Photoactivatable Prodrug of Doxazolidine Targeting Exosomes

Publication Date (Web): January 31, 2019. Copyright © 2019 American Chemical Society. Cite this:J. Med. Chem. XXXX, XXX, XXX-XXX ...
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Photoactivatable Prodrug of Doxazolidine Targeting Exosomes Ryo Tamura, Alla Balabanova, Samuel A Frakes, Austin Bargmann, Jan Grimm, Tad H Koch, and Hang Hubert Yin J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01508 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 1, 2019

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Photoactivatable Prodrug of Doxazolidine Targeting Exosomes Ryo Tamura1,2,3, Alla Balabanova1, Samuel A. Frakes1, Austin Bargmann1, Jan Grimm3, Tad H. Koch1, Hang Yin1,2,4* 1Department

of Chemistry and Biochemistry and 2BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA

3Molecular

Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

4School

of Pharmaceutical Sciences, Tsinghua University-Peking University Joint Center for Life Sciences, Tsinghua University, Beijing 100082, China

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Abstract Natural lipid nanocarriers, exosomes, carry cell signaling materials such as DNA and RNA for intercellular communications. Exosomes derived from cancer cells contribute the progression and metastasis of cancer cells by transferring oncogenic signaling molecules to neighbor and remote premetastatic sites. Therefore, applying the unique properties of exosomes for cancer therapy has been expected in science, medicine and drug discovery fields. Herein, we report that an exosome-targeting prodrug system, designated MARCKS-ED-Photodoxaz, could spatiotemporally control the activation of an exquisitely cytotoxic agent, doxazolidine (doxaz), with UV light. MARCKS-ED peptide enters a cell by forming a complex with exosomes in situ at its plasma membrane and in the media. MARCKS-ED-Photodoxaz releases doxaz under near UV irradiation to inhibit cell growth with low nanomolar IC50 values. The MARCKS-EDPhotodoxaz system targeting exosomes and utilizing photochemistry will potentially provide a new approach for the treatment of cancer, especially for highly progressive and invasive metastatic cancers.

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Introduction Extracellular vesicles (EVs) are significant nanocarriers in cell-to-cell communications to deliver bioactive molecules including DNA, RNA and proteins, which otherwise could be easily degraded in plasma without EVs. EVs consist of exosomes with diameter of 30-150 nm and microvesicles with diameter of 100-1000 nm. Exosomes are released by exocytosis after being formed in endosome, and microvesicles are produced by direct budding from the plasma membrane.1–4 EVs have been intensely studied in biomedical research including cancer growth and metastasis, immune system modulation, and drug delivery system development.5–8 Exosomes derived from cancer cells deliver oncogenic molecules to tumor microenvironments and sites of future metastases. For instance, exosomes from cancer cells prepare the environment for incoming cancer cells in premetastatic sites.9,10 Furthermore, cancer cell-derived exosomes transfer signaling molecules to the immune system that modulate the immune response against cancer cells.7,11,12 Exosomes have been used for delivering therapeutic cargos such as small molecules, proteins and nucleic acids to disease sites in pre-clinical and clinical studies.6,8,13–15 We have previously reported an exosome-sensing peptide called MARCKS-ED, derived from the effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) protein.16 MARCKS modulates phospholipase C signaling by segregating phosphatidyl inositolbisphosphate (PIP2).17,18 MARCKS-ED comprises 25 amino acids, out of which the 13 basic amino acid residues and 5 phenyl alanine residues contribute most of the binding to negatively charged and highly curved lipid membrane (Table 1). MARCKS-ED binds exosomes with higher affinity than larger vesicles because of their characteristic externalized phosphatidylserine (PS) and a highly curved membrane.16,19–21 Exosomes in nature deliver bioactive molecules for cellto-cell communications, otherwise degraded in plasma. Therefore, exosomes are attractive drug 3 ACS Paragon Plus Environment

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delivery vehicles. The recent methods for applications of exosomes to drug delivery are based on isolating exosomes from cell cultures, followed by drug loading by electroporation, incubation or sonication.6,8,14,22 However, since cancer cells continuously release exosomes in tumor microenvironments that are eventually circulating through the vascular system, utilizing those exosomes minimizes potential unwanted side effects from heterogenous origins and does not require large scale production and isolation of pure exosomes.23–25 Here, we report a novel drug delivery technology with an exosome sensing peptide, MARCKS-ED, which binds exosomes in situ and delivers a cargo into cancer cells. We use an exquisitely cytotoxic agent derived from doxorubicin, doxazolidine (doxaz), as the anticancer therapeutic cargo (Fig. 1A). Doxorubicin (dox) is an FDA approved anticancer drug. However, dose dependent cardiotoxicity and acquisition of multidrug-resistance phenotype including elevated expression of p-170 glycoprotein (P-gp) hamper clinical efficacy.26–28 Doxaz is a dox-formaldehyde conjugate at the 3’-NH2 and 4’-OH groups. The IC50 values of doxaz to cancer cells are 1 to 4 orders of magnitude lower than dox and its potency is not affected by P170gp because doxaz is an uncharged DNA alkylating agent at sites of 5’-GC-3’ while dox is a cation at physiological pH that inhibits the function of topoisomerase II.29,30 However, the halflife of doxaz is approximately 3 min with respect to hydrolysis to dox under physiological conditions due to the unstable oxazolidine ring.29,31,32 Therefore, prodrugs with stable carbamylated amino groups, p-aminobenzyloxycarbonyl (PABC) groups, have been investigated for therapeutic applications of doxaz. We have reported two types of enzymatically cleavable prodrugs of doxaz: Pentyl PABC-Doxaz (PPD) and GaFK-PABC-Doxaz (see abbreviations for definitions of acronyms).33–35 PPD was designed to be activated by carboxylesterase 2 (CES2) expressed in liver, non-small-cell lung, colon, pancreatic, renal, and thyroid cancer cells. PPD is 4 ACS Paragon Plus Environment

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effectively cleaved by CES2 and inhibited the tumor growth in xenografted mouse models of liver and non-small-cell lung cancer cell lines.34 GaFK-PABC-Doxaz is the prodrug of doxaz, which is activated by proteolytic enzymes strongly associated with cancer progression and poor prognosis.35 PPD and GaFK-PABC-Doxaz are efficiently activated by enzymes, but those enzymes are also present to some extent in normal cells. Therefore, we utilized a photoactivatable linker to achieve a potentially selective prodrug activation with light inside cancer cells.

Results Design and Synthesis Doxaz is readily synthesized from dox in the presence of excess paraformaldehyde to form an oxazolidine ring at 3’-NH2 and 4’-OH of the daunosamine sugar ring. Additional formaldehyde links two doxaz molecules to yield doxoform (doxf). Doxf hydrolyzes to doxaz which hydrolyzes to dox in aqueous medium. The half-life of doxf under physiological conditions is approximately 1 minute and the half-life of doxaz is approximately 3 minutes.31 Previous studies showed that carbamoylation of doxaz at the oxazolidine ring extends the halflife and keeps the drug inactive.33–35 Therefore, we applied a similar prodrug strategy to protect active oxazolidine by carbamoylating the secondary amine. Doxaz is a powerful cytotoxic agent but can hydrolyze to much less toxic dox rapidly. Hence, unwanted side effects can be minimized after the prodrug is activated inside cancer cells such as escape to normal cells. The final construct consists of three parts: a very strong anticancer cargo (doxaz), an o-nitrobenzyl photolabile linker to achieve spatiotemporal prodrug activation, and MARCKS-ED peptide that binds exosomes in tumor microenvironments (Fig. 1B). We used a photolabile linker with a 5 ACS Paragon Plus Environment

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small PEG spacer to improve the water solubility and accessibility to the peptide. MARCKS-ED peptide has been shown to be a sensor of negatively charged phosphatidylserine (PS) and membrane curvature by both electrostatic interactions and the vertical insertion of phenylalanine into the lipid bilayer. Exosomes are natural vesicles with PS enriched, highly curved membranes, which are correlated with tumor progression and metastasis.2,36 For the first step to prepare the photolabile linker, intermediate 5 (Scheme 1) was synthesized using previously reported methods.37,38 Its carbonyl group was reduced with sodium borohydride to generate the alcohol 6 in good yield. Then the resulting alcohol was activated to intermediate 7 with 4-nitrophenyl chloroformate and DMAP. To prepare doxaz, dox free base was extracted from a clinical sample of dox hydrochloride and transformed to doxf in the presence of excess paraformaldehyde. Doxf was coupled to the linker 7 with a 51% yield using HOBt in wet DMSO. The trace of water in wet DMSO hydrolyzes doxf to doxaz, and the secondary amine of doxaz couples to the activated linker. The modest yield was presumably due to steric effects at the bulky secondary amine of doxaz. Photodoxaz-PEG-azide 8 shows two peaks on RP-HPLC because it is a mixture of two diastereomers (Fig. S1). Interestingly, normal phase column chromatography did not work to purify 8; therefore, reverse phase column chromatography or preparative HPLC was utilized. MARCKS-ED peptide and other peptides in this study were synthesized using a standard Fmoc solid phase peptide synthesis (SPPS) protocol.39 We previously reported that both L- and D- configurations of MARCKS-ED have PS- and curvature-sensing capabilities, but the isomer with D- residues is more resistant to proteolytic degradation than the isomer with Lresidues.16,19,20,40 For comparison here, we synthesized both L- and D- configurations of MARCKS-ED peptide, designated L-MARCKS-ED and D-MARCKS-ED, respectively. In 6 ACS Paragon Plus Environment

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addition, we also synthesized the scrambled sequence of MARCKS-ED peptide (MARCKS-Scr), a non-binding control peptide (C2BL3L), and a mutant with the critical lysines to aspartic acids and phenylalanines to alanines (MARCKS-Mut) (Table 1).16 Our initial attempt to conjugate the peptide to the linker 8 used a copper catalyzed click reaction by installing 4-pentynoic acid to the N-terminus, but this approach failed, perhaps because the metal catalyst was sequestered by the highly charged MARCKS-ED or because the relatively bulky peptide renders steric hinderance.41 Furthermore, the anthracycline and linker are sensitive to acidic and basic conditions as well as metal catalyzed reactions. Transition metals are known to form a complex with anthracyclines.42 Therefore, we decided to use a thiol-maleimide Michael addition for this conjugation since it is a well-established bioconjugation method such as the method used in antibody drug conjugate syntheses.43,44 To install a maleimide, DBCO-maleimide was reacted with the intermediate 8. This completely transformed 8 to photdoxaz-PEG-maleimide 9 (Scheme 2). Analytical RP-HPLC showed four peaks since 9 was formed as two sets of two diastereomers, generated by the non-regioselective click reaction (Fig. S1). The thiol was installed to the N-terminus of the peptides by coupling with S-trityl-3-mercaptopropionic acid using HATU, and the peptides were each released by TFA global deprotection and cleavage from the resin. Intermediate 9 was conjugated to the thiol group of the peptides in PBS or phosphate buffer under a nitrogen atmosphere. The final products were then purified by reverse phase column chromatography. The RP-HPLC showed one broad peak containing 8 isomers, two sets of regioisomeric diastereomers with four diastereomers per set, generated by the Michael addition (Fig. S1). To compare the efficacy of doxaz conjugate to the corresponding dox conjugate, dox was also reacted with intermediate 7 followed by the same coupling method to LMARCKS-ED. We succeeded in the syntheses of the compounds 1, 2, 3 and 4 (Schemes 2 and

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S1, and Fig. S2).

However, the Michael additions of MARCKS-Scr and -Mut were not

successful. Therefore, we synthesized Cy5 labeled peptides to explore the cellular uptake of these peptides. All peptides and peptide conjugates were completely water soluble. Interestingly, nanoparticle tracking analysis (NTA) showed that MARCKS-ED conjugates 1 and 2, and Cy5labeled L- and D-MARCKS-ED, form nanoparticles with a diameter of approximately 100 nm (Fig. S3). This nanoparticle formation potentially prevents proteolytic degradations and unwanted prodrug activations, and improves water solubility, a reasonable circulation half-life in blood, and tumor accumulation.45

Photolysis The long-term goal of our approach is that the prodrug can be spatiotemporally activated by internal light after the prodrug is taken up by cancer cells; thereby, unwanted side effects can be minimized. As a proof-of-principle of this approach, we first measured the relative rate of photolysis of photodoxaz-PEG-azide 8 using a monochromatic laser. We used intermediate 8 for the quantum yield measurements instead of the final products 1, 2, 3, and 4 because of its ease of preparation and better solubility in the solution of the measurement. Intermediate 8 in a solution of 65% 15 mM phosphate buffer and 35% acetonitrile was irradiated with an Omnichrome HeCd monochromatic laser (12 mW, 325 nm) for 30 min. Stirring was achieved by continuous pipetting, and the optical density at 325 nm assured total absorption through 10% conversion. The amounts of dox released was monitored by RP-HPLC (Fig. S4, and S5). The quantum yield (Φ) was calculated using the time at which approximately 10% of dox was released. Figure 2 shows plots of dox released by UV light irradiation as a function of irradiation time. More than 95% of the prodrug was activated by 30 min of UV irradiation with a quantum yield (Φ) of 8 ACS Paragon Plus Environment

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0.025 (Table S1), agreeing well with previously reported quantum yields for analogous photocleavage reactions.46 Further, we irradiated the peptide conjugates, 1, 2, 3 and 4, for 15 min with a UV black lamp apparatus, which emits 0.8 mW/cm2 of UV light at the surface of a 96 well plate in the region 320-400 nm with maximum intensity at 350 nm as measured with a power meter (Fig. S6). The change in solvent from 35% acetonitrile to aqueous culture medium as well as the change from the azide intermediate to the final prodrug could affect the quantum yield. We suspect the effect is not dramatic and not so relevant to the prodrug development at this stage. RP-HPLC analyses showed approximately 50% of prodrug was activated (Fig.S7 A, B and C). L-MARCKS-Photodox 4 showed 70% photolysis (Fig. S7 D). This difference might derive from the bulky tertiary carbamate of doxaz prodrugs that potentially decreases the efficiency of the photoreaction. Unlike dox carbamates, doxaz carbamates are known to exist as a mixture of slowly interconverting chair/twist boat conformations of the amino sugar ring.47

Growth Inhibition of MDA-MB-231 Breast Cancer Cells We successfully showed the photolysis efficacy of our reagents. Next, we investigated the growth inhibition potency to cancer cells under UV irradiation. For the growth inhibition assessments, we used the same UV light apparatus used for photolysis experiments described above (Fig. S6). A 96-well plate was placed 12 cm away from the light source. The power of the light at this position was approximately 0.8 mW/cm2 (max. wavelength at 350 nm) over the area of the portion of the 96-well plate used as measured by a Scientech power meter. To test the phototoxicity of the UV light, we measured its effect on cellular growth of MDA-MD-231 cells. We found that 15 min of UV irradiation showed minimal effects on cellular growth (Fig. S8). Next, we investigated the cytotoxicities of the prodrugs, 1, 2, 3 and 4, under 15 min of 9 ACS Paragon Plus Environment

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near UV irradiation to MDA-MB-231 cells in culture. The prodrugs were incubated for 6 h at various concentrations in the media with 10% FBS. After cells were washed with PBS twice, they were irradiated with UV light for 15 min. Following incubating at room temperature for 15 min, PBS washing twice and addition of fresh media, the cells were grown until the untreated cells reach 80% confluency or 5 days. For the negative control without UV light, the 96-well plate containing the cells was wrapped with aluminum foil to prevent the activation by any ambient UV light. The cell densities were measured by crystal violet staining. The growth inhibition curves and IC50 values are reported in Fig. 3 and Table 2, respectively. L-MARCKSED-photodoxaz 1 showed significant cytotoxicity with UV black light even though it was inactive without UV black light (IC50: with UV = 0.015 µM, without UV = 2.5 µM). The Dconfiguration of the MARCKS-ED conjugate 2 (IC50: with UV = 0.042 µM, without UV = 2.0 µM) also showed a comparable result, but the IC50 with UV light was slightly higher than with the L-configuration. By contrast, C2BL3L conjugate 3 (IC50: with UV = 1.3 µM, without UV = 11 µM) was 87-fold less toxic than 1 under UV light. Interestingly, L-MARCKS-ED-photodox 4 (IC50: with UV = 1.2 µM, without UV = 6.5 µM) was 80-fold less toxic than 1 under UV light even though they differ by only a single carbon. This result supports the oxazolidine ring of doxaz being essential for high cellular growth inhibition.48 The IC50 values of 1 and 2 are comparable to PPD and GaFK-PABC-Doxaz with enzymatic activation.33–35

Cellular Uptake Both L- and D- configurations of MARCKS-ED-photodoxaz, 1 and 2, showed good potency against cancer cell growth under UV black light irradiation but were essentially inactive in the absence UV black light irradiation. Subsequently, we explored the cellular uptake 10 ACS Paragon Plus Environment

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properties of the MARCKS peptides. First, the compounds 1, 2, 3 and 4, were incubated with MDA-MB-231 cells for 6 h, which is the same condition for the growth inhibition assay above. Following PBS washing, trypsinization and centrifugation, the cellular uptake of the compounds was analyzed by flow cytometry (Fig. 4). Interestingly, D-MARCKS-ED-photodoxaz 2 had approximately 2- to 3-fold higher cellular uptake than the L-counterparts, 1 and 4. C2BL3L conjugate 3 had the lowest cellular uptake. Confocal microscopy also demonstrated cellular uptake of 1 and 2 (Fig. 5). Since 10 µM of 1 and 2 for 6 h incubation was too toxic, we decreased the exposure to 5 µM for 3 h. The images clearly showed the uptake of the peptides, and some peptides appeared on the plasma membrane (Fig. 5 A and B). Since the fluorescence intensity from the anthracycline is not sufficient for in depth analysis of cellular uptakes, we subsequently explored uptake using Cy5-labeled peptides. Although Dox is fluorescent, its fluorescence intensity was insufficient for the experiment. Hence, Cy5, which has a higher molar absorptivity and fluorescence quantum yield, was substituted for Dox. All peptides in Table 1 were labeled with Cy5-carboxylic acid using the standard HATU coupling. 49 MDA-MB-231 and Human Mammary Epithelial Cells (HuMEC), both cell lines known to uptake exosomes, were incubated with 1 µM of the peptides for 1, 3, or 6 h. 50,51 The fluorescence intensities from cells were then measured by confocal microscopy and quantified by ImageJ software. Both MDA-MB-231 and HuMEC cells showed a similar trend with respect to the uptake of photodoxaz peptide conjugates (Fig. S9 A and B). The uptake of DMARCKS-ED was significantly greater than L-MARCKS-ED. By contrast, C2BL3L and MARCKS-Mut did not show cell uptake at all. Interestingly, MARCKS-Scr showed a similar uptake with the wild type peptide. To see the correlation with exosomes, MDA-MB-231 cells were incubated with 1 µM L- or D-MARCKS-ED peptide with the exosomes labeled with 11 ACS Paragon Plus Environment

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fluorescence dye for 6 h (Fig. S10). Confocal microscopic analyses showed co-localization of the peptide and exosomes for both L- and D- peptides (Fig. S11 and S12 A and B). The higher magnification 3D images showed that both the peptide and exosomes are co-localized outside of the nucleus (Fig. S13). The time course experiment with continuous imaging for 1 h after addition of the peptide and exosomes showed that they are gradually associating with the plasma membrane, and some aggregations consisting of MARCKS-ED and exosomes appear on it upon cell entry (Fig. 6 and S14 and supporting videos). The complexation of the peptide and exosomes may also take place in media because we previously reported that MARCKS-ED peptides bind exosomes and liposomes in a biological solution.16 Further, quantification of cellular uptakes of Cy5-D-MARCKS-ED and cell-mask labeled exosomes showed the decrease in fluorescence intensity from exosomes when it was co-incubated with the peptide while the cellular uptake of peptide is not affected by exosomes. This result is presumably due to energy transfer from the cell-mask dye on exosomes to the peptide that supports the co-localization of the peptides and exosomes in the cells (Fig. S15).

Discussion In this work, we developed a strategy of using photoactivatable prodrugs, DoxazMARCKS-ED conjugates, to target selectively cancerous cells. The final constructs 1, 2, 3 and 4 were successfully synthesized as shown in Scheme 2 and Fig. S2. Interestingly, the compounds 1 and 2 form nanoparticles in PBS, presumably due to self-assembly of hydrophobic caged-doxaz and hydrophilic highly charged peptide. This is potentially beneficial for the stability in plasma by protecting the construct from unwanted activation and/or metabolic reduction of the nitro group.52 Photolysis experiments showed the quantum yield of cleavage of photodoxaz-PEG12 ACS Paragon Plus Environment

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azide is 2.5%. This low yield may be advantageous for protecting the prodrug from unnecessary photoactivation by ambient near UV light. However, since UV light permeability in tissue is limited, further modification of linker to improve photolysis efficiency is probably needed.53 Some photoactivable linkers with high efficiency have been reported during the last decades.54 For this application, photocleavage efficiency is defined as the product of the quantum yield of reaction and molar absorptivity at the wavelengths of light available.55 For example, Menge and Heckel reported that a coumarin protecting group is 17 times more effectively uncaged than 2-(onitrophenyl)-propyl caging group due to higher molar absorptivity at comparable wavelengths.56 Therefore, improving the photocleavage efficiency is the focus of our efforts for the success of future in vivo experiments. Furthermore, a light delivery method will also be important for in vivo applications because external UV light cannot permeate human skin and tissue permeability is limited.53,57 Ibsen et al. utilized a LED fiber-optic system to deliver UV light to deep tumor tissue in mouse experiments and showed successful prodrug activation by inserting a fiber optic to the tumor.58,59 Less invasive methods were also reported using radioactive species. For instance, Kotagiri et al. reported an innovative way to generate near UV light inside tumors with Cerenkov radiation (CR) generated by clinically relevant radionuclides. They demonstrated that titanium dioxide (TiO2) nanoparticles are excited by CR and induce photodynamic therapy. 60 In addition to the TiO2 nanoparticles, other nanoparticles and photosensitizers were reported for CR induced photodynamic therapy. 61–63 Further, Ran et al. applied CR to activate o-nitrobenzyl ether-caged luciferin in vivo.64 All of these previous studies pointed to the direction that the photoactivation of a prodrug may be a viable method for clinical applications. In our current studies, we showed that both L- and D-MARCKS-ED-photodoxaz, 1 and 2, were successfully activated by UV light with low ~nM IC50 values while toxicity was 13 ACS Paragon Plus Environment

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significantly lower without UV light irradiation. On the other hand, L-MARCKS-ED-Photodox 4 was 80-fold less toxic than 1 under UV irradiation even though the difference is only one carbon. These results clearly showed doxaz is more effective than dox. As a comparison, the C2BL3L control 3 was 87-fold less toxic than 1 likely because it did not get into cells well as we anticipated. Interestingly, D-MARCKS-ED 2 has more cell penetrating capability than LMARCKS-ED 1 but it is slightly less toxic to cancer cells. We have previously reported that DMARCKS-ED peptide is roughly 6 times more resistant to proteolytic hydrolysis than LMARCKS-ED-peptide.40 We speculated that the stability of the peptide might contribute to the higher cell uptake of D-MARCKS-ED. Furthermore, A more stable nanoparticle formulation of the prodrug may be beneficial if photoactivation to doxaz occurs in the nanoparticle and if the more hydrophobic environment of the nanoparticle slows the rate of hydrolysis of doxaz to dox at sites remote from its DNA target. We designed the prodrug to target the exosomes secreted from cancer cells and deliver the therapeutic cargo into the cells. Since cancer cell-derived exosomes correlate with cancer progression and metastasis, our approach will potentially inhibit cancer growth, progression and metastasis caused by exosomes. Nakase et al. reported that cationic lipids or peptides could enhance exosome delivery to cytosol because polycationic peptides or polymers have cell membrane penetrating capability that help exosomes enter cells.65,66 We hypothesized that MARCKS-ED peptide binds the PS enriched highly curved membrane of exosomes and forms an exosome-peptide complex in situ that localizes on the plasma membrane and enters the cell. Co-incubation of the peptides with fluorescence labeled exosomes clearly showed co-localization in cells. Furthermore, the time-course imaging experiments showed that MARCKS-ED peptide and exosomes co-localize on the plasma membrane and subsequently enter the cell. These 14 ACS Paragon Plus Environment

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experiments imply that they were taken up together by the same mechanism. However, we cannot rule out other possible mechanisms of peptide uptake. For example, cell-penetrating peptides (CPPs) such as the TAT peptide derived from the human immunodeficiency virus type 1 (HIV-1) and the penetratin peptide derived from the amphiphilic Drosophila Antennapedia homeodomain possess unique cell penetrating properties though the exact mechanism of uptake has not been completely understood. In general, CPPs are polycationic peptides and believed to enter the cells by direct penetration of plasma membrane or endocytosis pathways.67 As MARCKS-ED peptide is polycationic, it could enter the cells by a similar manner with CPPs. Exosome targeting is a new strategy for drug delivery. Some exosome-sensing probes have been reported that potentially support our approach. For example, Carney et al. developed a cyclic peptide, LXY30, that targets α3β1 integrin on exosomes and demonstrated that LXY30 detects exosomes and even differentiate cancer cell-derived exosomes from noncancerous exosomes. Furthermore, the exosome uptake to cancer cells was inhibited by LXY30 in a dose dependent manner.68 Similarly, Ghosh et al. demonstrated that the venceremin peptide, targeting heat shock proteins on exosomes, captures and precipitates exosomes. 69 Although those studies were aimed for detecting exosomes, the exosome-sensing probes could be used to modify the surface of exosomes as well as to deliver therapeutic cargos likewise as we demonstrated in this study.

Conclusion In summary, we successfully developed the photoactivatable prodrug of an exquisitely cytotoxic anticancer agent, doxaz, targeted with a novel cell-penetrating-peptide derived from

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the effecter domain of MARCKS protein. MARCKS-ED-photodoxaz easily enters cancer cells and with UV irradiation releases the exquisitely toxic but short-lived DNA cross-linking agent, doxaz, to inhibit cancer cell growth. MARCKS-ED peptide co-localizes with exosomes on the plasma membrane to enter the cell. Since exosomes are highly correlated with cancer progression and metastasis, our method may provide a new approach for development of targeted anticancer therapies. Experimental 1. General All chemicals were purchased from Sigma Aldrich (St. Louis, MO), unless otherwise noted. Clinical samples of doxorubicin hydrochloride were gifted by FeRx, Inc. (Aurora, CO). All reagents and amino acids for solid phase peptide synthesis were purchased from ChemPep Inc. (Wellington, FL). All NMR spectra were taken at 400 MHz on a Varian Inova (Palo Alto, CA) or 400 MHz Bruker Avance-III (Billerica, MA) spectrometer in deuterated solvents purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). Chemical shifts are reported in δ values of ppm and were standardized by the residual solvent peak in MestReNova NMR software (Mestrelab Research, Santiago de Compostela, Spain). Thin layer chromatography (TLC) was performed on Merck Kieselgel 60Å F254 plates and visualized with a UV lamp at 254 nm. All peptides were synthesized using a CEM Liberty Microwave Peptide Synthesizer via solid phase Fmoc peptide chemistry (Matthews, NC). The resin used was a H-Rink-Amide-Chem matrix with a loading capacity of 0.45 mmol/g, purchased from PCAS BioMatrix Inc (Saint-Jean-surRichelieu, Quebec Canada). UV-vis spectroscopy was performed with a Beckman Coulter DU 730 Life Science UV/Vis Spectrophotometer (Brea, CA). High resolution mass spectrometry was performed on a Waters Synapt G2 HDMS (q-TOF) instrument (Milford, MA). Low resolution 16 ACS Paragon Plus Environment

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LC-MS was performed on an Agilent 6120 (Q-MS) instrument (Santa Clara, CA). Analytical and semi-preparative reverse phase HPLC was performed on an Agilent 1200 series instrument (Santa Clara, CA). Normal phase and reverse phase column chromatographies were performed on a Biotage Isolera Flash System (Charlotte, NC) using Sorbent Technologies Silica gel 60 Å (230-240 mesh) (Norcross, GA) and C18 column, respectively. The purity of compounds was evaluated via HPLC and 1H NMR. All tested compounds are ≥95% purity, unless otherwise noted. The molar extinction coefficients were dox (480 nm) 11,500 M-1cm-1 and Cy5 (646 nm) 250,000 M-1cm-1.

2. Synthesis Dox free base preparation A clinical sample of doxorubicin-HCl (40 mg) was dissolved in 10 mL of MeOH in a separatory funnel. After 10 mL of saturated NaHCO3 (Aq.) was added to adjust the pH to 8.5, the dox free base was extracted with 20 mL of chloroform three times. The combined organic layers were dried with sodium sulfate and chloroform was removed via rotatory evaporator. The compound was dried in vacuo and stored in a -20 °C freezer.

Doxaz and DoxF Synthesis Dox free base was dissolved in deuterated chloroform, followed by addition of 10 equiv of prilled paraformaldehyde. The reaction was monitored by 1H NMR. After 2 or 3 days stirring, all

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doxorubicin was converted to doxoform. After the reaction mixture was filtered, the solvent was removed by rotatory evaporator and the doxoform was dried in vacuo. 35

N-(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)-2-[4-(1-hydroxyethyl)-2-methoxy-5nitrophenoxy]acetamide (6). Compound 5 (1.61 g, 3.43 mmol) was dissolved in MeOH (15 mL). To this solution was added sodium borohydride (0.519 g, 13.7 mmol) slowly at 0 °C. The resulting solution was warmed to room temperature and stirred for 5 h. The reaction was quenched by adding saturated NH4Cl (Aq.). The product was extracted with ethyl acetate three times. The combined organic phase was washed with brine and dried over sodium sulfate, and the solvent was removed by rotatory evaporator. The crude product was purified by normal phase silica gel flash column chromatography eluting with 5% MeOH in DCM (v/v) to give product 6 (1.2 g, 72% yield) with the following spectral properties: 1H NMR (400 MHz, DMSO-d6) δ 8.09 (t, J = 5.7 Hz, 1H), 7.52 (s, 1H), 7.39 (s, 1H), 5.53 (d, J = 4.4 Hz, 1H), 5.32 – 5.20 (m, 1H), 4.60 (s, 2H), 3.93 (s, 3H), 3.61 – 3.47 (m, 10H), 3.44 (t, J = 5.8 Hz, 2H), 3.41 – 3.35 (m, 3H), 3.29 (q, J = 5.8 Hz, 2H), 1.37 ppm (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 167.06, 153.49, 145.40, 138.97, 138.53, 109.65, 109.28, 69.71, 69.67, 69.58, 69.49, 69.17, 68.80, 67.92, 63.86, 56.04, 49.88, 40.02, 39.81, 39.60, 39.39, 39.18, 38.97, 38.76, 38.24, 25.04 ppm. ESI-MS: [M+H]+ calcd 472.2043, found 472.2043: [M+Li]+ calcd 478.2126, found 478.2123. NMR assignments appear in Figure S16.

1-(4-{[(2-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}ethyl)carbamoyl]methoxy}-5-methoxy-2nitrophenyl)ethyl 4-nitrophenyl carbonate (7).

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Compound 6 (0.282 g, 0.562 mmol) was dissolved in THF (10 mL) with activated 4Å molecular sieves. 4-Nitrophenyl chloroformate (0.170 g, 0.843 mmol), DMAP (0.0068 g, 0.056 mmol) and triethylamine (0.114 g, 1.124 mmol) were added. The resulting mixture was stirred at room temperature for 16 h. Ethyl acetate and water were poured into the reaction mixture, followed by washing with saturated NaHCO3 (Aq) five times. The organic phase was washed with brine and dried with sodium sulfate, and the solvent was evaporated. The crude mixture was purified by normal phase silica gel flash column chromatography eluting with 5% MeOH in DCM (v/v). The fractions were collected, evaporated and dried to give 7 (0.322 g, 90% yield) with the following spectral properties: 1H NMR (400 MHz, DMSO-d6) δ 8.29 (d, J = 9.3 Hz, 2H), 8.12 (t, J = 6.0 Hz, 1H), 7.57 (s, 1H), 7.53 (d, J = 9.3 Hz, 2H), 7.23 (s, 1H), 6.28 (q, J = 6.4 Hz, 1H), 4.66 (s, 2H), 3.99 (s, 3H), 3.61 – 3.47 (m, 10H), 3.44 (s, 2H), 3.35 (s, 4H), 3.28 (d, J = 5.7 Hz, 2H), 1.73 ppm (d, J = 6.5 Hz, 3H); 13C NMR (101 MHz, DMSO) δ 166.88, 154.98, 153.68, 151.13, 146.51, 145.12, 139.40, 131.09, 125.32, 122.54, 109.55, 108.93, 72.92, 69.70, 69.66, 69.57, 69.49, 69.16, 68.80, 67.69, 56.40, 49.87, 40.03, 39.82, 39.61, 39.40, 39.19, 38.98, 38.77, 38.27, 21.11 ppm; ESI-MS: [M+H]+ calcd 637.2106, found 637.2111. NMR assignments appear in Figure S17. Photodoxazolidine-PEG-Azide (8). Compound 7 (0.159 g, 0.250 mmol) and HOBt hydrate (0.045 g, 0.294 mmol) were dissolved in DMSO (6 mL). After the mixture was stirred at room temperature for 10 min, doxaz (0.0817 g, 0.147 mmol) in DMSO (6 mL) was added. The resulting mixture was stirred at room temperature for 2 days. The reaction was monitored by analytical HPLC. The unreacted doxaz was hydrolyzed to dox by adding 1X phosphate buffer (10 mL) and the unreacted 7 was quenched by adding by 1 equiv of D-glucosamine and DIEA, and 2 equiv of HOBt. Following overnight stirring, DCM (20 mL) was added and the organic phase was washed with water three times. The 19 ACS Paragon Plus Environment

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crude mixture was purified by reverse phase column chromatography using a C18 column eluting with H2O and ACN with 0.1 % TFA (v/v) to give 8 (0.08 g, 51% yield) with the following spectral properties: NMR spectra with assignments appear in Figure S18. ESI-MS: [M-H]- calcd 1051.3499, found 1051.3470.

Photodoxazolidine-PEG-Maleimide (9). Compound 8 (0.0398 g, 37.8 μmol) and DBCO-maleimide (0.0194 g, 58.5 μmol) were combined in DMSO (4.5 mL). The resulting mixture was stirred at room temperature for 22 h. To the reaction mixture were added DCM and water, followed by washing with water three times and brine, drying with sodium sulfate, and evaporation of solvent. The crude mixture was purified by normal phase silica gel column chromatography eluting with 90% (v/v) DCM and 10% (v/v) MeOH to give 9 (0.045 g, 81% yield) with the following spectral properties: NMR spectra with assignments appear in Figure S19; ESI-MS: [M+H]+ calcd 1480.5109, found 1480.5135.

Photodoxorubicin-PEG-azide (10). Compound 6 (0.035 g, 0.055 mmol) and HOBt hydrate (0.011 g, 0.074 mmol) were dissolved in DMSO (2 mL). To this mixture was added dox free base (0.02 g, 0.037 mmol) in DMSO (2 mL). The resulting mixture was stirred at room temperature overnight and monitored by analytical HPLC (method 1). To the reaction mixture was added 20 mL of DCM. The resulting solution was washed with water three times and dried over anhydrous sodium sulfate. The crude mixture was purified by reverse phase column chromatography using a C18 column eluting with H2O and ACN with 0.1 % (v/v) TFA to give 10 (0.026 g, 69% yield) with the following spectral 20 ACS Paragon Plus Environment

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properties: NMR spectra with assignments appear in Figure S20; ESI-MS: [M+Na]+ calcd 1063.3396, found 1063.3369.

Photodoxorubicin-PEG-Maleimide (11). Compound 10 (0.0130 g, 0.0124 mmol) and DBCO-maleimide (0.0064 g, 0.015 mmol) were dissolved in DMSO (1.5 mL). The resulting mixture was stirred at room temperature for 22 h. The reaction was monitored by analytical HPLC (method 1). After chloroform was added to this mixture, it was washed with water three times and dried over anhydrous sodium sulfate. The crude mixture was purified by normal phase silica gel column chromatography eluting with 95% (v/v) DCM and 5% (v/v) MeOH to give 11 (7.2 mg, 39 % yield) with the following spectral properties: NMR spectra with assignments appear in Figure S21; ESI-MS [M+H]+ calcd. 1468.5109, found 1468.5056.

General Method for peptide-thiol conjugation. L-MARCKS-ED-SH. (L-configuration) L-MARCKS-ED peptide (sequence: KKKKKRFSFKKSFKLSGFSFKKNKK) (0.1 mmol) was synthesized by standard Fmoc solid phase peptide synthesis. The peptide on resin (0.1 mmol), strityl-3-mercaptopropionic acid (0.069 g, 0.2 mmol) and HATU (0.077 g, 0.2 mmol) were combined and dissolved in DMF (8 mL). After the solution reached a homogeneous appearance, DIEA (0.0516 g, 0.4 mmol) was added. The resulting mixture was stirred at room temperature overnight. The peptide cleavage from the resin and universal deprotection were carried out with

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TFA cocktail: TIPS (100 μL), H2O (250 μL), EDT (250 μL), and TFA (9.4 mL). The reaction was left at room temperature for 3 h. After filtering out resin, cold diethyl ether was added and the precipitated peptides were collected by centrifugation (4500 xg). The crude peptide was purified by semipreparative RP-HPLC using method 2. The pure product showed the following spectral properties; ESI-MS: calcd. [M+3H]3+ 1056.6, [M+4H]4+ 792.7, [M+5H]5+ 634.4, [M+6H]6+ 528.8, [M+7H]7+ 453.4, [M+8H]8+ 396.9, found [M+3H]3+ 1056.6, [M+4H]4+ 792.7, [M+5H]5+ 634.4, [M+6H]6+ 528.8, [M+7H]7+ 453.4, [M+8H]8+ 396.9.

D-MARCKS-ED-SH. (D-configuration) The peptide with each amino acid residue having the D-configuration was similarly prepared. It showed the following mass spectral properties: ESI-MS: calcd. [M+2H]2+ 1584.4513, [M+3H]3+ 1056.6368, [M+4H]4+ 792.7296, found [M+2H]2+ 1584.4550, [M+3H]3+ 1056.6370, [M+4H]4+ 792.7276.

C2BL3L-SH C2BL3L-SH peptide was similarly prepared with the following mass spectral properties. ESIMS: calcd. [M+1H]+ 1339.6, [M+2H]2+ 670.3, found [M+1H]+ 1339.6, [M+2H]2+ 670.3.

General method for thiol-maleimide Michael addition for synthesis of 1, 2, 3 and 4. The peptide-thiol conjugate (0.050 g, 0.016 mmol) was dissolved in degassed 1X PBS (pH=7.4, 10 mL) under a nitrogen atmosphere, to which photodoxaz-maleimide (0.028 g, 0.019 mmol) in 22 ACS Paragon Plus Environment

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DMF (1 mL) was added dropwise. The reaction was left in the dark with a nitrogen atmosphere at room temperature overnight. The crude product was purified by reverse phase column chromatography using a C18 column eluting with 60% (v/v) H2O with 0.1% (v/v) TFA and 40% (v/v) ACN with 0.1% (v/v) TFA to give the pure product. The fractions containing the product were lyophilized. Purity was more than 95% (RP-HPLC) eluting with method 1 and monitoring at 210, 254, 280 and 480 nm (Figures S35, S36, S37 and S38). L-MARCKS-ED-Photodoxazolidine (1). 7% yield. ESI-MS:C223H325N51O56S, calcd. [M+3H]3+ 1550.1388, [M+4H]4+ 1162.8561, [M+5H]5+ 930.4864, [M+6H]6+ 775.5733, [M+7H]7+ 664.9211, [M+8H]8+ 581.9319, found [M+3H]3+ 1550.1439, [M+4H]4+ 1162.8579, [M+5H]5+ 930.4872, [M+6H]6+ 775.5684, [M+7H]7+ 664.9202, [M+8H]8+ 581.9130. Frag. 1: C194H297N50O43S, calcd. [M+4H]5+ 810.7760, [M+5H]6+ 675.8133, [M+6H]7+ 579.4114, [M+7H]8+ 507.1100, [M+8H]9+ 450.8756, found [M+4H]5+ 810.6526, [M+5H]6+ 675.7117, [M+6H]7+ 579.3247, [M+7H]8+ 507.0339, [M+8H]9+ 450.5086. Frag. 2: C202H308N51O47S, [M+4H]5+, calcd. 847.8120, found 847.6667. Frag. 3: C202H309N51O48S, [M+5H]5+, calcd. 851.4120, found 851.2692. D-MARCKS-ED-Photodoxazolidine (2). 25% yield. ESI-MS: calcd. [M+3H]3+ = 1550.1388, [M+4H]4+ = 1162.8561, [M+5H]5+ = 930.4864, [M+6H]6+ = 775.5733, [M+7H]7+ = 664.9211, found [M+3H]3+ = 1550.1199, [M+4H]4+ = 1162.8431, [M+5H]5+ = 930.4782, [M+6H]6+ = 775.5732, [M+7H]7+ = 664.9173. Frag. 1: C194H297N50O43S, calcd. [M+5H]6+ 675.8133, [M+6H]7+ 579.4114, [M+7H]8+ 507.1100, [M+8H]9+ 450.8756, found [M+5H]6+ 675.7092, [M+6H]7+ 579.1815, [M+7H]8+ 507.0352, [M+8H]9+ 450.8104. 23 ACS Paragon Plus Environment

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Frag. 2: C202H308N51O47S, [M+5H]6+, calcd. 706.6767, found 706.5542. Frag. 3: C202H309N51O48S, [M+6H]6+, calcd. 709.6767, found 709.5551. C2BL3L-Photodoxazolidine (3). 34.6 % yield. ESI-MS: C128H163N25O46S, calcd. [M+2H]2+ 1410.5546, [M+3H]3+ 940.7056, found [M+2H]2+ 1410.5590, [M+3H]3+ 940.7059. Frag. 1: C99H135N24O33S, [M+2H]3+ calcd. 740.6446, found 740.9850. Frag. 2: C107H146N25O37S, calcd. [M+1H]2+ 1203.7700, [M+2H]3+ 802.8467, found [M+1H]2+ 1203.509, [M+2H]3+ 802.6745. L-MARCKS-ED-Photodoxorubicin (4). 26.6% yield. ESI-MS: C222H325N51O56S, calcd. [M+3H]3+ 1546.1388, [M+4H]4+ 1159.8561, [M+5H]5+ 928.0864, [M+6H]6+ 773.5733, found [M+3H]3+ 1546.1353, [M+4H]4+ 1159.8542, [M+5H]5+ 928.0833, [M+6H]6+ 773.5701. Frag. 1: C194H297N50O43S, [M+5H]6+ 675.8133, [M+6H]7+ 579.4114, [M+7H]8+ 507.1100, found [M+5H]6+ 675.7104, [M+6H]7+ 579.3233, [M+7H]8+ 507.0322. Frag. 2: C201H308N51O47S, [M+5H]6+, calcd. 704.6733, found 704.5548. Frag. 3: C201H309N51O48S, [M+6H]6+, calcd. 707.6750, found 707.5566.

Associated Content Supporting information The supporting Information is available free of charge on the ACS Publications website at DOI: Molecular Formula Strings (CSV).

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Supporting figures, tables and videos, experimental, assignments of proton NMR signals on spectra, mass spectra, HPLC chromatograms, and sets of NMR spectra (PDF).

Author Information Corresponding Author: Dr. Hang Yin School of Pharmaceutical Sciences, Tsinghua University, Beijing 100082, China E-mail: [email protected]

Acknowledgements This work was funded by the National Key R&D Program of China (No. 2017YFA0505200), National Institutes of Health (NIH R01CA183953), and National Natural Science Foundation of China (Nos. 21825702 and 21572114). The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core. Spinning disc confocal microscopy was performed on Nikon Ti-E microscope supported by the BioFrontiers Institute and the Howard Hughes Medical Institute. We would like to thank Dr. Benjamin L Barthel, Mr. Thomas Price Kirby and Dr. Peter Cogan for helpful discussions.

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Abbreviations ACN = acetonitrile, DBCO = dibenzocyclooctyne, DCM = Dichloromethane, DIEA = N,Ndiisopropylethylamine, DMF = dimethylformamide, DMSO = dimethyl sulfoxide, Dox = doxorubicin, DMAP = 4-(dimethylamino)pyridine, Doxaz = doxazolidine, DoxF = doxoform, ED = effector domain, EDT = Ethane ditiol, EM = exact mass, ESI-MS = Electrospray Ionization Mass Spectrometry, EtOH = ethanol, Fmoc = fluoronylmethyloxycarbonyl, FBS = fetal bovine serum, HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate, HOBt = hydroxybenzotriazole, HPLC = High Performance Liquid Chromatography, IC50 = half maximal inhibitory concentration, MALDITOF-MS = Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry, MARCKS = myristoylated alanine-rich C kinase substrate protein, MeOH = methanol, NMR = Nuclear Magnetic Resonance, NTA = Nanoparticle Tracking Analysis, PBS = phosphate buffered saline, Photodoxaz = Photodoxazolidine, Photodox = Photodoxorubicin, PS = phosphatidylserine, RT = room temperature, SDS = Sodium dodecyl sulfate, TFA = Trifluoroacetic acid, TIPS = triisopropylsilane, PPD = Pentyl PABC-Doxaz , GaFK-PABCDoxaz = N-(N-(N-acetylglycyl-D- alanyl-L-phenylalanyl-L-lysyl)-p-aminobenzyloxycarbonyl)doxazolidine.

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Figure 1. (A) Interconversion of doxorubicin (dox), doxazolidine (doxaz) and doxoform (doxf) by reaction with formaldehyde. IC50 values are with a dox-resistent NCI-RES/Adr cell line.31 (B) MARCKS-ED-photodoxazolidine consisting of three parts: anticancer agent (doxaz), photolabile linker, and MARCKS-ED peptide, and release of doxaz with near UV light. 32 ACS Paragon Plus Environment

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Table 1. The peptide sequences studied in this work. Red is a cationic amino acid. Blue is phenylalanine. Peptide

Sequence

L-MARCKS-ED KKKKKRFSFKKSFKLSGFSFKKNKK D-MARCKS-ED KKKKKRFSFKKSFKLSGFSFKKNKK MARCKS-Scr

KKKGKKNSSKKFFFFSKFKLSRKKK

MARCKS-Mut

DDDDDRASADDSADLSGASADDDDD

C2BL3L

GGDYDKIGKNDA

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O

NO2

O O

OH

NaBH4/MeOH

H N

O

O

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NO2

N3

O

72 %

O

H N

O

O

O

O

5

O

O

6 NO2

O Cl

O 2N

O

O

O

DMAP/Et3N/THF

O

NO2

O

90 %

OH

Doxaz O

O

O

O

O

H N

O

N3

HOBt/DMSO 51%

7

O OH OH

O

O

OH O

O O

O

N O NO2

O

O O

O

O

N3

O

N H 8

Scheme 1. Synthesis of photodoxaz-PEG-azide 8 as a mixture of two diastereomers. The structure numbering system for 8 appears in Fig. S18.

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N3

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O O

N

O N H

O OH

OH

O

O N

OH

O

OH O

O

O

O

N

O

DBCO-maleimide

O

8

NO2 DMSO O

O

O

OH

O OH

OH O

O

O O

O

O HS NO2

O

O O

O

N

N

N H

R

N

N

HN O

O

N

O

9

O

N

N

N H

O

OH

O

O

O

O

O

N O

N

O

N H

N O

1 R = L-MARCKS-ED: KKKKKRFSFKKSFKLSGFSFKKNKK 2 R = D-MARCKS-ED: KKKKKRFSFKKSFKLSGFSFKKNKK 3 R = C2BL3L: GGDYDKIGKNDA

HN O O

N

O O

S

O

O

NH

OH

4: L-MARCKS-ED-Photodox R = KKKKKRFSFKKSFKLSGFSFKKNKK

O

HN R

Scheme 2. Synthesis of phtodoxaz-peptide conjugates. The click reaction gives 9 as a mixture of two regioisomers that are each a mixture of two diastereomers. The final products, 1, 2, 3 and 4, are mixtures of two sets of regioisomers with four diastereomers per set. Only one regioisomer is shown.

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Figure 2. Photolysis of Photodoxaz-PEG-Azide 8. The compound 8 was irradiated by a monochromatic laser (12 mW, 325 nm). Dox released was monitored by RP-HPLC (480 nm). The quantum yield (Φ = 0.025) was calculated using the time at which approximately 10% of dox was released (t = 60 s). Experiments were triplicated. Error bar is standard deviation.

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Figure 3. Results of growth inhibition by the Photodoxaz-peptides. (A) L-MARCKS-EDPhotodoxaz. (B) D-MARCKS-ED-Photodoxaz. (C) C2BL3L-Photodoxaz. (D) L-MARCKS-EDPhotodox. Red dots are with black light UV irradiation (320-400 nm with maximum intensity at 350 nm). Black dots are without UV irradiation. Experiments were biologically independent triplicated. Error bar is standard deviation.

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Table 2. IC50 values of Photodoxaz-peptide conjugates. IC50 values are average of triplicates. SE is standard error of the mean. Compound L-MARCKS-ED-Photodoxaz D-MARCKS-ED-Photodoxaz C2BL3L-Photodoxaz L-MARCKS-ED-Photodox

Irradiation IC50 ± SE (µM) +hν -hν +hν -hν +hν -hν +hν -hν

0.015 ± 0.008 2.5 ± 0.4 0.042 ± 0.010 2.0 ± 0.6 1.3 ± 0.1 11 ± 1 1.2 ± 0.4 6.5 ± 0.7

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Fold Δ 1.0 170 2.8 130 87 730 80 430

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Figure 4. Cell uptake of Photodoxaz peptides. Uptake was monitored by flow cytometry. MDA-MB-231 cells were incubated with the respective compound (1µM) for 6 h. Fluorescence intensities were normalized to the untreated cell (control). Each error bar is the standard deviation of the mean fluorescence intensity. Experiments were biologically independent triplicated.

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Figure 5. Photodoxaz uptake images. (A) L-MARCKS-ED-Photodoxaz 5 µM for 3 h. (B) DMARCKS-ED-Photodoxaz 5 µM for 3 h. (C) Untreated cells. 40x magnification. Scale bar 20 µm.

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Figure 6. Co-localization of D-MARCKS-ED and exosomes. Cy5-D-MARCKS-ED was incubated with exosomes for 1 h. Images were taken without washing cells to see the colocalization on the plasma membrane. Scale bar is 20 µm.

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