Intratumoral Visualization of Oxaliplatin within a Liposomal

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Intratumoral visualization of oxaliplatin within a liposomal formulation using X-ray fluorescence spectrometry Hidenori Ando, Amr S. Abu Lila, Masao Tanaka, Yusuke Doi, Yasuko Terada, Naoto Yagi, Taro Shimizu, Keiichiro Okuhira, Yu Ishima, and Tatsuhiro Ishida Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00762 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Molecular Pharmaceutics

Intratumoral visualization of oxaliplatin within a liposomal formulation using X-ray fluorescence spectrometry

Hidenori Ando,†,# Amr S. Abu Lila,†,‡,§ Masao Tanaka,† Yusuke Doi,† Yasuko Terada,‖ Naoto Yagi,ǁ Taro Shimizu,† Keiichiro Okuhira,† Yu Ishima,† and Tatsuhiro Ishida †,*

†

Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical

Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505, Japan ‡

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig

University, Zagazig 44519, Egypt §

Department of Pharmaceutics, College of Pharmacy, Hail University, Hail 81442, Saudi

Arabia ǁ

Japan Synchrotron Radiation Research Institute (JASRI), 1-1-1 Kouto, Sayo, Hyogo

679-5198, Japan #

Department of Organ Anatomy and Nanomedicine, School of Medicine,

Yamaguchi University, 1-1-1, Minami-kogushi, Yamaguchi 755-8505, Japan

*Corresponding author: Tatsuhiro Ishida, Ph.D. Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505, Japan TEL: +81 88-633-7260 FAX: +81 88-633-7259 E-mail: [email protected]

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Micro-synchrotron radiation X-ray fluorescence spectrometry (µ-SR-XRF) is an X-ray procedure that utilizes synchrotron radiation as an excitation source. Micro-SR-XRF is a rapid, non-destructive technique that offers mapping and quantification of metals and biologically important elements in cell or tissue samples. Generally, the intratumor distribution of nanocarrier-based therapeutics is assessed by tracing the distribution of a labeled nanocarrier within tumor tissue, rather than by tracing the encapsulated drug. Instead of targeting the delivery vehicle, the present study employed µ-SR-XRF to visualize the intratumoral microdistribution of oxaliplatin (l-OHP) encapsulated within PEGylated liposomes. Tumor-bearing mice were intravenously injected with either l-OHP containing PEGylated liposomes (l-OHP liposomes) or free l-OHP. The intratumor distribution of l-OHP within tumor sections was determined by detecting the fluorescence of platinum (Pt) atoms, which are the main elemental components of l-OHP. The l-OHP in liposomal formulation localized near the tumor vessels and accumulated in tumors in greater concentrations compared with the free form, which is consistent with the results of our previous study that focused on fluorescent labeling of PEGylated liposomes. In addition, repeated administration of l-OHP liposomes substantially enhanced the tumor accumulation and/or intratumor distribution of a subsequent dose of l-OHP liposomes, presumably via improvements in tumor vascular permeability, which is also consistent with our previous results. In conclusion, µ-SR-XRF imaging efficiently and directly traced the intratumor distribution of the active pharmaceutical ingredient (API) l-OHP encapsulated in liposomes within tumor tissue. Micro-SR-XRF imaging could be a powerful means for estimating tissue distribution and even predicting the pharmacological effect of nanocarrier-based anticancer metal compounds.

Keywords: Intratumor distribution, Liposomes, Micro-synchrotron radiation X-ray fluorescence spectrometry (µ-SR-XRF), Oxaliplatin, Tumor sections


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Introduction Platinum-based therapeutics refers to a specific class of cytotoxic agents that are derived from the element platinum. Despite being one of the most effective classes of chemotherapeutics, their clinical use is hampered, at least in part, by their toxicity profiles as well as an unfavorable pharmacokinetic profile manifested by non-specific accumulation and/or relatively short blood circulation times.1,2 Accordingly, considerable effort has been devoted to overcoming these drawbacks. One of the most intriguing strategies to circumvent these shortcomings has been the utilization of drug delivery technologies to engineer novel platinum drug formulations. Currently, nanoparticle-encapsulated platinum formulations, such as polymeric micelles,3-5 dendrimers,6 liposomes,2,7 nanogels,8 lipid-based nanoparticles,9 and PLGA microspheres,10 have been adopted to reduce toxicity and improve overall therapeutic efficacy.11 Clinically, several platinum-based therapeutics, such as cisplatin-encapsulating micelles (NC-6004, NanoCarrier; Phase III in Japan) and DACH-platinum micelles (NC-4016, NanoCarrier; Phase II in USA), are being investigated. Oxaliplatin (l-OHP) is a third-generation platinum analogue that exerts a potent therapeutic effect against several gastrointestinal cancers. Previously, we reported that l-OHP-containing PEGylated liposomes (l-OHP liposomes) showed superior tumor growth inhibition, compared with free l-OHP.12,13 In order to gain insight into the mechanism underlying the potent antitumor efficacy of l-OHP liposomes, the relationship between the therapeutic effect and l-OHP accumulation/distribution within tumor tissue was also investigated. The preferential intratumor accumulation of l-OHP liposomes via enhanced permeation and retention (EPR) is predominantly responsible for tumor growth suppression that is superior to that of free l-OHP.14 In the same context, we have also demonstrated that metronomic S-1 dosing synergistically augments the therapeutic efficacy of liposomal oxaliplatin, presumably via permitting deep penetration/accumulation of l-OHP liposomes into a tumor mass.12 Nevertheless, in these studies the intratumor accumulation of l-OHP liposomes was correlated to the



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accumulation of the delivery vehicle (PEGylated liposomes) within tumor tissues, rather than the encapsulated drug (l-OHP) itself, which might not exactly reflect the actual accumulation of encapsulated l-OHP within tumor tissue. Accordingly, the FDA has strongly recommended the adoption of analysis/imaging techniques to trace active pharmaceutical ingredients (API) in pharmaceuticals, rather than the delivery vehicle, prior to the approval of nanocarrier-based therapeutics. Micro-synchrotron radiation X-ray fluorescence spectrometry (µ-SR-XRF) is a well-established technique that is used to identify and quantify the chemical elements present in a sample with high spatial resolution by detecting specific X-ray fluorescence after exposure to a primary X-ray. µ-SR-XRF traces not only the chemical elements that are originally present in animal tissues such as K, Na, Zn, Mg, and Fe, but also the elements composing drugs such as Pt and Au.15-17 Accordingly, in the present study, we utilized µ-SR-XRF to map the spatial distribution of l-OHP encapsulated in PEGylated liposomes within tumor tissue. By the aid of µ-SR-XRF imaging, the intratumor distribution of l-OHP in liposomal formulation can be imaged by detecting Pt atoms, which is a main component of l-OHP. This imaging technique is expected to provide valuable information that can be used to predict the intratumoral distribution of the API (l-OHP) encapsulated in PEGylated liposomes, which can reflect the overall therapeutic efficacy of l-OHP liposomes. In this study, therefore, we traced the intratumoral distribution of Pt, the main elemental component of l-OHP, via µ-SR-XRF following the intravenous injection of either l-OHP liposomes or free l-OHP into tumor-bearing mice. In addition, the tumor accumulation/distribution of l-OHP was observed following repeated dosing with l-OHP liposomes. The results of the present study demonstrated the feasibility of using µ-SR-XRF for tracing the spatial distribution of the API, namely l-OHP, encapsulated in PEGylated liposomes within tumor tissue.


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2. Materials and Methods 2.1. Materials Hydrogenated soy phosphatidylcholine (HSPC) and 1,2-distearoyl-snglycero-3-phosphoethanolamine-n-[methoxy (polyethylene glycol)-2000] (mPEG2000DSPE) were generously donated by NOF (Tokyo, Japan). Oxaliplatin (l-OHP) was purchased from the Yakult Pharmaceutical Industry (Tokyo, Japan). Cholesterol (CHOL) and Evans blue were purchased from Wako Pure Chemical Industries (Osaka, Japan). Tissue-Tek® O.C.T compound was purchased from Sakura Finetek Japan (Tokyo, Japan). FITC-labeled Lycopersicon Esculentum (Tomato) lectin (FITC-lectin) was purchased from Vector Laboratories (CA, USA). All other reagents were of analytical grade. 2.2. Cells and animals DLD-1 human colorectal carcinoma and C26 murine colorectal carcinoma were obtained from the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University). The cells were cultured in RPMI-1640 medium (Wako Pure Chemical Industries) supplemented with (10% of total) heat-inactivated fetal bovine serum (Corning, Corning, NY, USA), 100 units/ml penicillin and 100 µg/ml streptomycin (ICN Biomedicals, Irvine, CA, USA) in a 5% CO2/air incubator at 37 °C. BALB/c mice (male, 5 weeks old) and BALB/c nu/nu mice (male, 5 weeks old) were purchased from Japan SLC (Shizuoka, Japan). The experimental animals were allowed free access to water and mouse chow, and were housed under controlled environmental conditions (constant temperature, humidity, and a 12-h dark–light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of the University of Tokushima. 2.3. Preparation of l-OHP-containing PEGylated liposomes l-OHP-containing PEGylated liposomes composed of HSPC/CHOL/ mPEG2000-DSPE (2/1/0.1 molar ratio) were prepared using a reverse-phase evaporation method described earlier.18 Un-encapsulated l-OHP was removed by dialysis and the loaded l-OHP was determined using an atomic absorption photometer (Z-5700, Hitachi,



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Tokyo, Japan). The particle size of the resultant liposomes was 130 ± 11 nm, as determined with a Zetasizer Nano ZS (Malvern Instruments Ltd., WR, UK). The l-OHP concentration in the liposome formulation was adjusted to 1.01 mg l-OHP/ml. 2.4. Preparation of tumor-bearing mice DLD-1-bearing and C26-bearing tumor models were established by subcutaneous inoculation of DLD-1 cells (2 x 106 cells/mouse) at the flank region of BALB/c nu/nu mice and of C26 cells (2 x 106 cells/mouse) at the flank region of BALB/c mice, respectively. All animal experiments were initiated when tumors reached 50-100 mm3 in size. 2.5. Micro-synchrotron radiation X-ray fluorescence spectrometry (µ-SR-XRF) analysis DLD-1-bearing mice were intravenously injected with 1, 2 or 3 doses of either free l-OHP (4.2 mg/kg) or l-OHP liposomes (4.2 mg l-OHP/kg) once a week. At 24 h post injection, the mice were euthanized, and the tumors were excised and snap-frozen in Tissue-Tek® OCT compound by dry-iced acetone. Frozen samples were cut into sections with a thickness of 10 µm using a Cryostat (CM3050S, Leica Microsystems K.K., Tokyo, Japan) and were mounted on a polypropylene sheet. µ-SR-XRF was performed using the beamline (BL37XU) at SPring-8, operated at 8 GeV and ~100 mA. The tumor samples were irradiated with incident X-rays with an energy of 13.5 keV which was efficient in exciting Pt Lα line (Ex.: 9.441 keV), Pt Lβ line (Ex.: 11.069 keV), and Fe Kα line (Ex.: 6.398 keV). The fluorescence X-rays were measured using a Si solid-state detector under air at room temperature. Maximum imaging resolution of the µ-SR-XRF system was 0.5 µm square, and it took more than 7.2 h to obtain the 8 mm square whole tumor image (Excitation time: 1 s, resolution: 50 µm square). Each sample was mounted on an x-y translation stage. The fluorescence X-ray intensity was normalized by the incident X-ray intensity, I0, to produce a two-dimensional elemental map. 2.6. Evaluation of vascular permeability in tumors C26 tumor-bearing mice were intravenously injected with 3 doses of l-OHP liposomes (4.2 mg l-OHP/kg) once every 3 days. Three days after the last injection, the


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mice were intravenously injected with Evans blue (0.1 mg/100 µl/mouse), and 4 h later they were injected with FITC-lectin (0.1 mg/100 µl/mouse). At 3 min after the FITC-lectin injection, the mice were euthanized and tumor sections (10 µm thick) were prepared using a Cryostat. The sections were then mounted on MAS-coated slides (Matsunami Glass, Osaka, Japan). The fluorescence of Evans blue (Ex: 550 nm, Em: 610 nm) and FITC-lectin (Ex: 494 nm, Em: 518 nm) was observed with a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). Sixteen images from randomly selected sections were examined. The permeable area was calculated using the following formula: Permeable area (%) = Evans blue positive area (%) – FITC-lectin positive area (%) 2.7. Statistical analysis Statistical comparisons were evaluated by analysis of variance (ANOVA) using the Bell Curve software from Excel (Social Survey Research Information, Tokyo, Japan). The level of significance was set at **p < 0.01.



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3. Results 3.1. Mapping the intratumor distribution of l-OHP via µ-SR-XRF The microdistribution of l-OHP in tumor tissue was analyzed using µ-SR-XRF (Fig. 1). The distribution of l-OHP was detected by the signals of Pt atoms, and the blood distribution was identified by the signals of Fe atoms, which are present in hemoproteins. The images of l-OHP (Pt) and blood (Fe) were visualized simultaneously within the same tumor section. In the control (untreated) group, Pt was not detected in the tumor section. However, upon treatment with either free l-OHP or l-OHP liposomes, the Pt of l-OHP was clearly detected in each tumor section. The tumor accumulation of l-OHP tended to be higher in tumors treated with l-OHP liposomes, compared with tumors treated with free l-OHP. The localization of Pt (of l-OHP) within the tumor section with regard to tumor blood vessels (Fe) was slightly different between the treatments. Free l-OHP showed a wide distribution within the tumor section that was far from the Fe in the blood, which might have been a reflection of the small molecular weight of the free drug, which caused its distant diffusion in the blood vessels. On the other hand, the intratumor distribution of l-OHP in the liposome formulation seemed co-localized in the vicinity of the Fe in the blood. These results indicate that both forms of l-OHP are distributed around blood vessels, but the l-OHP in a liposome formulation is more highly accumulated in the tumor tissue compared with free l-OHP. 3.2. l-OHP accumulation in tumors following repeated doses of l-OHP liposomes We previously used fluorescent-labeled liposomes to show that sequentially administered l-OHP liposomes have diverse intratumor distribution patterns that are related to the chronological alteration of blood vessels.13,18 However, it is unclear whether or not the intratumor distribution patterns are truly related to the intratumor distribution of APIs, namely l-OHP, encapsulated in liposomes. In this experiment, the intratumor distribution of l-OHP following its repeated administration within liposomes was compared with that of free l-OHP using µ-SR-XRF (Fig. 2). For comparative quantification, the intensity scale of Pt was fixed at 255 as a maximum and 0 as a minimum among all samples. For tumors treated with free l-OHP, although 3 sequential


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doses of free l-OHP slightly increased the intratumor accumulation of the Pt in l-OHP, the repeated doses scarcely altered the distribution pattern of the Pt in l-OHP within the tumor section. On the other hand, repeated administration of l-OHP liposomes substantially elevated the intratumor accumulation of the Pt in l-OHP, despite the fact that prior treatment with a single liposomal l-OHP injection hardly changed the distribution of a subsequent second dose of liposomal l-OHP. These results are entirely consistent with our previous results using fluorescent-labeled liposomes,13,18 which indicated that repeated dosing of l-OHP liposomes markedly increases l-OHP accumulation in tumors. 3.3. Determination of the whole tumor distribution of l-OHP following the repeated administration of l-OHP liposomes In order to visualize an accurate spatial mapping of Pt (of l-OHP) within a whole tumor in tandem with its co-localization in blood, the intratumoral distribution of l-OHP and blood was determined using µ-SR-XRF under low magnification (Fig. 3). The l-OHP was detected via Pt intensity and the blood was traced via Fe intensity. Merged images of the Pt in l-OHP and the Fe in blood were created using free Image J software. Following the repeated injection of l-OHP liposomes, the Pt in l-OHP was widely distributed throughout the tumor tissue and was selectively localized along with the distribution of Fe in the blood. In addition, the Pt in l-OHP in the liposome formulation was preferentially distributed in the peripheral region of the tumor tissue, which was also rich in blood Fe (Fig. 3). These results revealed that, in the treatment with l-OHP liposomes, not only the liposome carrier but also encapsulated l-OHP is distributed in the peripheral region of the tumor tissue along with blood vessels. 3.4. Tumor vascular permeability in tumor vessels following repeated treatment with l-OHP liposomes As described above, we showed that sequentially administered l-OHP liposomes show diverse intratumor distribution patterns of not only liposomes but also encapsulated API, namely l-OHP, which is related to the chronological alteration of blood vessels.13,18 However, the underlying mechanism that causes such intratumor distribution patterns following repeated dosing of l-OHP liposomes has not been elucidated. Since



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tumor vascular permeability potentially affects liposomal distribution within the tumor tissue, Evans blue was employed to assess the effect of different treatments on tumor vascular permeability using C26-bearing tumor model. Tumor-bearing mice were intravenously injected with 3 sequential doses of l-OHP liposomes. Three days following the last injection, mice were injected with Evans blue and then FITC-lectin in order to stain the blood vessels. In the control (untreated) group, FITC-lectin-positive cells were frequently located in the C26 tumor tissue, indicating that tumor angiogenesis is highly induced in the C26-bearing tumor model. It was reported that, in the DLD-1 tumor tissue, CD31-positive cells that is one of marker of angiogenesis were highly induced in the tumor site.19 These findings presumably indicate that angiogenic capacity of C26-bearing tumor model is consistent with that of DLD-1-bearing tumor model. To gain further insight into the contribution of sequential liposomal l-OHP dosing on tumor vascular permeability, the blood-permeable areas were calculated using the images of Evans blue and FITC-lectin (Fig. 4B). The non-treated group showed restricted vascular permeability (permeable area was 16.5 ± 7.8%). By contrast, repeated dosing of l-OHP liposomes significantly enhanced the vascular permeability of tumors (the blood permeable area within the tumor section was 27.8 ± 10.9%). These results indicate that repeated administration of l-OHP liposomes increases the blood flow into tumor tissue.


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4. Discussion Platinum-based chemotherapy is a cornerstone among therapeutic regimens for patients with many types of tumors.20 However, the in vivo therapeutic efficacy of platinum-based drugs is adversely limited by inefficient delivery to target tissues and by severe drug-related side effects. Accordingly, many nanocarrier systems, such as polymeric nanoparticles,21 polymeric micelles,22 liposomes,7,18 and carbon nanotubes,23 have been adopted to enhance the therapeutic efficacy of platinum-based drugs while minimizing their related side effects. Nevertheless, evaluation of the accumulation/distribution of encapsulated drugs within tumor tissues is crucial in verifying the therapeutic potential of platinum-based drugs loaded onto nanocarrier systems in both preclinical and clinical situations. Therapeutic efficacy is routinely gauged by the tumor accumulation of nanocarrier systems, rather than by the accumulation of the encapsulated drugs, which might not reflect the actual accumulation levels, and/or might not grant therapeutic efficacy to the encapsulated drug. Premature drug release from delivery vehicles during transit to the target site might result in the tumor accumulation of empty drug carriers, and, therefore, estimation of the accumulation levels of encapsulated drugs could be flawed by the presence of such empty carriers. In addition, inefficient drug release from delivery vehicles that preferentially accumulate at the target site could result in the tumor accumulation of non-bioavailable drugs. Therefore, an evaluation of the delivery vehicle rather than the encapsulated drug might not correctly reflect the therapeutic potential of the loaded drug. Such an observation was encountered with the Stealth liposomal formulation of cisplatin (SPI-077) for intravenous administration. Despite the preferential accumulation of a liposomal drug concentration in the tumor tissues of animal experimental models, inefficient drug release from the liposomes led to a failure of the anticipated anti-tumor responses both in animal models and in humans and subsequently to a product withdrawal from Phase II clinical trials.24 Accordingly, the FDA has recommended the adoption of analysis/imaging techniques for the tracing of APIs in pharmaceuticals, rather than delivery vehicles, prior to the approval of



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nanocarrier-based therapeutics. Several imaging techniques, such as molecular imaging using positron emission tomography (PET),25-27 laser-based mass spectrometry imaging (MSI),28,29 X-ray adsorption spectroscopy (XAS), 30,31 or X-ray fluorescence (XRF), have been adopted to trace APIs in pharmaceuticals. Among them, XRF is considered one of the valuable tools for the direct monitoring of metals in biological samples. XRF can be conducted by using either conventional X-ray tube (XRF) 32-36 or, alternatively, a radioactive source such as synchrotron (µ-SR-XRF).37-39 µ-SR-XRF, in particular, represents a rapid non-destructive technique that can be implemented on small animals for mapping and quantification of metals and biologically important elements in tissue samples with extremely high sensitivity (detection limit down to 10 ppm (µg/g)) and resolution.37-39 Accordingly, in the present study, we adopted µ-SR-XRF to trace/visualize the distribution of API, namely l-OHP, encapsulated in liposome carrier within solid tumor tissues, rather than the liposome carrier itself. µ-SR-XRF at SPring-8 is operated with the accelerated energy of an electron beam at 8 GeV, which is the highest energy source in the world. The obtained mapping images were composed of square units along one side of a 0.5-µm minimum. These factors indicated that µ-SR-XRF imaging offered highly informative data about intratumor distribution/accumulation of l-OHP in liposome formulation that could reflect the therapeutic efficacy of encapsulated l-OHP. In the present study, µ-SR-XRF was used to visualize the intratumor distribution of l-OHP via the detection of Pt intensity. l-OHP in a liposome formulation is highly accumulated in tumors (Fig. 1). The repeated dosing of l-OHP liposomes markedly elevated the accumulation of l-OHP in tumors (Fig. 2). The distribution of l-OHP in liposome formulation along with blood in tumor tissue (Fig. 3) was accompanied by increases in tumor vascular permeability (Fig. 4). These findings confirmed the mechanism behind the tumor accumulation of l-OHP containing PEGylated liposomes following repeated injection and were consistent with our previous study tracing liposomes.18 Collectively, the present imaging technique could enable an accurate spatial mapping of l-OHP entities in l-OHP liposomes, rather than the liposome carrier, with


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extremely high sensitivity, which, in turn, could be exploited for the gathering of invaluable data regarding the pharmacological effect of l-OHP liposomes in tumor tissues. The present imaging technique can be used to visualize the distribution of l-OHP within a liposome formulation for either an entire tumor (Fig. 3) or a single cell (Figs. 1 and 2). It is well established that l-OHP encapsulated within liposomes inhibits tumor growth mainly by inhibiting DNA replication/transcription processes.40,41 Accordingly, µ-SR-XRF-mediated simultaneous determination of the intratumor distribution/co-localization of Pt derived from l-OHP and the phosphorous in the DNA backbone can be utilized to evaluate the therapeutic efficacy of l-OHP liposomes at the cellular level. In addition, µ-SR-XRF mapping is a valuable tool for determining the distribution and/or concentration of anticancer metal compounds within biological samples.36,38,42 Accordingly, using µ-SR-XRF to trace the intratumor distribution of l-OHP could also provide meaningful information regarding the therapeutic effect of l-OHP liposomes. µ-SR-XRF mapping is verified as a valuable tool for determining the distribution and/or concentration of anticancer metal compounds within biological samples. Ilinski et al.36 verified the feasibility of using µ-SR-XRF to detect clinical doses of cisplatin in either sensitive or resistant ovarian cancer cells. Furthermore, µ-SR-XRF can be utilized for the direct assessment of the interaction of anticancer metal compounds with specific biological targets. Davis et al.37 examined the simultaneous distribution of both Pt derived for cisplatin and phosphorous in the DNA backbone in tumor sections of A549 lung cancer cells treated with cisplatin. They revealed that Pt in cisplatin-treated cells was clearly co-localized with phosphorous, which suggested a main interaction between Pt-based drugs and cell DNA that, in turn, reflected the potent anticancer efficacy of cisplatin against A549 lung cancer cells. To the best of our knowledge, the present study is the first published utilization of µ-SR-XRF for the direct determination of the spatial intratumor distribution of anticancer metal compound l-OHP when encapsulated within a nanocarrier system.



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Conclusions In the present study, we used an informative imaging technique, µ-SR-XRF, to visualize the tumor tissue distribution of a platinum-based antitumor agent, l-OHP, within a liposome formulation. The µ-SR-XRF technique efficiently determined the spatial distribution of l-OHP within a liposome formulation on a scale that ranged from a single cell to an entire tumor. Accordingly, the adoption of µ-SR-XRF mapping for determining the intratumor distribution of nanocarrier-based anticancer metal compounds within human tissue biopsies could expand the utilization of this technique into personalized medicine for cancer therapies using nanocarrier-based anticancer metal compounds.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest exist.

Acknowledgements The authors are grateful for helpful advice from Mr. James L. McDonald in developing the English manuscript. The synchrotron radiation experiments were performed at the BL37XU or BL40B2 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2013B1730, 2014A1707, 2014B1810, 2014B1813, 2015A1860, 2015A1876, 2015B1811 and 2016A1307). This work was partially supported by the Japan Society for the Promotion of Science through a Grant-in-Aid for Scientific Research (B) (15H04639), by the Takahashi Industrial and Economic Research Foundation, by the Osaka Community Foundation, and through a research program for the development of an intelligent Tokushima artificial exosome (iTEX) from Tokushima University.


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Figure legends Fig. 1. Visualization of the intratumor distribution of l-OHP using µ-SR-XRF. DLD-1-tumor-bearing mice were intravenously injected with a single dose of either free l-OHP or l-OHP liposomes (4.2 mg l-OHP/kg). At 24 h post injection, the tumors were excised and the intratumor distribution of l-OHP was determined using µ-SR-XRF. l-OHP was detected via Pt intensity, and blood was detected via Fe intensity. The image shown is typical of 3 independent experiments.

Fig. 2. l-OHP accumulation in tumor tissue following the repeated administration of l-OHP liposomes. DLD-1-tumor-bearing mice were intravenously injected with 1, 2 or 3 doses of either free l-OHP (4.2 mg/kg) or l-OHP liposomes (4.2 mg l-OHP/kg) once a week. At 24 h post injection, the tumors were excised and 10-µm-thick sections were frozen. Intratumor distribution of l-OHP was determined using µ-SR-XRF. l-OHP was detected via Pt intensity. Maximal Pt intensity in the image was fixed at 255 and the minimum was set at 0 for all samples. The image shown is typical of 3 independent experiments.

Fig. 3. Total intratumor distribution of l-OHP after repeated doses of l-OHP liposomes. DLD-1-tumor-bearing mice were intravenously injected with 3 doses of l-OHP liposomes (4.2 mg l-OHP/kg) once a week. At 24 h post injection, the tumors were excised, and 10-µm-thick tumor-sections were frozen. Intratumor distribution of l-OHP was determined by µ-SR-XRF. l-OHP was detected via Pt intensity, and blood was detected via Fe intensity. The image shown is typical of 3 independent experiments.

Fig. 4. Tumor vascular permeability following the repeated administration of l-OHP liposomes. C26-tumor-bearing mice were intravenously injected with 3 doses of l-OHP liposomes (4.2 mg l-OHP/kg) once every 3 days. On day 3 after the last injection, the


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tumors were excised following injections of Evans blue (0.1 mg/0.1 ml/mouse) and FITC-lectin (0.1 mg/0.1 ml/mouse). Frozen tumor sections were prepared with a thickness of 10 µm. (A) The fluorescence of Evans blue (Ex: 550 nm, Em: 610 nm) and FITC-lectin (Ex: 494 nm, Em: 518 nm) was observed with a fluorescence microscope. (B) Permeable areas were calculated using a formula described in the section titled “Evaluation of blood perfusion and/or permeability in the tumor tissue.” The data were calculated using 16 independent graphical images for each treatment group (**P < 0.01).



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TOC/Abstract Graphics


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Graphical abstract 190x275mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 1 190x275mm (300 x 300 DPI)


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Figure 2 190x275mm (300 x 300 DPI)



Figure 3 190x275mm (300 x 300 DPI)


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Figure 4 190x275mm (300 x 300 DPI)


Intratumoral Visualization of Oxaliplatin within a Liposomal

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