDox-LTSL displayed considerable drug leakage, 34% Dox release after 30 min incubation and 45% after 2 h incubation at 37°C (Fig. 3B). These data showed that Gd-Dox-STL had greater stability under physiological conditions compared to Gd-Dox-LTSL. The time course of Dox release showed that Gd-Dox-STL gave about 88% of Dox release within 10 sec at

21



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42°C (Fig. 3C). The release pattern of Dox after 1 min heating at different temperature is very similar with that after 5 min heating.

In Vitro Magnetic Resonance Imaging. The aim of this study was to evaluate the capability of MRI for monitoring released drug from thermosensitive liposomes. T1 relaxation times of the samples were measured after 5 min heat exposure at 37°C or 42°C on a 4.7 T MRI scanner. Fig. 4A and 4B shows the T1-weighted image of Gd-Dox-STL and Gd-Dox-LTSL and quantification of the image intensity, and Table 2 indicates T1 values of Gd-BOPTA only, Gd-Dox-STL, and Gd-Dox-LTSL after 5 min incubation at 37°C or 42°C. T1 values decreased when Gd-BOPTA was released out of the liposome because Gd-BOPTA was freely able to shorten the longitudinal relaxation time of water. As expected, there was no significant difference in T1 relaxation time of Gd-BOPTA only group between samples incubated at 37°C and 42°C (Table 2.). For the thermosensitive liposomes, although T1 values of the 42°C samples of free Gd-BOPTA, Gd-Dox-STL and Gd-Dox-LTSL with 0.25 mM of initial GdBOPTA concentration were similar at 520 ± 13, 445 ± 8 and 437 ± 8 ms, respectively, the values of the samples incubated at 37°C were 510 ± 13, 1200 ± 55 and 786 ± 24 ms, respectively. T1 value of the Gd-BOPTA of the liposome at 42°C is recovered to the value of free Gd-BOPTA. These results demonstrated that Gd-Dox-STL showed excellent performance as a MR tracer for drug release.

22


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A

B

23



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C

D

Figure 4. A) T1 -weighted MR images of gadobenate dimeglumine (Gd-BOPTA) only, thermosensitive liposomes loaded with Gd-BOPTA and doxorubicin (Dox) and incorporating short chain elastin-like polypeptides (Gd-Dox-STL), and lysolipid-containing thermosensitive 24


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liposomes loaded with Gd-BOPTA and Dox (Gd-Dox-LTSL), after 5 min incubation at 37°C and 42°C. The gadolinium concentration was 0.25 mM for all samples. The signal intensity of T1-weighted image was quantified using Image J 1.48v. The error bar represents the standard deviation (n=3) (B). A brighter image reflects lower T1-relaxation time and more enhanced contrast. Relaxation rate (R1) versus concentration of Gd-BOPTA was plotted. Gd-Dox-STL (C) and Gd-Dox-LTSL (D) were incubated at 37°C and 42°C to release Gd-BOPTA and Dox. The resulting relaxivity (slope) value was 1.32 and 6.64 mM-1 s-1 for Gd-Dox-STL, 3.38 and 7.96 mM-1 s-1 for Gd-Dox-LTSL after 37°C and 42°C incubation, respectively.

Table 2. T1 values (± standard deviation) of Gd-BOPTA only, thermosensitive liposomes loaded with Gd-BOPTA and doxorubicin and incorporating short chain elastin-like polypeptides (Gd-Dox-STL), and lysolipid-containing thermosensitive liposomes loaded with Gd-BOPTA and Dox (Gd-Dox-LTSL) after incubation at 37°C and 42°C Samples

Free Gd-BOPTA

Gd-Dox-STL

Gd-Dox-LTSL

Temperature

37°C

42°C

37°C

42°C

37°C

42°C

T1 (ms)

510 ±13

520 ±13

1200± 55

445± 8

786± 24

437 ±8

The ability of Gd-BOPTA to provide MRI contrast enhancement at 37°C (body temperature) and 42°C (mild hyperthermia) is a critical factor for success for MRI tracking in combination with mild hyperthermia. Therefore, R1 was determined from T1 relaxation times obtained from the samples after incubation at different temperatures. T1 was also measured at various concentrations of Gd-BOPTA. Fig. 4C and D presents plots of relaxation rate (1/T1)

25



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for Gd-Dox-STL and Gd-Dox-LTSL as function of Gd-BOPTA concentration, which displayed linear relationships. Relaxivity values were estimated from the slopes of each line, which were 1.32 and 6.64 mM-1s-1 for samples of Gd-Dox-STL incubated at 37°C and 42°C, respectively, and the corresponding values for Gd-Dox-LTSL were 3.38 and 7.96 mM-1s-1, respectively. The relaxivity of free Gd-BOPTA was also plotted and identical as 5.40 mM-1s-1 at both temperatures (Supplemental fig. 3).

Pharmacokinetics and Biodistribution Studies. To assess the pharmacokinetics of Gd-DoxSTL and Gd-Dox-LTSL in a non-heated model, blood samples were taken as a function of time after administration of the liposomes, and Gd was measured by ICP-AES analysis. Blood concentration versus time curves were obtained by plotting the percentage of Gd initial dose per gram (% ID/g) over time. Fig. 5A showed that the plasma half-lives of Gd-Dox-STL and Gd-Dox-LTSL were 4.85 and 1.95 h, respectively. Additionally, the biodistribution of Gd was examined in a non-heated model to determine clearance pathway for Gd-Dox-STL and Gd-Dox LTSL (Fig. 5B). Organs were taken 24 h post-injection and analyzed by ICP-AES. Both Gd-Dox-STL and Gd-Dox-LTSL groups showed accumulation mainly in the liver and spleen, organs of the reticuloendothelial system. Accumulation was lowest in the heart and skeletal muscle. Gd-Dox-STL showed about 2.5-fold increased accumulation in tumor compared to Gd-Dox-LTSL.

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A

27



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B

Figure 5. A) Pharmacokinetics of gadolinium in thermosensitive liposomes loaded with GdBOPTA and doxorubicin and incorporating short chain elastin-like polypeptides (Gd-DoxSTL) (closed circles) and lysolipid-containing thermosensitive liposomes loaded with GdBOPTA and Dox (Gd-Dox-LTSL) (open circles). The liposomes were intravenously administrated (5 mg Dox/kg) to BALB/c mice. Data are mean ± S.D. (n = 3). B) Biodistribution of gadolinium after intravenous administration of Gd-Dox-STL and Gd-DoxLTSL in tumor-bearing mouse. These pharmacokinetics and biodistribution data were obtained from the non-heated mouse models. The relative dose ratio Dox/Gd-BOPTA for GdDox-STL and Gd-Dox-LTSL were 0.165 and 0.173, respectively.

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In Vivo Evaluation of Gd-BOPTA and Dox Release from Gd-Dox-STL by HIFU-Induced Mild Hyperthermia. In vivo experiments were performed to assess the capability of Gd-DoxSTL combined with HIFU-induced hyperthermia for targeted delivery of Dox and the theranostic potential to monitor drug delivery efficacy by MRI. The results of ICP-MS analysis showed that no gadolinium was detected in tumor specimens from either side of two mice in the treatment group, which was probably due to individual variability in intra-tumoral drug retention as observed in a similar previous study.36 Therefore, the treatment group was subdivided into two groups according to the presence of gadolinium in the tumor, which eventually resulted in three groups (i.e., group A: treatment group with a response (i.e. gadolinium accumulation), n = 4; group B: treatment group with no response, n = 2; group C: control group, n = 3) or six sub-groups. There was no drug- or procedure-related mortality of the animal during the experiments. As shown in Table 3 and Fig. 6 and 7, the right side tumors treated with HIFU hyperthermia in group A demonstrated gadolinium accumulation (97.6 ± 37.5 µg/g of tissue), and the other tumors showed no accumulation. Values from both the MRI (∆R1, 0.13 ± 0.02/s) and fluorescence assays (Dox, 347.5 ± 56.5, arbitrary unit) of the right side tumors in group A were significantly greater than values from tumors on the contralateral side in group A and tumors in group C (p < 0.05). As for comparisons with of group A with group B, the values were greater in group A, however, the differences were not statistically significant (p = 0.064). This was probably due to small number of samples in group B. These results suggested that Gd-Dox-STL, as a theranostic agent, was able to deliver Dox primarily to the tumor targeted by HIFU-induced mild hyperthermia, and its delivery could be successfully monitored by MRI. 29



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Figure 6. T1 maps of the tumor areas overlaid on anatomical MR images of the mouse bearing bilateral tumors in in-vivo experiment. Left and right columns represent baseline and post-therapy MR images, respectively. High intensity focused ultrasound (HIFU) therapy was 30


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applied only to the right side tumor (lower image, right column). T1 value response after therapy was more prominent in the HIFU-treated tumor than the non-treated tumor (3820 ms to 3032 ms vs. 3522 ms to 3274 ms, respectively).

B

A

31



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C

Figure 7. ∆R1 and the amount of gadolinium or doxorubicin (Dox) accumulation in the present/absent of high intensity focused ultrasound (HIFU) treatment. A) ∆R1, B) amount of gadolinium accumulation, C) amount of doxorubicin accumulation.

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Table 3. The experimental values of in vivo MR imaging and HIFU-induced mild hyperthermia ∆R1 of MRI

Gadolinium accumulation

Doxorubicin accumulation

p-value*

Group

p-value*

(/s)

Group A

HIFU -

n =4

0.033±0.017

(Gd-STL +, response +)

HIFU +

n =4

0.133±0.020

Group B

HIFU -

n =2

0.030±0.003

(Gd-STL +, response -)

HIFU +

n =2

0.007±0.029

Group C

HIFU -

n =3

0.010±0.004

(Gd-STL -)

HIFU +

n =3

0.005±0.022

(µg/g of tissue) 0.043** NA 0.064 0.064 0.034** 0.034**

(arbitrary unit)

ND

245.8±9.2

97.6±37.5

374.5±56.5

ND

236.0±9.0

ND

241.5±4.5

ND

227.0±3.2

ND

236.3±3.5

Values represent mean ± standard error of mean. ND: not detected, NA: not available, * Comparisons with HIFU+ tumors in group A, Mann-Whitney test ** Statistically significant

33


0.043** NA 0.064 0.064 0.034** 0.034**


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DISCUSSION There are several benefits of drug tracking through in vivo imaging using thermosensitive nanocarriers loaded with both an anticancer drug and a contrast agent. First, the treatment duration can be adjusted in situ through direct visualization of the drug biodistribution. If the intended site is sufficiently heated and the area is large enough to cover the whole tumor, the real-time assessment of the thermally stimulated area can provide useful information to determine the level of drug release at specific regions. By adjusting the focus of the thermal probe in situ, the drug can be released evenly throughout the whole tumor site. It prevents the need of additional treatments caused by insufficient drug release in advance. Second, in situ monitoring of drug delivery provides a prediction of potential therapeutic response and a basis for future therapy planning of the individual patient through evaluation of the drug accumulation pattern for that individual’s tumor. Third, the coupling of a MR-guided HIFU system and thermosensitive liposomes loaded with drug and MR contrast agent could offer on-site verification of therapeutic effectiveness which may contribute to reducing the steps necessary for evaluating therapeutic responses. We previously reported the development of thermosensitive liposomes incorporating short chain elastin-like polypeptides (STL). STL are highly stable in plasma at 37°C compared to LTSL. Drug encapsulated in STL was rapidly released from the liposome after mild hyperthermia (42°C).34 A gadolinium-based contrast agent and Dox were both loaded into LTSL that was reported to have low stability in serum-containing media due to dissociation of the lysolipid from the liposomal bilayer.31 We considered that the high stability of STL makes a benefit for co-loading of drug and imaging agent probably thanks to their stable bilayer. 37-39

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In this study, we prepared thermosensitive liposomes encapsulating Gd-BOPTA and Dox (Gd-Dox-STL). For Gd-BOPTA loading to STL, we tried two procedures, IL at the hydration step and AL after extrusion (Supplemental Fig. 1). Although AL procedure showed high loading efficiency of Gd-BOPTA compared to IL (Table 1), the drug release profile after AL was very poor (Supplemental Fig. 2). The AL method probably reduced the stability of lipid bilayer membrane of liposome because Gd-BOPTA seemed to be loaded in the inner core, and also inserted into the lipid membrane. As a result, the IL procedure was used for preparing liposomes for the following experiments in this study. The size and morphology of Gd-Dox-STL displayed typical liposome characteristics. The liposomes were 123 ± 12 nm with a spherical shape and bilayer structure (Fig. 2). The in vitro Dox release profile of STL at different temperatures was comparable to LTSL, but STL was more stable at physiological temperatures (37-39°C). The comparison of the serum stability between Gd-Dox-STL and Gd-Dox-LTSL revealed that STL could be a suitable formulation for MRI monitoring because Gd-BOPTA and Dox can be maintained inside liposome due to the high stability (Fig. 3B). Cholesterol, one of the membrane components in STL, was incorporated to provide the stability to phospholipid bilayer, because the surface packing effect of cholesterol enhances the rigidity of membranes.40 Even after Gd-BOPTA loading, it was shown that the stability was well maintained. In addition, incorporation of the ELP-lipid conjugate augmented the membrane disruption of STL in the gel to liquid-crystalline phase transition, through the conformational change of ELP at temperatures produced by mild hyperthermia.41-42 In order to monitor drug delivery and release from thermosensitive liposomes, it would be advantageous if the difference of T1 relaxation time between physiological temperature and mild hyperthermia were large. When Gd-BOPTA is encapsulated inside the inner core of 35



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liposomes, water diffusion is limited across the lipid bilayer; as a result, Gd-BOPTA is prevented from shortening the T1 relaxation of water. On the other hand, once Gd-BOPTA is released from the liposome, it reduces the T1 value, affecting the contrast of the MR image.30,36,43,44 T1-weighted image of Gd-Dox-STL and Gd-Dox-LTSL with the same initial concentration of Gd-BOPTA (0.25 mM) were obtained. The differences in the T1-weighted image of Gd-Dox-STL between 37°C and 42°C was larger than that of Gd-Dox-LTSL (Fig. 4A), as shown by T1 values in Table 2. The relaxivity values for Gd-Dox-STL at 37°C and 42°C were 1.32 and 6.64 mM-1s-1, respectively, a 5-fold increase after release of the contrast agent from the liposomes. However, the relaxivity for Gd-Dox-LTSL had only about a 2-fold increase, showing 3.38 and 7.96 mM-1s-1 for 37°C and 42°C, respectively (Fig. 4C and D). Gd-BOPTA only control samples had almost identical values (5.40 mM-1s-1) for the relaxivity after 37°C and 42°C incubations, as expected (Supplemental Fig. 3). The performance for MRI monitoring of Gd-Dox-STL originated from its stability at 37°C under physiological conditions. The relaxivity of Gd-Dox-LTSL at 37°C was 3.38 mM-1s-1, 2.5-fold higher than Gd-Dox-STL (1.32 mM-1s-1) supporting that Gd-BOPTA leakage likely occurred from the liposomal membranes of Gd-Dox-LTSL at 37°C. The pharmacokinetics of gadolinium in a non-heated model showed that Gd-Dox-STL is about 2.5 times more stable than Gd-Dox-LTSL in the bloodstream (Fig. 5A). This was completely consistent with the in vitro studies including serum stability and MR imaging. In addition, the biodistribution results in a non-heated model at 24 h post-injection indicated that removal of the liposomes from the bloodstream through the reticuloendothelial system (RES) into the liver and the spleen. This could result in slow excretion of the liposomes through the hepatobiliary system (Fig. 5B). The accumulation of the Gd-Dox-STL in tumor tissues is 36


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higher than Gd-Dox-LTSL, probably due to their relative stabilities in vivo and increased circulation time with longer half life (Fig 5). It is important that the differential tumor accumulation of Gd-Dox-STL can provide flexible application time for HIFU treatment in clinic. By considering these pharmacokinetics and biodistribution results of each formulation, an appropriate protocol of heating can be optimized and selected. In terms of mechanism of drug release, LTSL undergoes rapid drug triggering by intravascular release when HIFU is immediately applied after injection.45 However, STL possessing stable property and also efficient drug release profile, can be used for interstitial drug release. For this reason, direct comparison of our biodistribution results of STL and LTSL appears to have little meaning. Moreover, we would not think that our biodistribution data acquired from a non-heated tumor model have enough relevance on how much drug would accumulate in the relative organs and the tumor because biodistributions, especially that of LTSL, would be likely to be quite different when heat is applied. Taking into consideration the fact that STL has the property stated above, two-step HIFU treatment might be more effective.In this strategy, the first HIFU can betreated immediately after STL administration and second HIFU can be applied after several hours post-injection for enough lag time to give high intratumoral acculumation.46 In vivo assessment using the mouse tumor model showed the potential of Gd-Dox-STL as a theranostic agent when combined with HIFU-induced mild hyperthermia therapy and MRI monitoring. Our results showed that HIFU is able to deliver the anticancer drug specifically to the intended tumor and MRI was an effective way to demonstrate the efficacy of drug delivery, even though drug retention in the tumor was seen only in four of six experimental cases probably due to individual variability in tumor vascularization or necrosis. Although we 37



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performed HIFU therapy and MRI examination separately in our experiments due to the limitations of our equipment, if MR-guided HIFU devices (Sonalleve MR-HIFU system, Philips Healthcare, Vantaa, Finland; Exablate system, InSightec, Haifa, Israel) are adopted, these procedures can be conducted simultaneously. In addition, for the MR-guided HIFU devices, there are no technical limitations to using mild hyperthermia localized to the target tumor.47 Therefore, the clinical application of these results to the treatment of cancers arising from organs amenable to ultrasound such as prostate, liver, pancreas, breast, or thyroid, may be a reality in the near future. In addition, the simultaneous use of gadolinium contrast agents with HIFU therapy has been proven safe in humans,48 which seems to further support the value and clinical relevance of our results.

CONCLUSIONS We successfully developed thermosensitive liposomes encapsulating an MRI tracking agent and an anticancer drug and incorporating a short chain elastin-like polypetide (Gd-DoxSTL). These liposomes were highly stable at 37°C under physiological conditions and released drug rapidly and completely at 42°C after HIFU-induced mild hyperthermia. We compared the performance between lysolipid-based thermosensitive liposomes (LTSL) and our liposomes (STL), including the in vitro drug release profile, serum stability, T1 relaxation time, relaxivity (∆R1), in vivo pharmacokinetics, and biodistribution. The Dox release pattern and T1 relaxation time at 42°C were comparable for both liposomes. However, the stability, T1 relaxation, and R1 value of Gd-Dox-STL at 37°C were increased relative to Gd-Dox-LTSL. This was probably due to membrane leakage in LTSL at 37°C under physiological conditions as the half-life of LTSL was found to be 1.9 h. STL has a longer circulation time (half-life = 38


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4.9 h) in vivo taking advantage of its high stability at 37°C, which reduced premature drug leakage prior to reaching at the target site. In vivo Dox and Gd-BOPTA accumulation in the tumor showed that Gd-Dox-STL is a promising formulation for theranostics, when combined with HIFU-induced mild hyperthermia, therapy and MRI monitoring for the treatment of cancers arising from the organs accessible to ultrasound.

ASSOCIATED CONTENT Supporting Information Procedures of co-encapsulation of gadobenate dimeglumine and Dox into thermosensitive liposomes (Gd-Dox-STL); Dox release profiles of Gd-Dox-STL prepared by Gd-BOPTA additional loading (AL) after extrusion; Relaxivity (ΔR1) plot of gadobenate dimeglumine as a function of gadolinium concentration at 37°C or 42°C. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (H.R.K.), [email protected] (Y.K); Notes The authors declare no competing financial interest. †

Current address: Therapeutic Strategic Unit, Asia-Pacific R&D, Sanofi, Daejeon, 305-807,

Korea

ACKNOWLEDGEMENTS 39



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This research (in vivo study) was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (#2011-0006504).

ABBREVIATIONS Gd-BOPTA, gadobenate dimeglumine; Dox, Doxorubicin; STL, short-chain elastin-like polypeptide-incorporating thermosensitive liposomes; ELP, elastin-like polypeptide; LTSL, lysolipid-containing thermosensitive liposomes; Gd-Dox-STL, gadobenate dimeglumine and doxorubicin co-encapsulated elastin-like polypeptide-incorporating thermosensitive liposome; Gd-Dox-LTSL, gadobenate dimeglumine and doxorubicin co-encapsulated lysolipidcontaining thermosensitive liposomes; HIFU, high-intensity focused ultrasound; ICP-AES, Inductively Coupled Plasma Atomic Emission Spectroscopy

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