Rhamnolipid nanoparticles for in vivo drug delivery and photodynamic therapy

BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 19 (2019) 12 – 21

Original Article

nanomedjournal.com

Rhamnolipid nanoparticles for in vivo drug delivery and photodynamic therapy Gawon Yi a, b , Jihwan Son a, b , Jihye Yoo a, b , Changhee Park a, b , Heebeom Koo a, b, c,⁎ a Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea c Catholic Photomedicine Research Institute, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Revised 28 February 2019

b

Abstract Herein, we report the development of self-assembled nanoparticles using rhamnolipid, a biosurfactant. Rhamnolipid is produced by Pseudomonas aeruginosa, and has an amphiphilic structure that is suitable for the formation of a nanoparticle shell. These rhamnolipid nanoparticles were loaded with pheophorbide a (Pba), a hydrophobic photosensitizer. The resulting nanoparticles had about 136.1-nmdiameter spherical shapes and had excellent water solubility without aggregation for one month. These nanoparticles showed fast uptake into SCC7 tumor cells and induced photodynamic damage upon laser irradiation. After intravenous injection to SCC7 tumor-bearing mice, their long blood circulation time and high accumulation in tumor tissue were observed in real-time fluorescence imaging. Upon laser irradiation, these rhamnolipid nanoparticles showed complete tumor suppression by photodynamic therapy in vivo. These promising results demonstrate the potential of rhamnolipid nanoparticles for drug delivery, and suggest that further attention to rhamnolipid research would be fruitful. © 2019 Elsevier Inc. All rights reserved. Key words: Rhamnolipid; Nanoparticle; Drug delivery; Photodynamic therapy; Pheophorbide a

Nanoparticles have captivated the interest of researchers in the biomedical field. 1,2 Advances in nanotechnology have resulted in a plethora of useful nanoparticles, many of which have shown promising outcomes in imaging and drug delivery. 3,4 Many useful characteristics of nanoparticles have driven the intense interest in nanomedicine. Their small size makes them suitable for injection to the human body by syringe. 5 With rationally-designed surfaces, they can escape both renal clearance in the kidneys and removal by the reticuloendothelial system in liver and spleen, resulting in prolonged blood circulation compared to small molecules. 6,7 In

Abbreviations: Pba, pheophorbide a; Pba-RL-NPs, Pba-loaded rhamnolipid nanoparticles; NIRF, near-infrared fluorescence; XTT, 2,3-bis-(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide. Conflicts: Authors declare no conflict of interest. This work was supported by Basic Research Program (2016R1C1B3013951) through the National Research Foundation of Korea (NRF) funded by the Korean Government (Ministry of Science, ICT, & Future Planning). ⁎Corresponding author at: Department of Medical Lifescience, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea. E-mail address: [email protected] (H. Koo).

particular, nanoparticles have shown higher accumulation in angiogenic tissues, like tumors, by penetrating through the fenestrate structure of their blood vessels and persisting in these tissues due to slow lymphatic drainage, a phenomenon known as the enhanced permeation and retention (EPR) effect. 8,9 Small molecule drugs or dyes can be loaded or conjugated to nanoparticles to be used for improved imaging or targeted therapy. 10 Nanoparticles have been developed using a broad range of materials including polymers, lipids, silica, iron, gold, and graphene. 11 Recently, inorganic nanoparticles have been spotlighted due to their unique properties, but organic nanoparticles are relatively advantageous for clinical applications. 12 One facile method for forming organic nanoparticles is using selfassembly of amphiphilic molecules, which play a role as surfactants. The hydrophobic parts of such molecules form an inner core with organic solvents or oil that can carry hydrophobic drugs. 13 The hydrophilic parts of these molecules are in contact with the surrounding water and form an outer shell. In this way, they can lower the surface tension between hydrophobic and hydrophobic spaces and stabilize nanoparticle structure. 14 Phospholipids, pluronic polymers, the tween series, and sorbitan

https://doi.org/10.1016/j.nano.2019.03.015 1549-9634/© 2019 Elsevier Inc. All rights reserved. Please cite this article as: Yi G, et al, Rhamnolipid nanoparticles for in vivo drug delivery and photodynamic therapy. Nanomedicine: NBM 2019;19:12-21, https://doi.org/10.1016/j.nano.2019.03.015

G. Yi et al / Nanomedicine: Nanotechnology, Biology, and Medicine 19 (2019) 12–21

stearates are widely used as surfactants in the food, cosmetic, and pharmaceutical industries. Biosurfactants are surfactant molecules that originate from living cells. Researchers have been interested in biosurfactants due to their environmental friendliness, diversity, and facile production at large scale. 15 Emulsan, lecithin, sophorolipid, and rhamnolipid are representative biosurfactants, and they have been utilized by companies such as TeeGene Biotech, Saraya Co., AGAE Technologies, Jeneil Biosurfactant Co., Henkel, and Cognis Care Chemicals. 16 Particularly, rhamnolipid has been used for oil recovery, agriculture, cleaning, cosmetics, and pharmaceutics. 16 It is mainly produced by Pseudomonas aeruginosa, and is composed of a rhamnose sugar molecule containing β-hydroxy fatty acids. In 2016, Rademann’s group introduced the idea that rhamnolipid can encapsulate hydrophobic drugs. 17 They showed that rhamnolipid nanoparticles are not toxic in cell viability tests and are useful for skin penetration during ex vivo analysis. However, the therapeutic efficacy of rhamnolipid nanoparticles in drug delivery has not been evaluated in vivo until now. In this paper, we prepared rhamnolipid nanoparticles with flax seed oil cores, and loaded them with pheophorbide a (Pba), a photosensitizer we chose as a model drug. After evaluation of the size and stability of these Pba-loaded rhamnolipid nanoparticles (Pba-RL-NPs), their cellular uptake and photodynamic efficacy were observed in a SCC7 (mouse squamous cell carcinoma) cell line. For in vivo tests, tumor-bearing mice models were prepared with SCC7 tumor cells, and the biodistribution and tumor tissue accumulation of Pba-RL-NPs were observed by real-time nearinfrared fluorescence (NIRF) imaging after intravenous injection. Finally, photodynamic therapy in vivo was performed in these same mice models, and the therapeutic result upon laser irradiation was analyzed.

Method Materials Rhamnolipid was purchased from AGAE Technologies (Corvallis, OR, USA). Pheophorbide a (Pba) was purchased from Frontier Scientific Inc. (Logan, UT, USA). Flax seed oil was obtained from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 and dimethyl sulfoxide (DMSO) was purchased from Samchun (Seoul, Gangnam-gu, Korea). Xylene and ethyl alcohol were purchased from Duksan (Seongnam, Gyeonggido, Korea). Optimal cutting temperature (O.C.T) compound was purchased from Sakura Finetek (Tokyo, Japan). Singlet Oxygen Sensor Green reagent (SOSG) and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide) was purchased from Invitrogen (Carlsbad, CA). Phenazine methyl sulfate (PMS) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Paraformaldehyde (4%) and formalin (10% ) solutions were purchased from Biosesang (Seongnam, Gyeonggi-do, Korea). Eosin Y and Mayer’s Hematoxylin were purchased from Cancer Diagnostics, Inc. (Durham, North Carolina, USA). Dulbecco’s phosphatebuffered saline (DPBS), Roswell Park Memorial Institute

13

(RPMI) medium and fetal bovine serum (FBS) were purchased from Biowest (Nuaille, France). Antibiotic-antimycotic solution and 0.05% Trypsin-EDTA were purchased from Gibco-BRL (Grand Island, NY, USA). Preparation of rhamnolipid nanoparticle containing pheophorbide a Pheophorbide a-loaded rhamnolipid nanoparticles (Pba-RLNP) were fabricated by a traditional oil in water (O/W) emulsion method. We dissolved 10 mg of rhamnolipid in 2.4 ml of a 2% (v/v) glycerol/water solution. Pheophorbide a (Pba, 3 mg) and flax seed oil (50 mg) were dissolved in 100 μl of DMSO and this solution was added to the RL solution slowly. The mixture was then dispersed under sonication by a C505 probe sonicator (Sonics; Newtown, CT, USA) for 10 min. Unloaded Pba was removed by dialysis (MWCO: 13KD) in distilled water for 1 hour. Characterization of nanoemulsion The size and zeta potential of Pba-RL-NPs were measured at 25 °C in PBS (pH 7.4) using a Zetasizer (Nano ZS90; Malvern Instruments, Malvern, UK) and Zetasizer software (version 7.12). To observe the morphology of NPs, we used transmission electron microscopy (TEM) with negative staining using 2%(w/ v) uranyl acetate solution. To measure the amount of Pba loaded in the NPs, they were completely dissolved in a detergent solution (DMSO: PBS: DW=5: 4: 1, 1% Triton X-100). Then, the amount of Pba in the solution was calculated based on the fluorescence of Pba (415/668 nm) and its standard curve between concentration and fluorescence (Figure S1). The fluorescence was detected using a synergy H1 Hybrid Multi-Mode Reader (Biotek Instruments, Inc., Winooski, YT, USA). To obtain the release profile of Pba from NPs, Pba-RL-NPs were placed in the dialysis membrane (MWCO: 13KD) and dialyzed under DPBS (pH 7.4). The external solution was obtained at predetermined time point and the florescence of Pba (415/668 nm) was measured by microplate reader. Cellular imaging All in vitro studies were performed with the SCC7 cell line (mouse squamous cell carcinoma line). SCC7 cells were obtained from the American Type Culture Collection (Rockvile, MD). The cell line was cultured in RPMI medium with 10% FBS and 1% antibiotic-Antimycotic at 37°C with 5% CO2. SCC7 cells were seeded in 24-well plates at 2 × 10 4 cells per well and grown for 2 days. After DPBS washing, cells were treated with different concentrations, from 1 to 4 μg/ml of Pba, of free Pba or Pba-RL-NPs in serum-free medium and incubated for 2 hours. Then, cells were washed with DPBS two times and fixed with 4% paraformaldehyde for 30 min at 4°C. Cells were then stained with 2 μg/ml of Hoechst 33342. After two more DPBS washes, cell imaging was performed with a Fluorescence Inverted Microscope IX 71 (Olympus, Tokyo, Japan). For confocal imaging, SCC-7 cells seeded into confocal dish for 10 × 10 4 cells / plate and grown for 2 days. Free Pba and Pba-RL-NPs (4 μg/ml of Pba concentration) in free medium were treated to cells and incubated for 2 hours. Then, cells were washed with DPBS for

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Figure 1. Synthesis and characterization of pheophorbide a-loaded rhamnolipid nanoparticles (Pba-RL-NPs). (A) Chemical structure of rhamnolipid and schematic illustration of Pba-RL-NPs. (B) Size of Pba-RL-NPs as a function of rhamnolipid concentration. Results represent mean ± S.E. (n = 3). (C) Size distribution and transmission electron microscopy (TEM) image of Pba-RL-NPs. Scale bar indicates 200 um. (D) Pba-RL-NPs size over 5 days. Results represent mean ± S.E. (n = 3). Images of free Pba (left) and Pba-RL-NPs (right) in PBS (pH 7.4).

twice and fixed with 4% paraformaldehyde for 30 min at 4°C. Cells were stained with 2 μg/ml of Hoechst 33342 for 15 minutes and cell imaging was obtained with a Confocal Laser Scanning Microscope (LSM800 w/Airyscan, Carl Zeiss, Germany). Singlet oxygen generation measurement Singlet oxygen generation from Pba-RL-NPs was analyzed by SOSG. Free Pba or Pba-RL-NPs (1 μg/ml of Pba concentration) was mixed with SOSG (0.06 μg/ml) in DPBS and irradiated with a 671-nm laser for different durations. 10% DMSO was mixed to release the loaded Pba and prevent aggregation. Then, SOSG fluorescence (488/525 nm) from all samples was measured by a synergy H1 Hybrid Multi-Mode Reader (Biotek Instruments, Inc., Winooski, YT, USA). XTT assay Viability testing of the SCC7 cells was performed by XTT assay. SCC7 cells were cultured in a 96-well plate at 0.5 × 10 4 cells per well and grown for 2 days. Then, the cells were treated with 100 μl of free Pba or Pba-RL-NPs (1-4 μg/ml Pba) in serum-free medium for 2 hours. After washing with DPBS two times, the medium was replaced with full growth medium. Each sample was then analyzed by the XTT assay to observe dark toxicity. Each well was treated with 25 μl of XTT solution (1 mg/ml in serum-free

RPMI medium containing 7.5 μg/ml of PMS). After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured by synergy H1 Hybrid Multi-Mode Reader (Biotek Instruments, Inc., Winooski, YT, USA). To test photodynamic effect in vitro, each well was irradiated with a 671-nm laser (0.3 W, 1 J) and analyzed similarly In vivo and ex vivo imaging All animal studies were approved by the Institutional Review Board of our university (approval no. CUMC-2016-0315-01). C3H/ HeN mice (3 weeks old, OrientBio, Seongnam city, Korea) were used for in vivo imaging. To establish a tumor model, 1 × 10 6 SCC7 cells in culture medium (100 μl) were injected subcutaneously into the left femoral region of the mice. When the tumors’ size grew to approximately 150 mm 3, free Pba or Pba-RL-NPs (2.5 mg/kg of Pba in 100 μl physiological saline, n = 3) were injected into the mice via the tail vein. Afterwards, mice were anesthetized by inhalation of isoflurane, and whole-body imaging was performed with an IVIS Lumina XRMS (PerkinElmer Inc., Waltham, Massachusetts, USA) set at 660/710 nm at 1, 3, 6, 12 and 24 hours after injection. All images were analyzed by Living Image 4.5 software (PerkinElmer Inc., MA). Blood samples (30 μl) were also obtained from the mice at the same time points. The collected blood was transferred to a 384HT plate and was mixed with detergent solution (DMSO: DW=4: 1,

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Figure 2. Cellular uptake of Pba-RL-NPs and singlet oxygen generation. (A) Fluorescence images of SCC7 tumor cells treated with free Pba and Pba-RL-NPs for an incubation time of 2 hours. Scale bar indicates 10 um. (B) Fluorescence intensity of Pba in SCC7 cells treated with different Pba concentrations. Results represent mean ± S.E. (n = 10). (C) Single oxygen generation from free Pba- and Pba-RL-NPs-treated cells in 10% DMSO in DPBS (pH 7.4) solution after laser irradiation for a predetermined time (n = 3).

2% Triton X- 100) before their fluorescence was measured with an IVIS Lumina XRMS. Twenty-four hours post-injection, tumor and major organs (heart, lung, liver, spleen and kidney) were dissected and imaged using IVIS Lumina XRMS similarly. The dissected tumors from the mice were prepared with O.C. T compound in a mold and stored at -80°C. After cutting the samples to 7-um thickness, tumor slices were washed with DPBS three times and stained with Hoechst 33342 (2 μg/ml). Subsequently, fluorescence images of the tissues were obtained by Fluorescence Inverted Microscope IX 71. To observe clearance route of free Pba and Pba-RL-NPs, the animals were euthanized 3, 6, 12, and 24 hours post-injection. After opening abdomen region, the fluorescence images of mice were obtained by IVIS Lumina XRMS with same condition. The major organs (heart, lung, liver, spleen, and kidney) and tumor were dissected and obtained ex vivo images by IVIS Lumina XRMS. After that, 50 mg of the dissected organs and tumor tissue were washed with PBS (pH 7.4) and minced with tissue grinder in 100 μl of Mammalian protein extract buffer solution. The minced tissues were grinded again using a C505 probe sonicator (Sonics; Newtown, CT, USA) for few seconds. Then, the 100 μl of detergent solution (1% Triton X-100, DMSO: PBS:

DW=5: 4: 1) was added to each sample and obtained images using IVIS Lumina XRMS. Photodynamic therapy in vivo For in vivo photodynamic therapy, we prepared SCC7 tumorbearing mice models with methods similar to those used in the imaging experiments. The mice were separated into three groups (saline, free Pba, and Pba-RL-NPs, n = 4), and each group was intravenously injected with 100 μl of the corresponding solution (100 μl physiological saline with 2.5 mg/kg of Pba for the Pba groups). The tumor region was treated with laser irradiation (671 nm, 2.3 W/cm 2) for a duration of 5 min at 12 and 24 hours after injection. The tumor size was measured each day with a digital caliper, and the tumor volume was calculated to be [(longest length of tumor) × (shortest length of tumor) 2 × 1/2]. The body weight of the mice was also monitored. According to the guidelines of the IACUC (Institutional Animal Care and Use Committees, Animal and Plant Quarantine Agency, Korea), when the tumor volume reached 2000 mm 3 or more, the corresponding animal was euthanized. At 14 days after injection, the remaining mice were euthanized, and their tumors were dissected for histological analysis.

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H&E staining The collected tumor tissue samples were fixed with 10% formalin solution for 2 days. Fixed tumor tissues were embedded in paraffin and sectioned at 4-um thickness. The sliced samples were stained with hematoxylin and eosin, and then observed using a Microscope Axio Imager A1 (Zeiss, Germany). Statistics The statistical significance of differences between groups was analyzed using one-way ANOVA. P values below 0.01 were considered significant.

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Results

We prepared rhamnolipid nanoparticles via the traditional oilin-water (O/W) emulsion method using flax seed oil, 18 and as a model drug, pheophorbide a, a near-infrared photosensitizer, was loaded into these nanoparticles (Figure 1, A). When we increase the concentration of rhamnolipid during fabrication, the size of the resultant nanoparticles decreased (Figure 1, B). At 4 mg/ml of rhamnolipid, the nanoparticle size reached about 136.1 nm, which was the particle size used in the following studies. The synthesized Pba-RL-NPs showed spherical shapes in transmission electron microscopy (TEM) images and had slightly negative surface charge with a zeta-potential value -34.5 mV due to the carboxylic acid groups of rhamnolipid (Figure 1, C). The size of the Pba-RLNPs did not change significantly over 5 days stored at pH 7.4, showing their high stability. When the nanoparticles were stored for a month at concentration of 0.5 mg/ml Pba in PBS (pH 7.4), no aggregation was observed, even though free Pba formed large aggregates under the same conditions (Figure 1, D). The loading efficiency of Pba in Pba-RL-NP was 91.5 ± 4.5%. When we tried to increase the feeding amount of Pba from 3 mg to 7 mg, the size increased to 164.5 nm and loading efficiency decreased to 80.14 % (Figure S2). Therefore, we used 3 mg Pba feeding condition for the following experiments. We attempted to analyze Pba release from the Pba-RL-NPs, but the concentration of the released Pba after one month was below the detection limit, demonstrating the excellent stability of the system. Cellular uptake of Pba-RL-NPs and singlet oxygen generation To observe cellular uptake of Pba-RL-NPs, SCC7 mouse squamous cell carcinoma cells were incubated with different concentrations of Pba-RL-NPs and free Pba for 2 hours at 37°C (Figure 2, A). Pba-RL-NPs-treated tumor cells showed intense fluorescence similar to the case of free Pba indicating fast cellular uptake. In confocal images with higher resolution, free Pba and Pba-RL-NPs were mainly located in cytosol (Figure 2, B). Considering that the main targets of PDT are generally subcellular organelles including mitochondria, their distribution in cytosol would be suitable to kill the cells upon laser irradiation. 19 Similar results were observed after cells were treated with various concentrations of Pba (from 1 to 8 μg/ml), with the signal increasing in a dose-dependent manner (Figure 2, C). To measure

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Concentraion of Pba (ug/ml) Figure 3. Cell viability test and photodynamic effect on cells. Cell viability based on XTT assay (A) in the dark and (B) after laser irradiation (671 nm, 0.3 W, 1 J). SCC7 cells were treated with free Pba and Pba-RL-NPs for 2 hours before irradiation. Results represent mean ± S.E. (n = 5).

the generated singlet oxygen upon laser irradiation, we used singlet oxygen sensor green (SOSG). 20 Both free Pba and Pba-RL-NP showed effective generation of singlet oxygen in 10% DMSO in DPBS (pH 7.4) solution when irradiated by a laser (Figure 2, D). It means that the photodynamic ability of the loaded Pba did not change during loading process. In vitro cytotoxicity test and photodynamic effect of Pba-RL-NPs By XTT test, we first evaluated cell viability of free Pba and Pba-RL-NPs in dark conditions at different concentrations of Pba (Figure 3, A). Free Pba and Pba-RL-NPs showed over 80% viability up to 4 μg/ml of Pba concentration, indicating no significant cytotoxicity. After irradiation by 671 nm laser at 0.3 W (1 J), the cell viability decreased due to the photodynamic effect in both groups (Figure 3, B). The cell death increased with increased Pba concentration, and free Pba and Pba-RL-NPs showed similar efficacy.

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Figure 4. In vivo biodistribution of free Pba and Pba-RL-NPs in SCC7 tumor-bearing mice after intravenous injection. (A) Real-time near-infrared fluorescence (NIRF) images of whole body distribution after intravenous injection of free Pba and Pba-RL-NPs. (B) Time-dependent fluorescence intensity analysis of a tumor site. Results represent mean ± S.E. (n = 3). *P b 0.01. (C) NIRF images of the blood samples drawn from the same mice. (D) Quantification of the NIRF intensity of blood samples in (C). * P b 0.01.

In vivo biodistribution of Pba-RL-NPs in SCC7 tumor-bearing mice

circulation times and enhanced tumor-targeting via the EPR effect.

To analyze in vivo biodistribution, free Pba and Pba-RL-NPs (2.5 mg/kg of Pba) were intravenously injected into SCC7 tumor-bearing mice. We acquired real-time NIRF images at predetermined times with IVIS Lumina XRMS system, and the whole body images were overlaid with X-ray images. 20 In the mice treated with Pba-RL-NPs, the NIRF signal from the tumor site increased gradually up to 12 hours post-injection, showing efficient delivery of Pba to tumor site (Figure 4, A). In contrast, free Pba-treated mice showed fast excretion of Pba, and the signal from the tumor site was weaker than that from the Pba-RLNPs-treated group. The NIRF signal from the tumor sites of PbaRL-NP-treated mice was about 4.43 times higher than those from free Pba-treated mice demonstrating the superior tumor-targeting ability of Pba-RL-NP (Figure 4, B). The NIRF images and the fluorescence intensity of blood samples acquired from same groups of mice showed a longer blood circulation time for PbaRL-NPs than for free Pba (Figure 4, C and D). These results demonstrate that Pba-RL-NP successfully provided long blood

Ex vivo imaging of major organs and tumor tissues At 24 hours after injection, major organs and tumor tissues were dissected, and their ex vivo NIRF images were obtained. The images exhibited that Pba-RL-NP-treated mice showed higher fluorescence intensity in all organs (heart, lung, liver, spleen, and kidney) and tumors than free Pba-treated mice (Figure 5, A). When we compare the NIRF signal in the dissected tumors, the signal from Pba-RL-NPs-treated mice were 4.7 times higher than that from free Pba-treated mice (Figure 5, B). We also analyzed frozen sections of the excised tumor tissue of each group via fluorescence microscopy (Figure 5, C). Pba-RL-NPtreated tumor samples showed intense Pba fluorescence which was not observed in the case of free Pba-treated tumors. In accordance with the in vivo results, the ex vivo data showed that Pba-RL-NPs delivered Pba to tumor site efficiently after intravenous injection. We also performed animal experiments to analyze the clearance pathway of free Pba- and Pba-RL-NPs.

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Figure 5. Ex vivo imaging of Pba-RL-NPs in SCC7 tumor-bearing mice after intravenous injection. (A) Ex vivo NIRF images of the dissected tumors and major organs (heart, lung, liver, spleen, and kidney) of SCC7 tumor-bearing mice 24 hours post-injection of free Pba and Pba-RL-NPs. (B) NIRF intensity analysis of tumor tissue and major organs in (A). *P b 0.01. (C) NIRF images of the sliced tumor tissue in (A).

At 3, 6, 12, and 24 hours after i.v. injection of free Pba- and PbaRL-NPs, the mice were analyzed in vivo and ex vivo (Figure 6). As shown in the data, they were secreted from body by both renal (kidney) and hepatic pathway (liver). The fluorescence intensities of the grinded organs from free Pba- and Pba-RL-NPsinjected mice also showed similar trends (Figure S3). In vivo photodynamic therapy by Pba-RL-NPs and histological analysis. To evaluate therapeutic efficacy of Pba-RL-NPs, we injected free Pba or Pba-RL-NPs into the same mice model intravenously and treated them with laser irradiation (671 nm, 2.4 W/ cm 2 , 5 min) 12 and 24 hours post-injection (n = 4). The sizes of tumors were measured by a digital caliper every day for two weeks. The different growth pattern could be evaluated at P

value below 0.1 by one-way ANOVA. Pba-RL-NP-treated mice showed complete suppression of tumor growth 14 days, unlike the other groups (Figure 7, A and B). The saline-treated control and free Pba-treated groups showed rapid tumor growth, and had to be euthanized 11 and 13 days post-injection, respectively, due to animal ethics concerns regarding large tumor sizes. The body weights of the mice showed no significant difference between groups within the experimental period demonstrating low toxicity of Pba-RL-NPs in vivo (Figure 7, C). After that, the dissected tumor tissues were stained with H&E (Figure 7, D). The tumor tissues from saline- and free Pba-treated groups showed normal tumor tissue structure, whereas tumor tissues from Pba-RL-NP-treated group exhibited a region destroyed by photodynamic therapy. These results demonstrated that the improved tumor-targeting ability of Pba-RL-NPs resulted in the enhanced therapeutic efficacy in vivo.

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Discussion Despite extensive research efforts focused on nanomedicine, only a few nanoparticles are currently used in clinical applications, which shows that it is still important to develop and test a new nanomaterials for medical applications. Amphiphilic surfactants are a major component of selfassembled nanoparticles, and various materials have been used as surfactants. However, biosurfactants like rhamnolipid have been rarely used in nanoparticle research even though they have established commercial uses in markets such as cosmetics, agriculture, and pharmaceuticals. 16 To the best of our knowledge, our research is the first intravenous injection of rhamnolipid nanoparticles for drug delivery and photodynamic

therapy in vivo. On the basis of these results, we expect that more applications for rhamnolipid nanoparticles will be explored in the future. In this paper, we selected Pba, a photosensitizer, as our model drug because the intrinsic fluorescence of photosensitizers is useful for both imaging as well as photodynamic therapy. 21,22 The near-infrared fluorescence of Pba enabled non-invasive whole body imaging of Pba-RL-NPs in a tumor-bearing mice model and allowed for real-time analysis of their biodistribution. Pba-RL-NPs may prove to be useful for simultaneous imaging and therapy, or theranostics by a single material. 23,24 However, the utility of RL-NPs is not restricted to photosensitizers. The use of RL-NPs for the drug delivery should be generalizable to almost any hydrophobic drug. In vivo and ex vivo imaging data

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Time (days) Figure 7. In vivo photodynamic therapy using Pba-RL-NPs. (A) Tumor images from SCC7 tumor-bearing mice 14 days post-injection of saline, free Pba and Pba-RL-NPs (2.5 mg/kg of Pba) after laser irradiation (671 nm, 2.4 W/cm 2, 5 min). (B) Tumor growth after photodynamic therapy. Results represent mean ± S. E. (n = 4). * P b 0.01. Arrow means laser irradiation. # means early sacrifice due to large tumor size. (C) Relative body weight change of the mice in (A). (D) Histological analysis of the tumor tissues after photodynamic therapy that were stained with H&E. Scale bar indicates 100 um.

showed that the amount of Pba-RL-NPs in tumor tissue gradually increased to 12 hours post-injection. It is different with that of free Pba that decreased from 3 hours may due to the fast secretion from body. Long circulation time and increase of tumor accumulation is general situation of NPs based on EPR effect, so that we expect that these RL-NPs can be applied to delivery of other anticancer drugs to tumor in future. In summary, we developed new nanoparticles using rhamnolipid, a representative biosurfactant. After loading the nanoparticles with the photosensitizer Pba, the size of the resulting Pba-RL-NPs was about 136.1 nm, and they were stable in aqueous suspension for at least one month. Pba-RL-NPs showed fast cellular uptake in SCC7 tumor cells and killed the cells upon laser irradiation. After intravenous injection to SCC7 tumor-bearing mice model, PbaRL-NPs demonstrated long blood circulation times. Real-time NIRF imaging indicated 4.7 times higher tumor tissue accumulation of Pba-RL-NPs compared to free Pba. Upon laser irradiation of the tumor tissue, Pba-RL-NPs showed complete suppression of tumor growth by photodynamic therapy in vivo. These overall results demonstrated that rhamnolipid nanoparticles are highly useful for drug delivery, which will likely expand the application of rhamnolipids to biomedical fields in future.

Acknowledgments This work was supported by Basic Research Program (2016R1C1B3013951) through the National Research Foundation of Korea (NRF) funded by the Korean Government (Ministry of Science, ICT, & Future Planning). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.nano.2019.03.015.

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