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Zebrafish: A Visual Model to Evaluate the Biofate of Transferrin Receptor-targeted 7Peptide-decorated Coumarin 6 Micelles Ye Li, Xiaoning Song, Xiang Yi, Ruibing Wang, Simon Ming-Yuen Lee, Xueqing Wang, and Ying Zheng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12809 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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ACS Applied Materials & Interfaces

Zebrafish: A Visual Model to Evaluate the Biofate of Transferrin Receptor-targeted 7Peptide-decorated Coumarin 6 Micelles Ye Li 1, Xiaoning Song2, Xiang Yi3, Ruibing Wang1, Simon Ming-Yuen Lee1, Xueqing Wang2*, Ying Zheng1* 1

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China

2

Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

3

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina, USA

*To whom correspondence should be addressed: 1. Ying Zheng, Ph.D. Institute of Chinese Medical Sciences, University of Macau Tel: (853) 88224687; Fax: (853) 28841358 E-mail: [email protected] 2. Xueqing Wang, Ph.D. School of Pharmaceutics, Peking University Tel: 86-10-82805935; Fax: 86-10-82805935 E-mail: [email protected]

Keywords:

Zebrafish,

7Peptide,

Transferrin

receptor,

poly-(ethylene

glycol)-block-poly (ε-caprolactone) micelles, Ligand density, Biological barriers.

ACS Paragon Plus Environment


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Abstract In the present study, the zebrafish was explored as an in vivo model to assess the biofate of transferrin receptor (TfR)-targeted coumarin 6 (C6) micelles across various biological

barriers.

Three

7peptide

(7pep)-decorated

poly-(ethylene

glycol)-block-poly (ε-caprolactone) (PEG-b-PCL) micelles loaded with fluorescence coumarin 6 (7pep-M-C6) with different ligand densities were constructed with particle sizes between 30-40 nm. Whole-mount immunostaining revealed that the expression level of TfR in the retina, brain and intestine increased along with development stage. Compared to unmodified micelles, 7pep-M-C6 demonstrated higher uptake efficiency in the larval zebrafish. Pre-inhibition of TfR with 7pep implicated the TfR-mediated endocytosis pathway in the uptake of 7pep-M-C6. Confocal images of the larval zebrafish eye and brain showed the efficient delivery of C6 across the retinal pigment epithelial to the ganglion cell layer and the significant accumulation of C6 in all brain tissues, respectively, which plateaued when the ligand density was 10%. To investigate the intestinal distribution of C6, micelles were administered to adult zebrafish via gavaging. Notably, 7pep-M-C6 enhanced the transport of C6 across the villi and increased its aggregated in the basolateral membrane of the intestine. After the oral administration of 7pep-M-C6, C6 accumulated in the eye and brain. Förster Resonance Energy Transfer (FRET) analysis suggested that intact 7pep-modified micelles could enter the epithelial cells of the intestine, brain and eye after oral administration in adult zebrafish. In conclusion, zebrafish could be used as a model for in vivo visual assessment of the


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biofate of TfR-targeted drug delivery systems.

Introduction Based on the identification of membrane receptors, such as transferrin receptor (TfR), epidermal growth factor receptor, folate receptor and integrin receptor, which are expressed on the membranes of cells/tissues for the active transport of nutrients, different types of ligands have been synthesized and decorated on the surfaces of nanoparticles

to

enhance

drug

cellular

uptake

through

endocytosis.1-3

Ligands-modified nanoparticles have been reported in vivo to enhance oral drug absorption, and improve drug therapeutic efficacy for cancer treatment, such as glioma and neuroblastoma.4-6 However, these nanoparticles may acquire protein corona once interacting with biological fluids in vivo, which may change their physicochemical properties and biological fate, therapeutic efficacy or even toxicity.7-8 When the nanoparticles are modified with ligands, the protein corona may affect the specific reaction between ligands and receptors on cells.9 Therefore, a thorough understanding of the dynamic process during the in vivo delivery of targeted nanoparticles is important. To this end, in the present study, we developed and validated zebrafish as an in vivo model for the visual evaluation of the biofate of transferrin receptor-targeted 7peptide (7pep)-decorated coumarin 6 (C6) micelles. Reflecting its whole body transparency, zebrafish could be used for tracking fluorescence probe and drug delivery systems (DDSs) in real time.10 Moreover, the structure and function of biological barriers, including the blood retinal barrier (BRB),



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blood brain barrier (BBB) and gastrointestinal barrier, are similar to that of mammals at certain levels.11 For larval zebrafish, exposed to nanoparticles, the main uptake route was via oral delivery into the intestine. Nanoparticles could accumulate in the intestine and reach the cardiovascular system.10 In addition, adult zebrafish have been utilized to provide additional information about the uptake, distribution, excretion of nanoparticles.12 Our previous studies have demonstrated zebrafish as a simple and dynamic model to simultaneously assess the transport of DDSs, such as nanocrystals, nanoparticles and cucurbituril complexes, across several biological barriers.13-15 TfR, a trans-membrane receptor, is expressed on several biological barriers in the intestine, brain, eye, lung, etc..16 For zebrafish, tfr1 was detected using RT-PCR and whole-mount in-situ hybridization at 12 hours post fertilization (hpf), and tfr1b likely functions

for

iron

acquisition.17-18

The

7pep

(Histidine-Alanine-Isoleucine-Tyrosine-Proline-Arginine-Histidine, HAIYPRH), is a peptide obtained through phage display that exhibits high affinity to TfR with a unique binding site.19 In our previous study, 7pep-conjugated poly-(ethylene glycol)-block-poly (ε-caprolactone) (PEG-b-PCL) micelles were constructed for oral drug delivery, and its endocytosis, intracellular trafficking and transcytosis were investigated in Caco-2 cell model.20 The results demonstrated that 7pep-conjugated PEG-b-PCL micelles loaded with C6 (7pep-M-C6) could specifically interact with gastrointestinal endothelial cells, thereby increasing the transport and altering the transport pathway of the delivery system. Herein, we utilized 7pep-M-C6 with different ligand densities (2.5%, 10% and


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20%) as a targeted drug delivery system. C6 is a typical hydrophobic model drug with auto-fluorescence that could be monitored in vivo via fluorescence or confocal microscopy. Micelles without 7pep modification (M-C6) were used as a control. First, we systemically validated TfR expression at different development stages and tissues of zebrafish. Subsequently, the biofate of these micelles in larvae and adult zebrafish were investigated. The efficacy of micelle delivery in the retina, brain and intestine were compared using ultra-microtome and confocal imaging. The in vivo integrity of functional micelles for oral delivery was explored in adult zebrafish using Förster Resonance Energy Transfer (FRET) technology.

Materials and Methods Materials

mPEG3000-b-PCL2500

(Mw/Mn=1.09)

N-hydroxysuccinimidyl-PEG4000-b-PCL2500 Advanced

Polymer

Materials

(Mw/Mn=1.30) Inc.

and were

(Montreal,

supplied

by

Canada).

Histidine-Alanine-Isoleucine-Tyrosine-Proline-Arginine-Histidine (HAIYPRH; 7pep) was obtained from GL Biochem Peptide Ltd. (Shanghai, China). Coumarin-6 (purity>98%), 1-phenyl-2-thiourea (PTU), low gelling temperature agarose, ethyl 3-aminobenzoate methanesulfonate (MS-222) were obtained from Sigma Aldrich (St. Louis, MO, USA). 3,3’-Dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine (DiI) and Hoechst 33342



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were purchased from Invitrogen (Carlsbad, CA, USA). Anti-transferrin receptor antibody (ab84036) and Goat anti-rabbit IgG H&L (Alexa Fluor® 568) (ab175471) were purchased from Abcam, UK. All other chemical regents were of analytical grade or above.

Zebrafish husbandry and breeding

Wild-type zebrafish were used in the present study. The procedures for zebrafish culture, breeding, embryos collection, embryonic and larval culture were performed under standard procedures.21 Briefly, a light (14 h) and dark (10 h) cycle was used to raise female and male zebrafish. For maximal embryo production, mature male and female (2:3) zebrafish were transferred into a 1-L breeding tank and separated using a mesh screen (2 mm) the night before breeding. At the onset of the light cycle, male and female zebrafish initiated breeding behavior. All fertilized embryos were gathered at the beginning of the next light cycle. After eggs collection at 0-2 hpf, the embryos that developed normally were selected for the experiments. The collection of embryos, and the subsequent experiments were all performed in E3 medium (13.7 mM NaCl, 540 µM KCl, 25 µM Na2HPO4, 44 µM KH2PO4, 300 µM CaCl2, 100 µM MgSO4, and 420 µM NaHCO3, pH 7.4) at 28.5 ° C. Ethical approval for the animal experiments was granted through the Animal Research Ethics Committee, University of Macau.

TfR whole-mount immunostaining

Immunohistochemistry was performed using zebrafish at 1, 4 and 7 days post fertilization (dpf). Briefly, zebrafish were fixed in 4% paraformaldehyde in PBS for


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15 min, rinsed with PBST, and subsequently, the fixed samples were treated with H2O2 diluted in KOH (6%) for 5 to 10 min. The samples were then blocked (2% BSA in PBST) for 2 h at room temperature. Rabbit polyclonal anti-transferrin receptor antibody (1:100 diluted in blocking buffer) was used as the primary antibody and incubated with the samples overnight at 4 °C. Subsequently, the samples were washed 6 times with PBST (30 min each), followed by incubation with secondary antibody (1:1000 diluted in blocking buffer, ab175471, Abcam). After incubation, the samples were washed 3 times with PBST. For the control group, zebrafish were directly incubated with secondary antibody, and the other processing steps were conducted as described above. Fluorescent images were captured using a fluorescence microscope (Olympus IX81 Motorized Inverted Microscope, Tokyo, Japan) with a digital camera (DP controller, Soft Imaging System, Olympus). The images were recorded by keeping the parameters of the camera, such as exposure time and sensitivity, constant throughout the imaging process. Except for capturing the fluorescence images, the samples were also used to prepare the frozen sections. Zebrafish at 4 or 7 dpf were embedded in Tissue Freezing Medium (Leica, Germany), a convenient specimen matrix for cryostat sectioning, and frozen in -80°C for microtome slicing. The whole body was sectioned into 8-µm sections in a transverse orientation using a microtome (Leica, Germany) and thaw-mounted onto adhesion microscope slides. The sections were subsequently stained with 2 µg/mL Hoechst, and the coverslip was mounted with a drop of antifade mounting medium and sealed. The sections were observed using confocal laser



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scanning microscopy (CLSM).

Preparation and characterization of micelles and functional micelles

Unmodified micelles and functional micelles were prepared using a film hydration method according to Du et al..20 Briefly, to prepare the micelles (M-C6), C6 and mPEG3000-b-PCL2500 were dissolved in 4 mL acetonitrile. The liquid was subsequently evaporated through vacuum rotary evaporation at 60 °C to form a thin film. The obtained film was hydrated in E3 medium, followed by sonication for 5 min. Subsequently, the solution was filtered through a 0.22-µm PVDF membrane. To synthesize 7pep-PEG-b-PCL, the 7pep was conjugated to the distal end of PEG of N-hydroxysuccinimidyl-PEG4000-b-PCL2500 through a reaction between the NHS and amino group. HPLC and UV-Vis spectrum were used to monitor the conjugation reaction. To prepare 7pep-M-C6, an equivalent amount of mPEG-b-PCL was replaced with 7pep-PEG-b-PCL, and the percentage of 7pep-PEG-b-PCL polymer in the total polymer content was 2.5, 10 and 20% (w/w), respectively. The final concentrations of the C6 and the polymer were 2 µg/mL and 2 mg/mL, respectively. The particle size and polydistribution index (PDI) were determined using dynamic light scattering (DLS) (Nano-Zetasizer, Malvern, U.K.).

Embryo and larval zebrafish exposed to 7pep-M-C6

The embryo (1 dpf) and larval zebrafish (4 and 7 dpf) were incubated with micelles (400 ng/ml of C6) for 30 and 60 min. For the receptor inhibition experiment, the zebrafish were pre-incubated with 7pep for 30 min prior to the addition of


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7pep-M-C6. The whole-body uptake was visualized and quantified using fluorescence microscopy and fluorescence spectrophotometry, respectively. After incubation, zebrafish in each treatment group were anesthetized with 0.04% MS-222 and washed three times with E3 medium. After lateral orientation in 1% low-melting agarose, the zebrafish were imaged using a fluorescence microscope equipped with a digital camera (Olympus IX81 Motorized Inverted Microscope). All camera parameters remained the same as described above among the different micelles-treated groups. The C6 in whole body tissues was quantified as previously reported.22 Briefly, 30 live larval zebrafish per treatment (three replicates per treatment) were collected, transferred from 12-well plates to 100-µm nylon filters (BD Falcon, USA) and washed with distilled water. Whole bodies were weighed after overnight drying with silica gel. After homogenization with water/methanol (1:1, v/v) and centrifugation, the fluorescence densities of C6 were measured. The whole-body uptake was expressed as ng of C6 per mg of body weight. For the 7pep inhibition, zebrafish larvae were pre-treated with 7pep for 30 min, and subsequently 7pep-M-C6 was added to the medium for 30 min. Zebrafish in each group were collected for C6 quantification.

Eye and brain distribution of 7pep-M-C6 in larval zebrafish

After incubation with 7pep-M-C6 for 1 h, larval zebrafish at 7 dpf were collected, washed, and fixed. Sections of the eye and brain tissues in a transverse orientation were prepared for CLSM imaging.



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Distribution of 7pep-M-C6 in adult zebrafish after oral administration

To study the intestinal absorption of the micelles, adult zebrafish was fasted for at least 24 h prior to the procedure, and placed in the MS-222 solution (5%) for anesthesia. When the zebrafish lost righting reflex, the fish was removed and placed in the groove of a wet sponge with the head slightly protruding from the sponge, and 22-G catheter tubing was used to open the mouth of the zebrafish, and subsequently the tubing was gently inserted until the tip was past the gills. Micelles (10 µl) were directly injected. Notably, during this procedure, it is necessary to ensure that the solution dose not exit via the gills or the mouth.23 Finally, the zebrafish was removed from the sponge and placed in a tank with fresh water. After different time periods, the zebrafish were collected, anesthetized on ice, and subsequently dissected. The tissues were separated, washed adequately three times with distilled water and fixed. Frozen sections (8 µm) were prepared and CLSM imaging was conducted as described above. M-C6 or 7pep-M-C6 solution (10 µl) was directly injected into the intestines of adult zebrafish, and after 1 h, zebrafish were collected, anesthetized on ice, and subsequently dissected. The intestine was separated, washed three times with distilled water and fixed. Frozen sections of intestine tissues were prepared and imaged using CLSM. For the eye and brain distribution of 7pep-M-C6 (10%) in adult zebrafish, M-C6 or 7pep-M-C6 (10%) solution (10 µl) was directly injected into the intestines of adult zebrafish. After 1, 2 and 4 h, the zebrafish were collected, anesthetized on ice, and


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subsequently dissected. The eye and brain was separated, washed three times with distilled water, and fixed. The frozen sections were prepared and imaged using CLSM as described above.

In vitro integrity monitoring using FRET

DiO and DiI, two fluorophores able to interact by FRET were loaded into 7pep-PEG-b-PCL micelles using a method similar to the preparation of 7pep-M-C6 with a ligand density of 10%. The final concentration of DiO and DiI was 2 mg/L. In the in vitro experiment, the fluorescence spectra of 7pep-DiO-DiI-Micelles in E3 medium or acetone were measured using an excitation at 470 nm and an emission scan from 480 to 700 nm. After different time periods (1, 2, and 4 h), the fluorescence spectra of 7pep-DiO-DiI-Micelles in E3 medium were detected. We also detected the fluorescence spectra of 7pep-DiO-DiI-Micelles in the hydrophobic environments (1 mg/mL albumin or 20% serum) for 1, 2 and 4 h. The extent of FRET was quantified using a FRET ratio (IDiI/(IDiO+IDiI)). IDiO and IDiI were the fluorescence intensities of DiO and DiI at 500 and 580 nm, using an excitation wavelength of 470 nm, respectively.

In vivo FRET imaging in intestine, eye and brain of adult zebrafish

7pep-DiO-DiI-Micelles (10 µl) were injected into the intestines of adult zebrafish directly. At 1, 2 and 4 h after administration, intestine, eye and brain were separated, fixed and 8-µm cryostat sections were obtained. Samples for FRET analysis were imaged using LSCM and a 63Plan Apochromat numerical aperture 1.4 oil objective.



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Scanning was performed on a line-by-line basis with the zoom factor set to 0.75. The gain for each channel was constant throughout the imaging process. Confocal images were acquired using an excitation wavelength of 488 nm and the emission wavelengths between 580 and 650 nm for FRET and between 500 nm and 530 nm for DiO detection with an acquisition mode of xyz. For lambda scan, the spectral analysis of the emission in each ROI between 500-700 nm using an excitation wavelength of 488 nm was collected at 10-nm intervals with an acquisition mode of xyλ. The data were captured and analyzed using Leica Application Suite X.

Statistical analysis

All experiments were conducted in triplicate. One-way ANOVA was used to evaluate statistical differences. The level of significance for all analyses was p < 0.05 or p < 0.01.

Results and Discussion Anti- TfR whole mount immunostaining of larvae zebrafish As a well-known drug delivery target, TfR expression was upregulated on the surfaces of many cancer cells,24 and this receptor was also expressed in appreciable amounts on the surfaces of enterocytes in the intestine.25 Therefore, TfR was used as a major drug delivery target, enabling the transcytosis of TfR-targeted nanoparticles across epithelial and endothelial cell barriers.26 Using PCR or whole mount in situ hybridization, several studies have suggested that TfR could be expressed in


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zebrafish.17, 27 The function of TfR for hemoglobin production was also confirmed in a zebrafish mutant.18,

28

Typically, larval zebrafish at 7 dpf have fully developed

functional biological barriers.11,

29-31

However, these experiments typically use

embryos up to 3 dpf, which do not reflect expression patterns with different development stages of zebrafish. To further investigate the expression level of TfR at different developmental stages of zebrafish and examine the distribution of this receptor in different tissues, we conducted an anti-TfR whole mount immunostaining. As shown in Figure 1A, there was no significant red fluorescence signal in the negative control group when the zebrafish was only treated with the secondary antibody at all developmental stages up to 7 dpf. However, as shown in Figure 1B, after treatment with both primary and secondary antibodies, there was obvious red fluorescence in the zebrafish, and the fluorescence intensity in the zebrafish body apparently increased from 1 to 7 dpf. The fluorescence was predominantly distributed in the trunk compared to the yolk sac at 1 dpf, and subsequently distributed in the whole body at 4 and 7 dpf. The functional gastrointestinal barrier with intestinal epithelium of zebrafish gradually matured until 5 dpf,31-32 while the expression level of TfR determined by the fluorescence intensity was obviously higher in the intestine of zebrafish at 7 dpf than at 4 dpf (Figures 1B and C). Further quantitative analysis of in vivo fluorescent imaging (Figure 1D) revealed that the fluorescence intensity of the zebrafish whole body at 7 dpf was 7.5 and 1.4-fold higher than that at 1 and 4 dpf, respectively. Moreover, the fluorescence intensity of the intestine of zebrafish at 7 dpf was 1.3-fold higher than that at 4 dpf. These results demonstrated that TfR is



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expressed as early as 1 dpf, and its expression level increased with development stage.

Figure 1. Brightfield and fluorescence images of zebrafish after anti-TfR whole mount immunostaining. A: Brightfield and fluorescence images of zebrafish in the control group after anti-TfR whole-mount immunostaining without primary antibody incubation. B: Brightfield and fluorescence images of zebrafish whole body (1, 4 and 7 dpf) after immunostaining. Red fluorescence presents the staining of TfR. C: Observation of digestive system of zebrafish at 7 dpf at high magnification. White dotted box represents the red fluorescence in the intestine. D: Fluorescence intensity of the zebrafish whole body at different developmental stages (left) and intestine at 4 and 7 dpf (right). A 10×ocular lens and 10×objective lens were used for imaging. Scale bars, 500µm. **p < 0.01 versus the control group (left) and 4 dpf intensity (right), ## p < 0.01 versus the fluorescence intensity of TfR at different development stages. Next, to determine the primary tissue localization of TfR, frozen sections of whole embryos at 4 dpf and whole larvae at 7 dpf were examined. As shown in Figures 2C and D, compared to the control groups (Figures 2A and B), different tissues of 4 and 7 dpf zebrafish showed specific TfR fluorescence intensity, indicating that TfR was extensively expressed in the intestine, eyes and brain and slightly expressed in the muscle. In addition, the fluorescence intensity displayed a relatively high level in the intestine (Figure 2 E), consistent with the results obtained from fluorescence imaging (Figure 1C). Particularly, TfR was expressed in the retina of zebrafish. The magnified CLSM image of sections of the eyes of 7 dpf zebrafish showed relatively high anti-TfR


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immunostaining in different retinal layers, including the photoreceptor cell layer (PCL), outer nuclear layer (ONL), outer plexiform layer (OPL) and inner nuclear layer (INL) (Figure 2E). According to a previous study, TfR is a cell surface receptor that binds and delivers iron into the cell via endocytosis or transcytosis. This receptor has been detected in the ganglion cell layer (GCL), INL, OPL, photoreceptors, retinal pigment epithelial (RPE) cells and choroid in rat and mice.33 Taken together, TfR demonstrated similar localization in the retinal cell layers of zebrafish at 7dpf compared with the distribution of TfR in the retinal cell layers of rodents.



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Figure 2. Transferrin receptor expression in different tissues of zebrafish at 4 and 7 dpf (horizontal plane), respectively. CLSM images of the intestine, eye, brain and muscle slices of zebrafish at 4 dpf (A) and 7 dpf (B) in the control group without primary antibody incubation. CLSM images of the intestine, eye, brain and muscle slices of zebrafish at 4 dpf (C) and 7 dpf (D) with both primary and secondary antibody treatment. Scale bars, 50 µm. (E) Magnified CLSM images of the intestine and eye slices of zebrafish at 7 dpf. Blue fluorescence was from Hoechst stained nucleus, the red fluorescence was from staining of TfR. PCL, photoreceptor cell layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 10 µm. In the present study, we demonstrated TfR expression in different tissues and at


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different development stages of zebrafish, providing an essential foundation to develop zebrafish as an in vivo model to evaluate the biofate of receptor-targeted micelles in a more complex, complete and dynamic organism.

Uptake of 7pep-M-C6 in embryonic and larval zebrafish in vivo

Synthesis of 7pep conjugated PEG-b-PCL polymer was conducted as previously described.20 The diagram of 7pep-M-C6 and M-C6 were shown in Figure 3A. C6 was encapsulated in the hydrophobic core, and 7pep was conjugated to the distal end of polymer. The average particle size of these micelles was between 30-40 nm with a PDI less than 0.3. In the uptake of 7pep-M-C6 in vivo, embryos (1 dpf) or larvae (4 and 7 dpf) were incubated with micelles for 30 and 60 min and subsequently observed using a fluorescence microscope. The fluorescence images of embryos after incubation with micelles are shown in Figure S1A, and embryos incubated with M-C6 were used as the control group. After incubation for 30 min, obvious fluorescence was observed in each treated group. Differences in the brightness between the embryonic chorions and embryos inside revealed that micelles could transport across the chorion barrier (Figure S1A). Notably, the fluorescence intensity increased with increasing incubation from 30 to 60 min. Similarly, as shown in Figure S1B, the fluorescence was distributed throughout the entire body of 4 dpf zebrafish after incubation for 30 and 60 min. Moreover, different fluorescence intensities were observed between the control and 7pep-M-C6-treated groups.



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Larval zebrafish at 7 dpf, which had fully developed biological barriers, including BBB, BRB and gastrointestinal barrier,31, 34 and expressed relatively high levels of TfR, were also exposed to 7pep-M-C6 for 30 and 60 min. As shown in Figure 3B, obvious fluorescence was observed throughout the entire body of larval zebrafish treated with M-C6 or 7pep-M-C6. Moreover, the fluorescence intensity apparently increased with increasing 7pep density from 0% to 20%. Additionally, this uptake enhancement was further confirmed by the uptake quantification results (Figure 3C). These findings suggest that the uptake of each 7pep-M-C6 was higher than that of M-C6 at 30 and 60 min. However, there was no significant difference between the micelles with 7pep 10% and 20% densities among the treated groups (p>0.05). To further confirm whether the increased uptake was facilitated by TfR-mediated transport, larval zebrafish were pretreated with 7pep as an inhibitor. As shown in Figure 3D, the uptake of 7pep-M-C6 at all ligand densities was significantly reduced compared to the non-7pep-treated group (p

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