Identification of Novel Medulloblastoma Cell-Targeting Peptides for

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Identification of novel medulloblastoma cell-targeting peptides for use in selective chemotherapy drug delivery Kristel Tjandra, Nigel McCarthy, Lu Yang, Alistair Laos, George Sharbeen, Phoebe A. Phillips, Helen Forgham, Sharon Marie Sagnella, Renee Megan Whan, Maria Kavallaris, Pall Thordarson, and Joshua A. McCarroll J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00851 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

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Journal of Medicinal Chemistry

Identification of novel medulloblastoma cell-targeting peptides for use in selective chemotherapy drug delivery Kristel C. Tjandra2,5, Nigel McCarthy1, Lu Yang1, Alistair J. Laos2,5, George Sharbeen4, Phoebe A Phillips2,4, Helen Forgham1,2,3, Sharon M Sagnella1, Renee M Whan2,6, Maria Kavallaris1,2,3*, Pall Thordarson2,5*, Joshua A McCarroll1,2,3*

1. Tumour Biology & Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, NSW, Australia, 2031. 2. Australian Centre for Nanomedicine, ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, UNSW Sydney, NSW, Australia, 2052. 3. School of Women’s and Children’s Health, Faculty of Medicine, UNSW Sydney, NSW, Australia, 2052. 4. Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, School of Medical Sciences, UNSW Sydney, NSW, Australia, 2052. 5. School of Chemistry, UNSW Sydney, NSW, Australia, 2052. 6. Biomedical Imaging Facility Mark Wainwright Analytical Centre, UNSW Sydney, NSW, Australia, 2052. * Corresponding authors.

Running title: Peptide-drug conjugates as a novel treatment for medulloblastoma.

Key words: Peptide phage display, medulloblastoma, drug-conjugates, doxorubicin. 1 ACS Paragon Plus Environment

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ABSTRACT Medulloblastoma is a malignant brain tumor diagnosed in children. Chemotherapy has improved survival rates to approximately 70%, however, children are often left with long-term treatment side-effects. New therapies which maintain a high cure rate while reducing off-target toxicity are required. We describe for the first time the use of a bacteriophage-peptide display library to identify heptapeptides which bind to medulloblastoma cells. Two heptapeptides which demonstrated high [E1-3(1)] or low [E1-7(2)] medulloblastoma cell binding affinity were synthesized. The potential of the peptides to deliver a therapeutic drug to medulloblastoma cells with specificity was investigated by conjugating E1-3(1) or E1-7(2) to doxorubicin (5). Both peptide-drug conjugates were cytotoxic to medulloblastoma cells. E1-3-doxorubicin (1) could permeabilize an in vitro blood-brain barrier and showed a marked reduction in cytotoxicity compared to free doxorubicin (5) in non-tumor cells. This study provides proof-of-concept for developing peptide-drug conjugates to inhibit medulloblastoma cell growth while minimizing offtarget toxicity.

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INTRODUCTION Medulloblastoma is the most common pediatric malignant brain tumor affecting approximately 20% of children diagnosed with brain cancer.1 Patients with medulloblastoma can be stratified into four different genetic subgroups (WNT, SHH, group 3 and group 4).1 Treatment comprises of surgery, chemotherapy and craniospinal irradiation.1,2 Five-year survival rates for this malignancy have markedly improved (60-80%), but children who survive the disease are often left with severe life-long debilitating side-effects from the treatment that can include hearing loss, cardiotoxicity, neurocognitive dysfunction and increased chance of secondary cancers.3 These toxicities have a negative impact on the quality of life of medulloblastoma patient survivors. There is an urgent need to develop new targeted therapies which are effective at killing medulloblastoma cells and are less toxic than conventional therapy to non-tumor cells. Traditionally targeted therapies for cancer involve the use of monoclonal antibodies covalently attached to cytotoxic drugs (Brentuximab veolotin, Trastuzumab-emtansine).4 However, high cost of manufacturing and potential for immune stimulation has led to the search for next-generation drug-conjugates which can selectively target tumor cells. Peptides provide an exciting opportunity as an alternate candidate to antibodies for targeted drug delivery because of their potential to interact specifically with a target protein with higher activity per mass ratio, relative ease of synthesis (either manually or automated) with well-established techniques and low manufacturing costs compared to recombinant monoclonal antibody production.5 Identification of peptide interactions with proteins, cell membranes or cellular organelles can be achieved through several well-established methods such as peptide microarrays, yeast-two hybrid systems or peptide bacteriophage-display.5,6 Among these methods, peptide bacteriophage-display offers a significant advantage in that it is high-throughput, allows screening of highly diverse 3 ACS Paragon Plus Environment


peptide libraries that are not limited to individual peptide synthesis (as in microarrays), and users have control over the composition of the library.7 The identification of target peptides using bacteriophage display relies on binding of bacteriophages onto accessible components of defined targets. These targets can include whole cells, organs, proteins or cell surface receptors and can be used in both in vitro and in vivo model systems.7 DNA encoding large variants of selected peptides (typically 7-16 amino acids long) are incorporated into a bacteriophage genome encoding a phage coat surface protein. This coat protein is responsible for the expression of the fusion gene which expresses the peptide sequence at the surface of the bacteriophage.8 Incubation with the target of interest allows for adherent bacteriophage carrying the peptide on its surface to physically interact with its target.8 These bound-phages are recovered for amplification in bacteria to enable further characterization. Using this method, sequence information of the peptides can be obtained, providing a valuable platform for engineering ligand-specific therapies. This technique has been successfully applied to identify a host of different small peptides which can bind to target cells with high affinity and specificity.9 Furthermore, these peptides can be chemically conjugated to a vast array of different molecules such as imaging agents, therapeutic drugs as well as the surface of nanoparticles to enhance targeted drug delivery to the cell type of interest.10 Limited studies have examined the potential of using a targeted approach to deliver therapeutic drugs with specificity to medulloblastoma cells. Herein, we describe the use of a bacteriophage display library to identify novel heptapeptides which bind to medulloblastoma cells. From this screen we selected two peptides for further characterization, one displayed high binding affinity to medulloblastoma cells, while the other had low binding affinity. Both peptides (1 and 2) were synthesized and chemically conjugated to doxorubicin (5). Our results demonstrated that the high binding peptide (1) when conjugated to doxorubicin (5) was able to alter its mode of cellular 4 ACS Paragon Plus Environment

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internalization into medulloblastoma cells compared to the free drug. More importantly, the peptide-doxorubicin conjugate (3) could penetrate an in vitro blood-brain barrier (BBB) and displayed a high degree of specificity for medulloblastoma cells compared to non-tumor cells.



RESULTS Identification of novel peptides which bind to medulloblastoma cells. Peptides with the ability to bind to the surface of medulloblastoma cells were identified by exposing cells to a bacteriophage peptide display library which comprises of a diverse population of 1.28 x109 random heptapeptides fused to the minor coat surface protein (pIII) of M13 bacteriophages. Three rounds of in vitro biopanning were performed to enrich for medulloblastoma-binding peptides. At the end of each round, phages with their displaying peptides bound to the surface of cells were recovered and amplified in E.coli for subsequent biopanning. Any unbound phages were removed by extensive washing. In the third round of panning the number of recovered phages had increased by 6-fold when compared to the first round indicating an enrichment of bacteriophages bound to the surface of medulloblastoma cells (Figure 1).

Figure 1. Recovery rate of medulloblastoma-binding bacteriophage clones after three rounds of biopanning. A graph showing a marked increase in the number of recovered bacteriophages bound to the surface of medulloblastoma cells after three rounds of biopanning.


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Thirty individual phage clones were randomly selected, and their DNA sequenced. Three clones lacked exogenous sequences; however, twenty-seven phage clones were correctly identified by DNA sequencing. The peptide sequence NERALTL (E1-8) appeared five times in twenty-seven clones, DHCRICH (E3-9) appeared four times, and LSNNNLR (E2-2), KLWTLYP (E2-7) appeared twice (Table 1). NERALTL (E1-8) has previously been identified using peptide phage display as having high binding affinity to Nerve Growth Factor-β (NGF-β) and to epoxy 11,12, while LSNNNLR (E2-2) was reported to bind to poly(dimethylsiloxane).13



Bacteriophage Clone # E1-1 E1-2 E1-3 E1-4 E1-5 E1-6 E1-7 E1-8 E1-9 E1-10 E2-1 E2-2 E2-3 E2-4 E2-5 E2-6 E2-7 E2-8 E2-9 E2-10 E3-1 E3-2 E3-3 E3-4 E3-5 E3-6 E3-7 E3-8 E3-9 E3-10

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Peptide Sequence

Frequency

No Insert No Insert FSRPAFL SVLGSLV SNPSVGH FANAKVN VGGLSHR NERALTL QWTSLTS LPILRSQ NERALTL LSNNNLR SHNTHTV HAMRAQP STSFWIT TMWSRVS KLWTLYP STSFWIT KVFLLPP NERALTL AHRLNTE SLSSVHD ADMPILH No insert KLWMIPN SLGTAHR HLSAESW DDAEDSG DHCRICH SMFSVWR

1 1 1 1 1 1 1 1 5 2 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 4 1

Table 1: Peptide sequences identified as having binding affinity to the cell surface of medulloblastoma cells. Table listing the peptide sequences obtained from 27 different bacteriophage clones isolated and amplified after the third round of biopanning on medulloblastoma cells.


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To measure cell binding affinity, we used an ELISA. As expected, all 27 individual phage clones demonstrated an affinity to the cell surface of medulloblastoma cells (Figure 2).

Figure 2. Binding affinity of recovered bacteriophage clones to the cell surface of medulloblastoma cells. Representative graph showing medulloblastoma cell-binding affinity for 27 different bacteriophage clones. Cells exposed to M13KE bacteriophage (no peptide insert) served as control. Each bar represents the mean ± S.D. of duplicate wells from duplicate experiments. Three phage clones: E1-3 (FSRPAFL), E1-4 (SVLGSLV) and E1-5 (SNPSVGH) appeared to have high binding affinity for medulloblastoma cells, while phage clones E1-7 (VGGLSHR), E2-9 (KVFLLPP) and E3-1 (AHRLNTE) had low binding affinity (Figure 2). These results were confirmed by exposing medulloblastoma cells to equal amounts (5x109 pfu) of purified E1-3 or E2-9 bacteriophage which were identified as having high or low binding affinity respectively. After a short incubation period (1 hour) the cells were subjected to extensive washes and any remaining bacteriophage bound to the surface of the cells was collected, amplified and counted using a plaque assay. As predicted, there was a significant increase in the number of E1-3 bacteriophage captured on the surface of medulloblastoma cells (69 % increase, p < 0.05) 9 ACS Paragon Plus Environment


compared to the E2-9 phage (Supplementary Figure 1). Based on these findings clone E1-3 was selected for further characterization.

Internalization of E1-3 peptide (1) into medulloblastoma cells. The seven-amino acid peptide (1) (FSRPAFL), which was displayed by bacteriophage clone E1-3 was synthesized and labeled with a FITC fluorescent dye at the C-terminal end. The peptide (1) was soluble and stable in water and was non-toxic to medulloblastoma cells, non-tumor human fibroblasts and primary human brain astrocytes (Figure 3A-C). Live cell confocal microscopy demonstrated that the E13 peptide (1) could bind to the surface of medulloblastoma cells in as little as 15 minutes posttreatment (Supplementary Figure 2). Flow cytometry and confocal microscopy confirmed that increasing concentrations of the E1-3 peptide (1) were able to bind to the surface and become internalized into medulloblastoma cells 24 hours post-treatment (Figure 3D and 3E). Together, these results demonstrate that the peptide phage display library was able to successfully identify a peptide that was non-toxic and could be internalized into medulloblastoma cells.


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Figure 3. E1-3 peptide (1) is non-toxic to cells and is internalized into medulloblastoma cells. A-C) Graphs showing the E1-3 peptide (1) at increasing concentrations is non-toxic to medulloblastoma cells (A), human fibroblasts (B) and primary human brain astrocytes (C) 72 hours post-treatment; n = 3 independent experiments. Each data point represents the mean ± S.E.M. D) Graph showing an increase in green fluorescent signal intensity in medulloblastoma cells 24 hours post-treatment with differing concentrations of FITC-labelled E1-3 peptide (1). Cells treated with non-fluorescent peptide served as control; n = 3 independent experiments. E) Confocal microscope images demonstrating internalization of increasing concentrations (0.25 μM, 25 μM and 100 μM) of fluorescently labelled E1-3 peptide (1) into medulloblastoma cells 24 hours post-treatment. Cells treated with no peptide served as control. Red = cell membrane, Blue = nucleus, Green = E1-3 peptide (1). Scale bar = 20 µm.



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Design and synthesis of peptide doxorubicin conjugates (3 and 4). Children that survive medulloblastoma are often left with long-term debilitating side-effects from exposure to chemotherapy drugs.1 This is primarily due to chemotherapy drugs having poor specificity for tumor cells which results in damage or death to normal healthy cells. To establish proof of concept for peptide-drug conjugates to increase selective chemotherapy drug delivery to medulloblastoma cells we chemically conjugated E1-3 peptide (1) (peptide with high binding affinity to medulloblastoma cells) and E1-7 peptide (2) (peptide with low-binding affinity to medulloblastoma cells) peptides to the chemotherapy drug doxorubicin (5) (Supplementary Figures 3 and 4). Doxorubicin (5) was chosen as a model drug in this study for its fluorescent nature which allows for convenient in vitro tracking and its well-established cytotoxic effect. The chemical steps required to produce both peptide-doxorubicin conjugates (3 and 4) are shown in Figure 4. Attachment of doxorubicin (5) to both peptides was achieved through an N-terminus conjugation via a glutarate ester linker that is susceptive to hydrolysis. This conjugation follows a modified procedure originally developed by Nagy et al.14 The synthesis process involves protection of daunosamine amine of doxorubicin (5) with an Fmoc group followed by the attachment of an ester linker through the ring opening of glutaric anhydride. Conjugation between the drug analog to the peptide proceeds via a peptide coupling procedure using O-(7azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as the coupling agent and diisopropylethylamine (DIPEA) the base. After chemical conjugation the Fmocprotecting group on the doxorubicin (5) was released using 10% piperidine in dimethylformamide solution.

Addition

of

pyridine

/

trifluoroacetic

acid

mixture

in

N,N-dimethylformamide neutralized the compound. The reaction procedure produced the desired


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doxorubicin-peptide conjugates E1-3 doxorubicin (3) and E1-7 doxorubicin (4) with yields of 53% and 62% respectively (Figure 4).

Figure 4. Schematic diagram showing the synthetic steps required to chemically conjugate E1-3 and E1-7 peptides (1 and 2) to doxorubicin (5).

Internalization of E1-3 peptide doxorubicin conjugate (3) into medulloblastoma cells. Next, we used LCMS/MS, MALDI-TOF/MS, confocal microscopy, flow cytometry and Fluorescence Lifetime Imaging Microscopy (FLIM) to examine the stability and the cellular internalization process of E1-3-doxorubicin conjugate (3) into medulloblastoma cells. E1-3



doxorubicin conjugate (3) demonstrated biological stability when exposed to human or mouse serum for up to 4 hours (Supplementary Figures 5–7, Supplementary Table 1). Both free doxorubicin (5) and E1-3 doxorubicin conjugate (3) were rapidly internalized into medulloblastoma cells in as little as 15 minutes post-treatment (Figure 5A-B). Interestingly, the pattern of internalization of E1-3 peptide doxorubicin conjugate (3) was markedly different compared to free doxorubicin (5) at 15- and 30-minutes post-treatment (Figure 5A-D). Free doxorubicin (5) is known to enter cells via passive diffusion12-13 and hence was mainly localized to the perinuclear and nuclear regions (Figure 5A, C and E). In contrast, the E1-3 peptide doxorubicin conjugate (3) displayed a punctate pattern of expression throughout the cell cytoplasm along with some perinuclear and nuclear expression (Figure 5B-D). The pattern of expression for both drugs appeared similar after 60 minutes post-treatment (Figure 5E-F). Flow cytometry confirmed that increasing amounts of E1-3 peptide doxorubicin conjugate (3) was able to accumulate into medulloblastoma cells over a 3 hour time period, with most of the doxorubicin (5) located within the nucleus (Supplementary Figure 8).


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Figure 5: E1-3 doxorubicin conjugate (3) is internalized into medulloblastoma cells. Representative confocal microscope images of medulloblastoma cells treated with freedoxorubicin (5) (images A, C, E) or E1-3 doxorubicin conjugate (3) (images B, D, F) at 15, 30 and 60 minutes. White arrows indicate punctate pattern of expression of E1-3 doxorubicin conjugate (3) within the cell cytoplasm. Scale bar = 50µm. Based on these results we hypothesized that the conjugation of the E1-3 peptide (1) to doxorubicin (5) alters the process of how the drug is internalized into medulloblastoma cells. To investigate whether E1-3 doxorubicin conjugate (3) was endocytosed into medulloblastoma cells, we treated cells with four commonly used endocytosis inhibitors: (i) genistein (caveolae uptake), (ii) cytochalasin D (actin polymerisation inhibitor / macropinocytosis), (iii) amiloride hydrochloride



(caveloae / macropinocytosis), and (iv) chlorpromazine hydrochloride (clathrin-mediated endocytosis).15 Cells were pre-treated with non-toxic concentrations of the inhibitors prior to incubation with free doxorubicin (5) or the E1-3 doxorubicin conjugate (3). Flow cytometry analysis demonstrated that cytochalasin D was able to have a small but significant inhibitory effect (>11% decrease, p < 0.01) on the uptake of E1-3 doxorubicin (3) (results not shown). All four inhibitors had no effect on the uptake of free doxorubicin (5) (results not shown). To validate these results, we repeated the above experiment using cytochalasin D and performed FLIM analysis to monitor E1-3 doxorubicin (3) and free doxorubicin (5) uptake and intracellular trafficking. FLIM is a highly sensitive technique used to measure intracellular doxorubicin (5) delivery.16 FLIM analysis revealed that free doxorubicin (5) and E1-3 doxorubicin (3) had a primary lifetime of ~4.3 ns (1, 90 %) and a secondary shorter lifetime of ~1.3 ns (2, 10 %) (Supplementary Figure 9). However, as the drug migrated from the cytoplasmic compartment to the nuclear compartment the 2 became the primary lifetime at 80 % (located in the nucleus) and 1 at only 20 % (located in the

cytoplasm). The shift towards shorter fluorescence lifetime has been previously reported and is thought to be the result of the induction of apoptosis and chromatin condensation in the nucleus (Supplementary Figure 9).17 Based on this information we were then able to examine the effect of cytochalasin D on the intracellular transport of E1-3 doxorubicin (3) and free doxorubicin (5). As expected, cytochalasin D showed no effect on the internalization of free doxorubicin (5) (Figure 6A-B). However, cells pre-treated with cytochalasin D and then exposed to E1-3 doxorubicin (3) had a 1 as the most prominent lifetime (64 %) and the shorter 2 as the secondary lifetime (36 %) (Figure 6A-B). This suggests that cytochalasin D was able to partially decrease the amount of E1-3 doxorubicin conjugate (3) localized to the nucleus of medulloblastoma cells.


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Collectively, these results illustrate that E1-3 peptide (1) when chemically attached to doxorubicin (5) can alter the cellular internalization of the drug compared to its free form.

Figure 6. E1-3 peptide (1) alters the cellular internalization of doxorubicin (5) into medulloblastoma cells. Representative FLIM images (A) and graph (B) showing a change in the pattern of cellular uptake and intracellular trafficking of E1-3 doxorubicin conjugate (3) compared to free doxorubicin (5) in the presence of the endocytosis inhibitor, Cytochalasin D; columns, mean (3 independent experiments); error bars, S.E.M.



Cytotoxic activity of E1-3 doxorubicin conjugate (3) in medulloblastoma cells. To determine whether the E1-3 doxorubicin conjugate (3) had a similar cytotoxic profile to that of free doxorubicin (5) used in the clinic, medulloblastoma cells were treated with equivalent concentrations of free doxorubicin (5) or E1-3 doxorubicin conjugate (3). Seventy-two hours posttreatment cell proliferation / viability was measured using an alamar blue cell viability assay. Both free doxorubicin (5) and E1-3 doxorubicin conjugate (3) proved to be effective at inhibiting medulloblastoma cell growth (Figure 7). There was only a moderate difference in the IC50 values for both free doxorubicin (5) (IC50 = 8.8 ± 1.31 nM) and E1-3 doxorubicin conjugate (3) (IC50 = 25 ± 1.22 nM). This was most likely due to the different process of cellular internalization for E1-3 doxorubicin conjugate (3) and / or cleavage of the peptide from the drug once inside the cell (Figure 7). The high binding affinity of E1-3 doxorubicin conjugate (3) to medulloblastoma cells was further illustrated when cells were treated with the same concentrations of E1-7 doxorubicin conjugate (4). As noted above, E1-7 peptide (2) was identified in our bacteriophage library screen as having low-binding affinity to medulloblastoma cells. This was confirmed with E1-7 doxorubicin conjugate (4) displaying a 5-fold reduction in cytotoxicity compared to E1-3 doxorubicin conjugate (3) (IC50 values: 130 ± 1.27 nM and 25 ± 1.22 nM respectively), and 14fold reduction in cytotoxicity compared to free doxorubicin (5) (IC50 values: 130 ± 1.27 nM and 8.8 ± 1.31 nM respectively) (Figure 7).


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Figure 7. E1-3 and E1-7 doxorubicin conjugate (3 and 4) cytotoxic activity in medulloblastoma cells. Graph showing the cytotoxic profile for free doxorubicin (5), E1-3 doxorubicin (3) and E1-7 doxorubicin (4) conjugates in medulloblastoma cells 72 hours posttreatment. Each dot-point represents the mean ± S.E.M. from 3 independent experiments.



E1-3 doxorubicin conjugate (3) blood-brain barrier (BBB) permeability. A major clinical challenge for the treatment of brain cancers including medulloblastoma is the presence of the blood-brain barrier (BBB).18 The BBB comprised of endothelial cells with tight gap junctions together with astrocytes, pericytes and perivascular macrophages limit the penetration of chemotherapy drugs to the brain.18 To examine whether the E1-3 peptide (1) when conjugated to doxorubicin (5) could enhance its penetration across the BBB we employed the use of an in vitro BBB model using methods described by Puech et al.19 In brief, human brain endothelial cells (HBEC-5i) were seeded onto the apical side of a cell-culture insert and incubated with conditioned medium collected from human primary brain astrocytes for 14 days to encourage the formation of a tight cell monolayer which expresses high levels of tight junction proteins and drug efflux pumps to limit the penetration of high molecular weight molecules (Figure 8A).19 Immunofluorescent staining confirmed that HBEC-5i cells growing in human brain astrocyte conditioned medium formed a tight monolayer of cells which expressed the tight junction protein Zonula Occludens (ZO-1) (Figure 8B). Moreover, high expression of the drug efflux pump P-glycoprotein (P-gp) was also observed (Figure 8B). We validated that the HBEC-5i cell monolayer was functional and could inhibit the penetration of fluorescently labelled high molecular weight dextran (DextranFITC 155 KDa) (Figure 8C). Having established the integrity of the endothelial cell barrier, free doxorubicin (5) (250 µM) or E1-3 doxorubicin conjugate (3) (250 µM) were added to the upper chamber of the inserts and at increasing time points (30 minutes, 60 minutes, 120 minutes) aliquots of media were collected from the bottom chamber. The amount of doxorubicin (5) permeabilized through the HBEC-5i monolayer was measured using fluorescence spectrometry. E1-3 doxorubicin conjugate (3) was able to cross the HBEC-5i monolayer (Figure 8D). Notably, its


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permeability efficiency was significantly higher compared to free doxorubicin (5) (36.93 ± 0.7 µM and 28.93 ± 0.2 µM respectively, p

Identification of Novel Medulloblastoma Cell-Targeting Peptides for

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