Enhanced Delivery of Radiolabeled Polyoxazoline ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

Article

Enhanced Delivery of Radiolabeled Polyoxazoline into Tumors via Self-Aggregation Under Hyperthermic Conditions Kohei Sano, Yuko Kanada, Katsushi Takahashi, Ning Ding, Kengo Kanazaki, Takahiro Mukai, Masahiro Ono, and Hideo Saji Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00441 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 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

Molecular Pharmaceutics

Enhanced

Delivery

of

Radiolabeled

Polyoxazoline

into

Tumors

via

Self-Aggregation Under Hyperthermic Conditions

The names, affiliations, and addresses of the authors Kohei Sanoa,b,¶,*, Yuko Kanadaa,¶, Katsushi Takahashib, Ning Dinga, Kengo Kanazakia,c, Takahiro Mukaib, Masahiro Onoa, Hideo Sajia ¶

These authors contributed equally to this work.

a

Department of Patho-Functional Bioanalysis Graduate School of Pharmaceutical

Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, Japan 606-8501 b

Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1

Motoyama Kitamachi, Higashinada-ku, Kobe, Japan 658-8558 c

Medical Imaging Project, Corporate R&D Headquarters, Canon Inc., 3-30-2

Shimomaruko, Ohta-ku, Tokyo, Japan 146-8501

* Corresponding author: Kohei Sano, PhD, Lecturer Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyama Kitamachi, Higashinada-ku, Kobe, Japan, 658-8558 Tel: +81-78-441-7540 Fax: +81-78-441-7541 E-mail: [email protected]

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

Table of contents graphic


Page 2 of 29

Page 3 of 29 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


ABSTRACT: In order to develop a radiopharmaceutical for internal radiotherapy that

had a high anti-cancer effect, while exposing normal tissues to low radiation levels, we synthesized a radiolabeled polyoxazoline (POZ), a thermo-responsive polymer, and established a novel drug delivery system for targeting tumors by accelerating the accumulation of the radiolabeled POZ via self-aggregation under hyperthermic (42-43°C) conditions. By living-cationic polymerization using 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, POZ derivatives (Et-IspPOZ) (10, 20, and 30 kDa) with lower critical solution temperatures (LCSTs) of 37-38°C were synthesized, which were soluble at the body temperature, but self-aggregated upon heat treatment (42-43°C). Next, the indium-111 (111In)-labeled Et-IspPOZ was prepared, and the effect of molecular weight and injected POZ dose on the accumulation of radioactivity in the tumors was investigated upon intravenous injection of probes under hyperthermic conditions in colon 26-bearing mice. The uptake of radioactivity in tumors was increased when the molecular weight of POZ was greater than 20 kDa, while it was independent of the injected POZ dose (4-40 nmol). The amount of radioactivity retained in the tumor did not change for up to 3 h after exposure to heat treatment was stopped. Furthermore, the tumor uptake of the Et-IspPOZ derivative with a LCST greater than 42°C was significantly lower than that of Et-IspPOZ, which had a LCST of 37-38°C, suggesting the involvement of the self-aggregation of POZ on tumor uptake. Finally, the intratumoral localization of fluorescence-labeled Et-IspPOZ was evaluated using in vivo confocal laser microscopy. Many bright fluorescence spots were observed in the heat-treated tumors nearby and within blood vessels. In conclusion, the high tumor uptake of radiolabeled Et-IspPOZ was elucidated under hyperthermic conditions; thereby, the possibility of developing a novel internal radiotherapy using radiolabeled



POZ derivatives was demonstrated.

KEYWORDS: internal radiotherapy, polyoxazoline, thermo-responsive polymer,

cancer, hyperthermia


Page 4 of 29

Page 5 of 29 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


INTRODUCTION

Internal radiotherapy is a therapy wherein cytotoxic radiopharmaceuticals that are administered intravenously or orally could get accumulated and retained in the tumor, leading to tumor damage because of exposure to a local high-dose of radio-irradiation. The basic requirements for developing suitable therapeutic radiopharmaceuticals for internal radiotherapy should be: (i) cancer-specific accumulation and (ii) high and long-term intratumoral retention of radioactivity. The usefulness of sodium iodide-131 (Na131I) for thyroid cancer therapy,1 strontium-89 chloride (89SrCl2) for pain relief for multiple bone metastases, and radium-223 chloride (223RaCl2) for bone-metastasized prostate cancer2 as tumor-targeted radiopharmaceuticals for internal radiotherapy have been well-recognized. The yttrium-90 (90Y)-labeled anti-CD20 monoclonal antibody (Zevalin) targeting non-Hodgkin’s lymphoma has also been used at the clinical stage; however, some serious adverse effects such as blood toxicity could frequently emerge.3 Therefore, the development of radiopharmaceuticals that could exhibit highly cancer-specific therapeutic effects is required. Polyoxazoline (POZ), a polypeptide that is readily synthesized chemically,4 exhibits tumor accumulation via enhanced permeability and retention (EPR) effects, and a relatively rapid clearance from the blood5 as compared to polyehtylene glycol (PEG).6 Furthermore, POZ can self-aggregate above a characteristic lower critical solution temperature (LCST)7 within the range of 10-90°C, which can be controlled by modifying the acyl groups on POZ.8,9 In our previous study, the usefulness of POZ derivatives (LCST: 25°C) labeled with yttrium-90 (90Y) (β--ray-emitter, 2.28 MeV; half-life, 64.0 h) for brachytherapy had been determined.10 The intratumoral injection of 90

Y-labeled POZ achieved marked tumor shrinkage and massive cytotoxic damage.



Based on these findings, in this study, we planned to develop radiopharmaceuticals for internal radiotherapy which are soluble at room and body temperatures, but self-aggregate upon heat treatment after intravenous injection (Figure 1). We deduced that this therapeutic strategy using a tumor-specific heat treatment (hyperthermia) could achieve the high accumulation of POZ-based probes in the tumor via self-aggregation and rapid clearance from the normal tissues, leading to a reduction in the side effects in normal tissues. Hyperthermia is a type of cancer treatment, in which the cancerous tissue is exposed to a high temperature (approximately 42.5°C) by irradiation with radiowaves, and heat-treated cancer cells could be damaged with minimal adverse effects in normal tissues.

Figure 1. The concept of using an injectable polymer (polyoxazoline; POZ) labeled with radiometals that are thermo-responsively self-aggregated in tumor tissues after intravenous injection under hyperthermic conditions.

Therefore, we synthesized POZ derivatives with LCST of 37-38°C (above the body temperature and below the hyperthermic temperature). We expected that the aggregation of POZ derivatives would happen only under hyperthermic conditions. Furthermore, we labeled the POZ derivatives with indium-111 (111In) (γ-ray-emitter, 245 and 171 keV; half-life; 67.9 h) instead of

90

Y (β--ray-emitter) to quantify the uptake of probes in


Page 6 of 29

Page 7 of 29 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


tumors by measuring the radioactivity levels. We investigated the effect of molecular weight and injected dose of POZ, heating time, and transition temperature on uptake of 111

In-labeled POZ in tumors. Moreover, the retention of radioactivity after stopping

exposure to heat was evaluated. Finally, the intratumoral localization of POZ was evaluated using confocal fluorescence microscopy in vivo.



EXPERIMENAL SECTION

Synthesis of

111

In-labeled POZ. POZ was synthesized as described previously.11 In

brief, methyl p-toluene sulfonate (1 eq.) was mixed with a mixture of 2-ethyl-2-oxazoline (0-200 eq.) and 2-isopropyl-2-oxazoline (200-400 eq.) in anhydrous acetonitrile (Table S1), and then stirred for 14 min at 140°C with exposure to microwave irradiations. The molecular weight (MW) was determined by gel permeation chromatography. The MWs of Et-IspPOZ-1, Et-IspPOZ-2, Et-IspPOZ-3, and Et-IspPOZ-4 were 11126 Da, 19595 Da, 27592 Da, and 20623 Da, respectively. Subsequently, ethylenediamine was added to the reaction mixture and stirred for an additional 7 min at 140°C under conditions of exposure to microwave irradiations. The unreacted ethylenediamine was removed by dialysis against methanol. Furthermore, the POZ derivatives (1 eq.) synthesized as described above were conjugated with p-SCN-Bn-DOTA (10 eq.), a metal chelator for labeling with radiometal (111In), in methanol for 36 h at room temperature. The unconjugated DOTA was purified by ultrafiltration using a size exclusion membrane unit (Amicon Ultra centrifugal filter units, molecular weight cut-off: 3 kDa, Merck Millipore, Co.) and by gel filtration (PD-10 Columns, GE Healthcare).

111

In-labeling was performed by incubating the

POZ-DOTA conjugate (0.09-0.23 nmol) with

111

InCl3 in acetate buffer (pH 6.0)

(300-600 µL) for 10 min at room temperature. After incubation, excess ethylenediaminetetraacetic acid was added and incubated for 5 min at room temperature and the buffer was exchanged for phosphate buffered saline (PBS, pH 7.4) by ultrafiltration to remove the un-chelated 111In. The radiochemical yield was calculated as the amount of radioactivity in the final products (111In-labeled POZ derivatives) expressed as the percentage (%) of starting radioactivity (111InCl3) used in the


Page 8 of 29

Page 9 of 29 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


radiochemical reaction. The radiochemical purity was analyzed by size-exclusion chromatography (PD-10 Column), as previously reported.10 The radioactivity in each fraction (1 mL/fraction) eluted with PBS was measured with a 2480 WIZARD2 automatic gamma counter (PerkinElmer, Inc., Waltham, MA). The LCSTs of the POZ derivatives were measured using a Zetasizer Nano (Malvern Instruments Ltd.), as reported previously.10 The change in polymer size (aggregation) was evaluated with heating at a rate of 1°C/5min.

Cell Culture and Animal Model. A mouse rectal cancer cell line, colon 26, was purchased from the Riken Bio Resource Center (Tsukuba, Japan). The cells were maintained according to protocols described in previous reports.6,12 Animal experiments were conducted in accordance with our institutional guidelines and were approved by the Kyoto University and Kobe Pharmaceutical University Animal Care Committee. Five-week-old male BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). The animals were housed in air-conditioned rooms under a 12-h light/dark cycle and allowed free access to food and water. Colon 26 (1×106 cells) cells that were suspended in 50-100 µL PBS were inoculated in the thighs of the hind limb of mice by subcutaneous administration. Experiments with tumor-bearing mice were performed approximately 7-10 days after inoculation.

Biodistribution Studies (Heating Protocol). Under isoflurane anesthesia, the tumor was immersed in a temperature-controlled water bath maintained at 42-43°C for 15 min before the intravenous injection of the probes. The tumor and rectal temperature were measured with a needle-type thermometer or thermography before isoflurane anesthesia,



Page 10 of 29

after isoflurane anesthesia, 15 min and 75 min after starting heat treatment.

Effect of Molecular Weight of POZ on Tumor Accumulation. In order to investigate the effect of molecular weight of POZ on tumor accumulation,

111

In-labeled

Et-IspPOZ-1 (10 kDa), Et-IspPOZ-2 (20 kDa), or Et-IspPOZ-3 (30 kDa) (each 4 nmol/150 µL/22.2 kBq/mouse) was intravenously injected into tumor-bearing mice 15 min after pre-heating, and mice were sacrificed 60 min after probes were injected (Figure 2A). The organs of interest, including the tumor and blood, were excised. The organ weights were determined and the radioactivity in the organs was measured using a 2480 WIZARD2 automatic gamma counter (PerkinElmer, Inc.). The biodistribution of 111

In-labeled POZ derivatives was calculated as the percentage of injected dose

(radioactivity) per gram of tissues. As a control group (non-heated group), tumor-bearing mice injected with probes were left at room temperature, and 60 min later, the biodistribution was evaluated, as mentioned above.

Effect of Injection Dose of POZ on Tumor Accumulation. In order to investigate the effect of the injected dose of POZ on tumor accumulation,

111

In-labeled Et-IspPOZ-2

(20 kDa) (4, 20, or 40 nmol/150 µL/22.2 kBq/mouse) was intravenously injected into mice 15 min after pre-heating, and mice were sacrificed 60 min after probe injection (Figure 2B). The biodistribution was evaluated, as mentioned above.

Effect of Heating Time on Tumor Accumulation of POZ. In order to investigate the effect of heating time on accumulation of POZ in tumors, 111In-labeled Et-IspPOZ-2 (20


Page 11 of 29 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


Heating

A with Heat

111In-Et-IspPOZ-1,

2, or 3 (4 nmol) injection

Biodistribution study

without Heat -15

0

60

111In-Et-IspPOZ-2 (4, 20, or 40 nmol) injection


(min)

Heating

B with Heat

without Heat Time (min) -15

0

(min)

60

Heating

C with Heat

111

In-Et-IspPOZ-2 (4 nmol) injection

without Heat Time (min) -15

D

0


30

60

111

Heating

with Heat Time (min) -15

In-Et-IspPOZ-2 (4 nmol) injection

0

120

(min)


60

240(min)

Figure 2. Outline of the biodistribution studies. Effect of (A) molecular weight of POZ, (B) injection dose of POZ, and (C) heating time on tumor accumulation. (D) Evaluation of intratumoral retention of POZ after stopping exposure to heat.

kDa) (4 nmol/150 µL/22.2 kBq/mouse) was intravenously injected into mice 15 min after pre-heating, and the mice were sacrificed 30, 60, or 120 min after receiving probe injections (Figure 2C). The biodistribution was evaluated, as mentioned above.



Evaluation of Intratumoral Retention of POZ After Stopping Exposure to Heat. The time-dependent intratumoral retention of POZ after stopping exposure to heat was evaluated.

111

In-labeled Et-IspPOZ-2 (20 kDa) (4 nmol/150 µL/22.2 kBq/mouse) was

intravenously injected into mice 15 min after pre-heating, and the intratumoral temperature was maintained for 60 min at 40-41°C; thereafter, mice were left at room temperature for 3 h (Figure 2D). The biodistribution was evaluated, as mentioned above.

Effect of Phase Transition Temperature on Accumulation of POZ in Tumors. In order to investigate the effect of phase transition temperature on the accumulation of POZ in tumors,

111

In-labeled Et-IspPOZ-2 (20 kDa, LCST: 38°C) or Et-IspPOZ-4 (20

kDa, LCST: 51°C) (4 nmol/150 µL/22.2 kBq/mouse) was intravenously injected into mice 15 min after pre-heating, and mice were sacrificed 60 min after probe injection. The biodistribution was evaluated, as mentioned above.

Fibered Confocal Fluorescence Microscopic Imaging Studies. The localization of POZ derivatives was investigated using a fibered confocal fluorescence microscopy system (Cellvizio® Endomicroscopy System, Mauna Kea Technologies, Paris, France) with a ProFlex UltraMiniO probe (field of view: 240 µm diameter, lateral resolution: 1.4 µm, depth of focus: 60 µm). The FITC-labeled Et-IspPOZ (approximately 27 kDa) was synthesized using a protocol similar to that for DOTA-conjugated POZ derivatives10. In brief, fluorescein-5-isothiocyanate (ThermoFisher Scienific Inc., Waltham, MA, USA) (5 eq.) instead of p-SCN-Bn-DOTA was reacted with POZ derivative (1 eq.).


Page 12 of 29

Page 13 of 29 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


A heating system that functioned on the basis of warm water circulation was constructed, as shown in Figure 3. The tumors were pre-heated at 42-43°C for 15 min, and FITC-labeled Et-IspPOZ (34 nmol/150 µL/mouse) was intravenously injected thereafter. The tumor temperature was maintained for an additional 60 min, followed by confocal fluorescence microscopy studies under a controlled temperature (42-43°C). Evans blue (20 mg/kg, 100 µL) and Hoechst33342 (1 mg/100 µL) were intravenously injected 10-30 min and 1 min before imaging studies, respectively. The imaging data were acquired at a scan rate of 12 frames/sec.

Figure 3. A heating system based on warm water circulation. (A) mouse was anesthetized using isoflurane, and warm water was circulated in the direction indicated by arrows. (B) Thermographic images during heat treatment of tumors.

Statistical Analysis. Data are expressed as the mean ± standard deviation values from a minimum of three experiments. For multiple comparisons, a one-way analysis of variance with post-test (Tukey-Kramer test) was used. P < 0.05 was considered to indicate a statistically significant difference.



Page 14 of 29

RESULTS Physicochemical Properties of

111

In-labeled POZ Derivatives. Although the size of

each POZ derivative was approximately 5 nm below LCSTs, the size was dramatically increased above LCSTs. The LCSTs of Et-IspPOZ-1, Et-IspPOZ-2, Et-IspPOZ-3, and Et-IspPOZ-4 were 37°C, 37°C, 37-38°C, and 51-52°C, respectively. The radiochemical purities of these probes were above 96% (Figure S1). The radiochemical yields were approximately 53-76%.

In Vivo Biodistribution Studies (Control of Tumor Temperature). The tumor and rectal temperatures were measured with a needle-type thermometer. After pre-heating at 42-43°C for 15 min, tumor and rectal temperatures measured in mice under isoflurane anesthesia were 40.6±1.0°C and 34.1±1.2°C, respectively. After heating at 42-43°C for 75 min, the tumor and rectal temperatures were 40.2±0.4°C and 34.7±0.6°C, respectively.

Effect of Molecular Weight of POZ on Tumor Accumulation. In heat-treated tumors, the amounts of

111

In-labeled Et-IspPOZ-1 (10 kDa), Et-IspPOZ-2 (20 kDa), and

Et-IspPOZ-3 (30 kDa) accumulated were 3.3±0.5, 7.8±2.6, and 5.8±1.6 % injected dose/g, respectively (Figure 4A). The significant increase in the uptake of

111

In-labeled

POZ derivatives in tumors was observed in Et-IspPOZ-2 (20 kDa) and Et-IspPOZ-3 (30 kDa) groups, as compared with that in the Et-IspPOZ-1 (10 kDa) group. On the contrary, in non-heat-treated tumors, the levels of uptake of

111

In-labeled Et-IspPOZ-1,

Et-IspPOZ-2, and Et-IspPOZ-3 in tumors were low, therefore there were significant differences between heat-treated tumors and non-heat-treated tumors when the


Page 15 of 29

molecular weight was more than 20 kDa. As the molecular weight of POZ increased, a slower blood clearance, higher hepatic uptake, and lower kidney uptake were observed (Table S2).

Radioactivity (%injected dose/g)

Heat heat no Noheat heat

*

12 10

*

8 6 4 2

12

n.s.

heat Heat no Noheat heat

10 8 6 4 2 0

0 10 20 30 Molecular weight (kDa)

C *

12

4 20 40 Injection dose (nmol/mouse)

heat Heat no Noheat heat

*

*

10 8

B Radioactivity (%injected dose/g)

A


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


*

6 4 2 0 30 60 120 30min 60min 120min Heating time (min)

Figure 4. Effect of molecular weight (A), injected dose of POZ derivatives (B), and heating time (C) on the accumulation of probes in tumors. (A) 4 nmol of 111In-labeled Et-IspPOZ-1 (10 kDa), Et-IspPOZ-2 (20 kDa), or Et-IspPOZ-3 (30 kDa) was injected (protocol: Fig. 2A); (B) 4, 20, or 40 nmol of 111In-labeled Et-IspPOZ-2 (20 kDa) was injected (protocol: Fig. 2B); (C) 111In-labeled Et-IspPOZ-2 (20 kDa, 4 nmol) was injected (protocol: Fig. 2C). The tumors were pre-heated at 42-43°C for 15 min and the probes were intravenously administered. Data were expressed as mean ± S.D values. *P < 0.05.

Effect of Injection Dose of POZ on Accumulation in Tumors. In order to evaluate the effect of the injected dose of POZ on accumulation in tumors, varied POZ doses (4, 20, or 40 nmol) were intravenously administered (Figure 4B). There was no significant difference in the uptake of

111

In-labeled Et-IspPOZ-2 in the heat-treated tumors,



Page 16 of 29

regardless of the injected dose. Significant differences were observed between the heat-treated group and the non-heat-treated group for each injected dose. As the injected dose of POZ increased, kidney uptake was deceased, although there was no marked difference on the accumulation of radioactivity in the other organs (Table S3).

Effect of Heating Time on Accumulation of POZ in Tumors. The amounts of 111

In-labeled Et-IspPOZ-2 accumulated were 5.0±1.0, 7.8±2.6, and 7.1±1.5% injected

dose/g in the tumors, when the tumors were given heat treatment at a temperature of 42-43°C for 30, 60, or 120 min, respectively (Figure 4C). The significant increase in the uptake of

111

In-labeled Et-IspPOZ-2 in tumors was observed in the 60 min groups

compared to that in the 30 min group. In unheated tumors, the level of

111

In-labeled

Et-IspPOZ-2 accumulated were low. In the all groups, there was a slight increase in the uptake of radioactivity in the liver, spleen, and lungs in the heat-treated group, as compared to that in the non-heat-treated group (Table S4).

Evaluation of Intratumoral Retention of POZ After Stopping Exposure to Heat. The amount of 111In-labeled Et-IspPOZ-2 accumulated in the tumors did not change at 0 or 180 min after stopping exposure to heat, respectively (Table 1). No significant decrease in radioactivity in the tumor was observed up to 180 min after stopping exposure to heat treatment.


Page 17 of 29 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


Table 1. Time-dependent intratumoral retention of POZ after stopping exposure to heat. 111

In-labeled Et-IspPOZ-2 (20 kDa) (4 nmol) was intravenously injected into mice 15 min after

pre-heating, and intratumoral temperature was maintained for 60 min at 40-41°C; thereafter, mice were left at room temperature for 0 or 180 min. Results are expressed as means(%ID) ± SD values.

0 min Blood 8.79 ± 0.95 Heart 3.76 ± 0.22 Lung 4.03 ± 0.87 Liver 1.98 ± 0.22 Kidney 25.91 ± 4.30 Stomach 2.73 ± 0.68 Intestine 2.75 ± 0.35 Pancreas 2.77 ± 0.32 Spleen 2.29 ± 0.55 Muscle 0.90 ± 0.23 Tumor 7.80 ± 2.60

180 min 0.73 ± 0.17 3.23 ± 0.78 1.52 ± 0.35 1.27 ± 0.11 26.64 ± 2.79 4.43 ± 0.71 3.53 ± 0.48 3.29 ± 0.21 2.34 ± 0.35 0.87 ± 0.13 6.67 ± 1.84

Effect of LCST on Tumor Accumulation of POZ. The tumor uptake of radioactivity from

111

In-labeled Et-IspPOZ-2 (20 kDa, LCST: 37°C) and

111

In-labeled Et-IspPOZ-4

(20 kDa, LCST: 51-52°C) was evaluated, in order to determine the effect of the LCST on accumulation of POZ in tumors (Figure 5). We expected that the

111

In-labeled

Et-IspPOZ-4 would not aggregate under hyperthermic conditions (42-43°C). In heat-treated tumors, the amounts of

111

In-labeled Et-IspPOZ-2 and Et-IspPOZ-4

accumulated were 7.8±2.6 and 2.7±0.5% injected dose/g, respectively, while the amounts of 111In-labeled Et-IspPOZ-2 and Et-IspPOZ-4 accumulated in non-heat-treated tumors were 4.3±1.3 and 2.0±0.0% injected dose/g, respectively. The accumulation ratios between heated and non-heated tumors were 1.83 and 1.35 for Et-IspPOZ-2 and Et-IspPOZ-4, respectively.


111

In-labeled


12


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

10

*#

8

Heat heat no Noheat heat

6 4 2 0 37 ℃ 37˚C

51 ℃ 51˚C

Figure 5. Effect of phase transition temperatures (Tts) on accumulation of probes in tumors. The POZ derivatives with Tts of 37°C and 51°C were intravenously injected 15 min after pre-heating, and tumor accumulation of probes was evaluated at 60 min after administering probe injections. *P

Enhanced Delivery of Radiolabeled Polyoxazoline ... - ACS Publications

Recommend Documents