Sn2 Lipase Labile Prodrugs and Contact-Facilitated Drug Delivery for


Nov 15, 2017 - Nanomedicine technologies have often proven unstable in vivo due to premature release of drug cargoes during circulation resulting in l...
0 downloads 9 Views 1MB Size


Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

Chapter 8

Sn2 Lipase Labile Prodrugs and Contact-Facilitated Drug Delivery for Lipid-Encapsulated Nanomedicines D. Pan,1 G. Cui,2 C. T. N. Pham,3 M. H. Tomasson,4 K. N. Weilbaecher,5 and G. M. Lanza*,6 1Departments

of Bioengineering, Materials Science and Engineering and Beckman Institute, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, United States 2Department of Medicine, Division of Cardiology, Washington University Medical School, St. Louis, Missouri 63108, United States 3Department of Medicine, Division of Rheumatology, Washington University Medical School, St. Louis, Missouri 63110, United States 4Department of Internal Medicine, Division of Hematology, Oncology and Blood and Marrow Transplantation, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, United States 5Department of Medicine, Division of Oncology, Washington University Medical School, St. Louis, Missouri 63110, United States 6Department of Medicine, Division of Cardiology, Washington University Medical School, St. Louis, Missouri 63108, United States *E-mail: [email protected]

The concept of achieving Paul Erhlich’s inspired vision of a “magic bullet” to treat disease is now materializing with select monoclonal antibody therapies, but this achievement is not well replicated by current nanomedicine clinical candidates. Nanomedicine technologies have often proven unstable in vivo due to premature release of drug cargoes during circulation resulting in low therapeutic delivery to targeted cells. Compounding this nanoparticle payloads that reach target cells are typically internalized within endosomes, contributing to further drug loss and diminished intracellular drug bioavailability. Historically, size limited extravasation of nanoparticles beyond the circulation followed

© 2017 American Chemical Society Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

by inhomogeneous and inadequate deep penetration into disease sites has been the major nanoparticle biological barrier. However, nanomedicines can function as excipients and prolonged release systems to favorably alter drug pharmacokinetics and volume of distributions for greater efficacy and lower toxicity. Sn2 phospholipid prodrugs in conjunction with a contact-facilitated drug delivery mechanism have been found to minimize premature drug diffusional loss during circulation and to increase target cell bioavailability. The Sn2 phospholipid prodrug approach has been applied equally well for vascular constrained lipid-encapsulated particles delivering anti-angiogenic therapies, such as fumagillin or docetaxel, and to micelles penetrating through inflamed endothelium into disseminated cancers, such as in multiple myeloma with anti-cMYC payloads. Innovations like Sn2 phospholipid prodrugs in combination with the contact-facilitated drug delivery mechanism are poised to contribute to the translational success of nanomedicines by increasing efficacy and safety for an array of poorly treated diseases.

Introduction Nanomedicine can offer alternative approaches to intractable medical problems by providing probes to detect and characterize disease based on the expression of cell surface biomarkers. Using the same platform technology when appropriate, therapeutic compounds can be more specifically homed to lesions. 1A diverse spectrum of nanotechnologies including solid metal particles, engineered viral systems, polymeric nanosystems, and various lipid-based particles has been researched for medical imaging and drug delivery applications (1). Lipid-based particles have inherently high biocompatibility, a general ease of lipid functionalization, and a “soft” compliant 3D morphology that contributed to early and continued clinical success. The best-known nontargeted translational example of liposomal technology is Doxil™, a pegylated liposomal formulation (2–5). Doxil™ elimination follows a typical bi-exponential curve characterized by a rapid distribution phase with a 2-hour half-life and a much slower beta-elimination rate (~45 hours t1/2) (3). By incorporating doxorubicin into the aqueous phase of liposomes, the systemic drug volume of distribution in patients was decreased from 254 liters to 4 liters. This constrained volume of distribution and extended circulatory half-life, markedly improved the efficacy and tolerance of doxorubicin. The success of Doxil™ and the coincident advent of monoclonal antibody technology helped fuel the current era of targeted particle-based drug delivery. One of the earliest biological barriers recognized in cell culture for ligand-targeted particles was their uptake and internalization within endosomes, which reduced the effectiveness of the compound (6). Innumerable approaches 190 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

to overcome endosomal related losses of therapeutic cargoes of drugs, oligonucleotides, and proteins have been explored including cell-penetrating peptides, such as Tat (transactivator of transcription) (7). However, these cationic peptides interacted with cell-associated glycosaminoglycans and payloads were subsequently internalized by and lost to endocytosis. Arginine-rich cationic peptides coupled directly to peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) offered improvement at non-cytotoxic doses (7), but this did not hold for cationic nanoparticles (8). In the context of siRNA and gene delivery, effective delivery of the nucleic acid-based therapy from endosomes in the cytosol to the nucleus has achieved some promising successes (9–26).

I. Contact-Facilitated Drug Delivery i. Mechanism In contradistinction to investigators seeking to internalize particles into cells for drug delivery, a novel approach called contact-facilitated drug delivery (CFDD) was developed (27). CFDD is a slow second order process dependent upon the persistent interaction of the phospholipid-encapsulated nanoparticle with target membrane surfaces. Figure 1 illustrates the transfer of rhodamine-coupled phosphatidylethanolamine, a particle membrane marker, into the outer cell membrane and then inner membranes of a C32 melanoma cell in culture (28). Close microscopic examination (inset) revealed streaming of the fluorescent phospholipid from the particle into cell membrane.

Figure 1. Contact facilitated drug delivery illustrated with rhodamine perfluorooctylbromide (PFOB) nanoparticle bound to C32 melanoma cell (transfected with Rab GFP endocytic markers). Reproduced with permission from reference (28). Copyright (2008) Elsevier Ltd. (see color insert) Electron microscopic examination captured the hemifusion complexation of the monolayer of a perfluorocarbon (PFC) nanoparticle (~250nm) with the bilayer of target cell membrane (29). Unlike ligand-receptor interactions that occur in equilibrium, the hemifusion of the particle with the cell membrane is irreversible and the surfactant entrapped drug payload is delivered directly into 191 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

the outer membrane. Translation of the phospholipid-drug complex into the outer membrane can occur at 4°C, independent of ATP, but the translation of the phospholipid-prodrug from the outer to the inner membrane leaflet requires ATP. Since the inner cell membranes, excluding mitochondrial membranes, are contiguous, drug is transported throughout the cytosol. CFDD delivers the “kiss of death” while circumventing endosomal particle internalization and drug payload losses. (Figure 2)

Figure 2. SEM showing perfluorooctylbromide (PFOB) nanoparticle hemifusion complex to C32 melanoma cell. Reproduced with permission from reference (29). Copyright (2008) American Chemical Society. (see color insert) ii. Preclinical Examples of Drug Delivery For some very hydrophobic drugs, such as fumagillin, the contact facilitated drug delivery mechanism proved effective in vivo, particularly for anti-angiogenic therapy, which was often pursued in combination with MR neovascular molecular imaging. Fumagillin is an anti-angiogenic agent, isolated from Aspergillus fumigatus, specific for proliferating endothelial cells through inhibition methionine aminopeptidase 2. Its water-soluble clinical analogue, TNP-470, was produced semi-synthetically, and effective in rodents. In patients, it possessed only anecdotal effectiveness even at high doses, which often were complicated by numerous toxicities, including neurocognitive dysfunction (30–32). The effectiveness of fumagillin as a nanomedicine was first shown In the Vx2 syngeneic rabbit tumor model using αvβ3-targeted perfluorocarbon (PFC) nanoparticles. Native fumagillin (0.049mg/kg) incorporated into the phospholipid surfactant was given in 3 serial minute doses to the animals, reducing tumor development and angiogenesis (33). This constituted a greater than 10,000-fold reduction of drug versus the water-soluble TNP-470 analogue used in clinical 192 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

studies (33). Figure 3a Similar results were obtained in hyperlipidemic NZW rabbits with early aortic atherosclerosis in which αvβ3-targeted fumagillin PFC nanoparticles provided an MR neovascular estimate of plaque progression, delivered effective fumagillin anti-angiogenic therapy, and provided quantitative follow-up of treatment response (34). Figure 3b In the serum transfer K/BxN mouse model of inflammatory arthritis, which expresses the T-cell receptor transgene KRN and the MHC class II molecule A(g7), serial αvβ3-targeted fumagillin PFC particles decreased arthritic score, ankle thickness, inflammation, proteoglycan depletion, and angiogenesis (35). Figure 3c In each example, cumulative fumagillin dosage was well below the high levels of TNP-470 (30mg to 60 mg/kg/dose) used in cancer patients with neurocognitive deficits (30, 36, 37).

Figure 3. A) 3D MR angiogenesis maps of control and integrin-targeted fumagillin NP in Vx2 model. B) Angiogenesis contrast before and 1 week after a fumagillin or control NPs in hyperlipidemic rabbits C). Decreased arthritic score and ankle thickness following targeted fumagillin in the serum transfer K/BxN model of inflammatory arthritis. Reproduced with permission from reference (33). Copyright (2008) Federation of American Societies for Experimental Biology, from reference (34). Copyright (2006) Wolters Kluwer Health/Lippincott Williams & Wilkins and from reference (35). Copyright (2009) Federation of American Societies for Experimental Biology. (see color insert) iii. Lipid-Dissolved Drug Instability Similar, to fumagillin, other hydrophobic drugs, such as paclitaxel, dissolved readily into the phospholipid membrane component of PFC nanoparticles and were retained during in vitro dissolution studies. However, when given systemically, these compounds were rapidly and prematurely lost from the PFC nanoparticle surfactant (38). Simultaneous pharmacokinetic analysis of the phospholipid-anchored homing ligands and gadolinium chelates, dissolved hydrophobic drugs, and perfluorocarbon core indicated that the functionalized lipid components were stably integrated into the particle membrane and tracked with the PFC core (unpublished), but the free chemotherapeutic agents 193 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

were prematurely leaked from the particles into blood. As a theranostic, the molecular imaging functions were outstanding, but systemic drug retention was compromised. Closer examination of fumagillin dissolved into the nanoparticle surfactant also revealed that despite its efficacy in preclinical models, it diffused into the circulation during particle transit (39). Perhaps only 10 to 20% of the initial fumagillin drug load was retained and delivered to targeted neovasculature, but the remaining low dose of this highly potent anti-angiogenic was effective in vivo. Although fumagillin has negligible off-target toxicity due to its biochemical specificity for proliferating endothelial cells, the general translational benefit for fumagillin and similar hydrophobic compounds was diminished. iv. Phospholipid Sn2 Prodrugs Following these results, prodrugs coupling the active ingredients to phosphatidyl ethanolamine were synthesized. This prodrug motif proved ineffective because the compounds were too large for the ATP dependent transfer of compound from the outer to inner cell membranes. This was a benefit for the rapid bioelimination of phospholipid anchored gadolinium chelates but problematic for drug delivery. A new series of phospholipid compounds were synthesized with the drug tethered to phosphatidyl ethanolamine through various short spacers with enzymatically labile regions, such as esters. These were generally ineffective due to low drug bioavailability caused low release rates from the particle surface or low uptake of the liberated drug into the target cells when compared with free drug alone. Consequently, a different approach to a phospholipid prodrug was imagined wherein the active pharmaceutical ingredient (API) is coupled to the Sn2 acyl position (i.e., stereospecific numbered hydroxyl group of the second carbon of glycerol) (39, 40). This design retains the advantages of contact facilitated drug delivery previously established and stably incorporated the phospholipid prodrug into surfactant membrane of nanoparticles during self-assembly (27). Moreover, with the active pharmaceutical ingredient (API) nestled within the protective hydrophobic environment, exposure to the surrounding media during circulatory transit was minimized. Upon docking to a cell surface receptor, contact mediated streaming of the Sn2 phospholipid prodrug into the outer leaflet of the target cell membrane was facilitated by hemifusion. Figures 1 and 2 ATP dependent translation to the inner lipid leaflet followed by rapid distribution throughout the intracellular membranes was achieved (29). In the cytosol, numerous lipases are present with the capacity to effectively liberate drug from the membrane, although specific enzymes and their trigger for activity have yet to be defined (39, 40). v. Background for Phospholipid Sn2 Prodrugs Although the concept of Sn2 phospholipid prodrugs had never been considered for targeted drug delivery in association with CFDD as pursued herein, precedent for the formation of Sn2 phospholipid prodrugs was reported by David Thompson et al (41) in the context of triggered release mechanisms. Later, 194 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

Thomas Lars Andresen and colleagues (42–52) pursued this approach to deliver chemotherapeutics as Sn2 prodrugs via untargeted liposomes. They hypothesized that increased liberation of phospholipases into blood by cancers could trigger the local release of the Sn2 linked compounds enriching drug bioavailability. Ultimately this strategy, which was the basis for a small European biotechnology start-up company, failed. These investigators noted that non-pegylated liposomes were resistant to phospholipase A2 (PLA2) drug liberation as compared to stealthy pegylated liposomes, which retained drug poorly. Unfortunately, in vivo simple liposomes were highly susceptible to intravascular destruction and bioelimination, and unsuitable for clinical translation. Physical chemical modelling suggested that pegylation undesirably enhanced water hydration around the particle and increased enzyme access to the Sn2 ester bonds leading to premature drug release by PLA2. The same lipid prodrugs incorporated into natural lipid membranes were resistant to water and enzyme penetration, protecting against Sn2 hydrolysis and premature drug loss (42–52). Targeted PFC nanoparticles, nanoemulsions and other lipid micelle particles subsequently discussed were adequately stable for rapid targeting in vivo without pegylation. In the case of the PFC particles, which are vascular-constrained by size (~200-250 nm), target saturation as evidenced by MRI molecular imaging of angiogenesis in cancer was achieved in less than 3 hours (53). Moreover, robust serial repeatability of this homing within the same animal over time allowed construction of precise Vx2 tumor 3D neovascular maps illustrating the progression of neovascular expansion (54). Protection of the drug within the outer membrane reduced water accessibility to the Sn2 ester and prevented premature drug loss until ligand-mediated binding and CFDD ensued. Via the membrane hemifusion process, the entire phospholipid prodrug is translocated into the outer membrane of the target cell in minutes and then into the internal cell membrane system.

II. Sn2 Phospholipid Prodrugs and Contact-Facilitated Drug Delivery i. Fumagillin as an Anti-Angiogenic Sn2 Prodrug In addition to premature drug loss and despite the promising preclinical nanomedicine results across different pathologies, the “druggability” of native fumagillin was inherently compromised by photochemical instability related to the conjugated decatetraenedioic tail and to reactive epoxide rings at the active site. To address these problems, an Sn2 fumagillin prodrug (Fum-PD) was envisioned and the compound was synthesized in a straightforward way in two steps involving saponification of fumagillin dicyclohexylamine salt to fumagillol and a subsequent esterification with oxidized lipid 1-palmitoyl-2-azelaoyl-sn glycero-3-phosphocholine (PAzPC) (39). (Figure 4)

195 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

Figure 4. Structure of fumagillin with sources of instability indicated. Synthetic strategy for the preparation of sn-2 fumagillin prodrug and development of site-targeted nanoparticles: saponification of fumagillin with MeOH : water (1:1), 35% NaOH; esterification with PAzPC, DCC/DMAP; preparation of lipid thin film from a phospholipids mixture of 98.7 mole% lecithin PC, 0.15 mole% of ανβ3-ligand conjugated lipid and 1.12 mole% of fumagillin prodrug; self-assembly by brief sonication and microfluidization, perfluorocarbon, glycerin, pH 6.5, at 20,000 psi for 4 minutes. Reproduced with permission from reference (39). Copyright (2012) Future Medicine Ltd. (see color insert) The anti-angiogenesis efficacy of αvβ3-Fum-PD nanoparticles was visualized with photoacoustic microscopic imaging (PA) of neovasculature in a subcutaneous Matrigel® rodent model (55). Mice implanted with Matrigel™ 18 days previously received either ανβ3-copper oleate in oil nanoparticle (ανβ3-CuNPs), nontargeted CuNPs, or ανβ3-CuNP preceded by 10 minutes with a competitive dose ανβ3-oil only NPs (1:1). As seen in Figure 5, at 0 min, forming vascular tubules were observed by the inherent PA contrast imparted by erythrocyte hemoglobin. Following ανβ3-CuNP injection, numerous incomplete vascular sprout offshoots largely devoid of erythrocytes were noted. These sprouts were previously characterized to be nonpolarized immature endothelial cells (ανβ3+, Tie-2-, CD31+) as opposed to formed microvessels with polarized endothelium, which were ανβ3-, Tie-2+, CD31+ (56). Animals given nontargeted-CuNPs had very little change in PA signal with minimal passive accumulation and slight blood pool enhancement. In the competition group, very little change in the PA signal from 196 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

the vascular tubules or sprouts was observed. Pretreatment with ανβ3-targeted NPs comprising oil core (no Cu oleate) blocked the receptors to ανβ3-CuNP binding and even precluded significant passive blood pool accumulation.

Figure 5. In vivo PA images of the MatrigelTM plug area implanted in 4 groups of mice at 18 days using αvβ3-CuNP. (A)-(B) Targeted CuNP group. The enhanced neovasculature by Cu oleate NPs are marked by arrows in B. (C)-(D): Nontargeted CuNP group. (E)-(F): Competition group: mice received a competitive dose αvβ3-oil only NP (1:1) 10 min before αvβ3-CuNP. (G)-(H): Fum-PD group: mice received αvβ3-CuNP with Fum-PD 11 and 15 days after the MatrigelTM implantation then αvβ3-CuNP w/o Fum-PD on day 18 for PA imaging. For all PA images, laser wavelength = 767 nm. Reproduced with permission from reference (55). Copyright (2015) Ivyspring International Publisher. (see color insert) In follow up, ανβ3-CuNP incorporating Fum-PD within the phospholipid membrane were administered on days 11 and 15 post Matrigel™ implant. Again on day 18, the amount of neovasculature observed by PA imaging was sparse and similar for all groups. Following ανβ3-CuNP injection control animals that received either drug free ανβ3-CuNP or nontargeted ανβ3-CuNP with Fum-PD had marked neovascular expansion. However, animals pre-treated with ανβ3-CuNP incorporating Fum-PD had negligible neovascular sprouting, only microtubules noted at baseline were appreciated after contrast injection. Dr. Rakesh Jain first suggested “Pruning” of neovasculature in a visionary manner and in this study the concept was visually apparent with PA microscopic imaging (57–59). ii. αvβ3-Fum-PD Nanoparticles Suppress Inflammation The K/BxN model of inflammatory arthritis is a polyarticular inflammatory arthritis resembling rheumatoid arthritis (RA) (60) . Serial i.v. injection of αvβ3-Fum-PD NP after K/BxN serum transfer in mice with established arthritis 197 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

attenuated disease progression at 8-fold lower doses than previously reported using native fumagillin (0.3mg/kg versus 2.4mg/kg) (35, 61). By comparison, inflammation progressed unabated in mice that received αvβ3-Ctrl NP. Figure 6. Clinical scores and histologic examination of arthritic paws revealed that αvβ3-Fum-PD NP limited inflammatory leukocyte recruitment into the inflamed paws, protected against bone erosions, and minimized cartilage damage.

Figure 6. αvβ3-Fum-PD NP suppressed inflammatory arthritis in the KRN model. αvβ3-targeted particle with or without drug were administered on days 2, 3, and 4 (arrows). Clinical changes in ankle thickness (A), arthritic score (B), and body weight (C) were monitored daily. Histology on day 7 for inflammatory cell number per high power field (HPF, D, E, J), erosions (F,G,K) proteoglycan depletion (H, I, L) *p<0.05, ** p<0.01, *** p<0.0001. Reproduced with permission from reference (61). Copyright (2012) Elsevier Ltd. (see color insert) Joint-associated inflammatory biomarkers VCAM-1 and ICAM-1 and major pro-inflammatory cytokines and chemokines (IL-1β, IL-6, MCP-1, and TNF-α) were all diminished with αvβ3-Fum-PD NP. While fumagillin is well known to impact the proliferation of neovascular endothelial cells, the direct impact of this mycotoxin on nonendothelial cell types was negligible (62, 63). In follow-up studies, the anti-inflammatory benefits of fumagillin or Fum-PD nanotherapy was mediated by local production of endothelial nitric oxide (NO). Fum-PD-induced endothelial NO modulated proximal macrophage inflammatory response through AMP-activated protein kinase (AMPK). NO-induced AMPK activation reduced mammalian target of rapamycin (mTOR) activity, increased autophagic flux, and 198 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

increased autolysosome formation in vivo. The increased macrophage autophagy flux elicited degradation of IkappaB kinase (IKK), suppression NF-kB-p65 signalling pathway and ultimately diminished inflammatory cytokine release (64). While fumagillin is an exceptional anti-angiogenic compound with secondary anti-inflammatory benefit, the mycotoxin is difficult to produce biologically or synthetically. From a safety perspective, the minimal off-target toxicity of fumagillin is highly desirable, but other highly potent chemotherapeutics delivered as Sn2 prodrug nanomedicines can offer potent anti-angiogenic and anti-inflammatory benefit with low off-target toxicity due to the small doses required and macrophage phagocytosis system clearance of excess nanoparticles. One class of compounds with these properties were the taxanes. iii. Docetaxel Sn2 Prodrug as an Anti-Angiogenic Therapy Free paclitaxel, a cytoskeletal drug that corrupts mitotic spindle assembly, chromosome segregation, and cell division was incorporated into lipid-encapsulated perfluorocarbon nanoparticles and studied in the Vx2 rabbit squamous carcinoma model as an antiangiogenic therapy. It was not effective due to significant premature drug loss in circulation, greater than fumagillin. To address this, docetaxel, a closely related taxane, was synthesized into an Sn2 prodrug (Dxtl-PD) (40). During a 3 day in vitro dissolution study in PBS with albumin or human plasma, Dxtl-PD incorporated into the surfactant of PFC nanoparticles released less than 6% of the prodrug cumulatively, indicating drug membrane stability (40). Moreover, spiking the dissolution buffer or plasma media with phospholipase A2 (PLA2) produced negligible release of the drug. These results are in contradistinction to approximately 10%/day loss of native paclitaxel from PFC nanoparticles reported under similar PBS-albumin buffer dissolution conditions (38). Furthermore, no passive prodrug transfer to erythrocytes through transient contact was measured when Dxtl-PD nanoparticles were incubated in whole blood with slow continuous agitation. Importantly, endothelial cells (2F2B) exposed to equimolar paclitaxel, paclitaxel prodrug, docetaxel, or docetaxel prodrug for 1 hour at varying doses and proliferation were assessed at 24, 48 and 72 hours post exposure with nearly identical biopotency response curves (40). (Figure 7) The anti-angiogenesis efficacy of the docetaxel Sn2 prodrug in αvβ3-targeted PFC nanoparticles (αvβ3-Dxtl-PD NP) was compared in the Vx2 rabbit tumor model with non-targeted Dxtl-PD NPs (NT-Dxtl-PD NP) and αvβ3-No Drug NPs. Animals were treated on days 9, 12, and 15 days post implant and molecularly imaged with MR (3T) on day 17. In the control rabbits, neovascularization was heterogeneously distributed along the tumor periphery with minimal signal in the core. αvβ3-Dxtl-PD NPs substantially reduced (p<0.05) MR detectable angiogenesis in the Vx2 tumor model. This strong anti-angiogenesis result of αvβ3-Dxtl-PD NP reversed the negative effects achieved previously with targeted particles incorporating native paclitaxel. To verify that the anti-neovascular effect of the Dxtl-PD NPs observed with MR molecular imaging was not attributable to the systemic premature release of taxane, a separate cohort of rabbits with the Vx2 tumor received serial Abraxane® treatments at the equivalent prodrug dose 199 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

and the response was compared to a saline control. MR molecular imaging of angiogenesis in this cohort on day 17 revealed no anti-angiogenic effect with Abraxane® (40).

Figure 7. Mouse vascular endothelial cells (2F2B) stimulated with angiotensin II (1nM) were treated with paclitaxel, docetaxel, paclitaxel prodrug, or docetaxel prodrug at 0.5 µg/ml, 1 µg/ml, 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml for 1 hour. Cultures were monitored for proliferation at 24, 48, and 72 hours. No difference (p= NS) in biopotency of the different taxanes forms was detected. Reproduced with permission from reference (40). Copyright (2014) Ivyspring International Publisher. (see color insert) iv. cMyc-Max Transcription Factor Antagonism in Myeloma Multiple myeloma (MM) is a malignancy of antibody over-production from a clone of plasma cells evolving from terminally differentiated B-lymphocytes. While recent therapeutic advances suggest future improvement, the current 5-year survival rate in patients with MM is less than 40% (65). MM is initially responsive to several classes of chemotherapy, (e.g. proteasome inhibitors, immunomodulatory drugs (IMiDs), and alkylating agents), but virtually all patients relapse and die from progressive disease. The b-HLHZIP transcription factor c-Myc (MYC) is a powerful oncogene activated in many types of cancer and is a central driver of myeloma development (66, 67). The transcription factor interacts with multiple signaling cascades, which makes it an attractive therapeutic target. Transformation by MYC is dependent 200 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

upon dimerization with the bHLHZIP protein MAX. MYC-MAX heterodimers bind to E-Boxes in the vicinity of target genes (68) to regulate their expression and to modulate numerous biological functions (69–71). Myc antagonism has been approached through anti-sense strategies (72), RNA interference (73), and interference with MYC-MAX dimerization using small molecules (74). Several small-molecule inhibitors of the MYC-MAX interaction were developed (74–78). However, the effectiveness of these compounds was diminished by rapid systemic metabolism, significant toxicity, poor cancer cell bioavailability and an inability of the drug to reach intratumoral inhibitory concentrations (77). An Sn2 phospholipid prodrug nanomedicine strategy was employed using a prototype compound (10058-F4) designated myc-inhibitor-1 prodrug (MI1-PD) (79, 80). The cytotoxic activities of the modified base compound, MI1, and the prodrug MI1-PD were evaluated and compared in human (H929 and U266) and mouse (5TGM1) multiple myeloma cell lines at concentrations ranging from 1.0 nM to 100 µM. MTT cell viability assay and showed that MI1-PD decreased cell viability substantially more (p<0.05) than free MI1. Similarly, MI1-PD induced significant apoptosis in the three MM cell lines, 92%, 91% and 91%, respectively, whereas apoptosis induced with MI1 was significantly less, 19%, 31% and 31%, respectively (p<0.05) (80). As with all targeted therapies, MI1-PD nanotherapeutic efficacy was dependent upon the specific binding of nanoparticle to target cells. In this regard, the suitability αvβ3-integrin and α4β1-integrin (VLA-4) target biomarkers evaluated in human and mouse MM cells showed potential utility for both targets for human MM in culture, but only α4β1-integrin in the mouse 5TGM1 line. The 5TGM1 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice (81–83). KaLwRij mice inoculated intravenously with 5TGM1 cells were treated intravenously with targeted (T) or nontargeted (NT) perfluorocarbon (200nm) or micellar (20nm) lipid-encapsulated particles incorporating MI1-PD (D) or no drug (ND) on days 3, 5, 7,10,12, and 14. Treatment groups were: 1) ND/NT 200nm PFC NP; 2) T/ND 200nm PFC NP; 3) T/D 200nm PFC NP; 4) NT/ND 20nm micelles; 5) T/ND 20nm micelles, and 6) T/D 20nm micelles (80). (Figure 8) Disease progression was monitored by survival and serial measurements of serum paraprotein on day 17. Free MI1 and free MI1-PD offered no survival benefit when given IV. Decreased serum immunoglobulin, a reflection of tumor burden, and increased animal survival (p<0.05) were only noted in the T/D 200nm PFC NP and T/D 20nm micelle groups (T/D 20nm: 52 days vs. 29 days, p=0.001). Control nontargeted drug or targeted no drug NP treatments regardless of particle size provided no benefit. These results illustrated this ultrasmall nanomedicine approach to MM using a potent MYC-MAX inhibitor. These data provide a foundation to reconsider MYC transcription factor antagonism as a viable therapeutic strategy in MM and more broadly further exemplify the potential of nanomedicine approaches utilizing Sn2 lipase labile prodrugs to overcome the poor solubility, poor systemic stability, poor biodistribution, or poor bioavailability that plague many therapeutic compounds.

201 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

Figure 8. Anti-MYC NPs prolong survival in a mouse model of multiple myeloma. (A) MTT studies in H929 (Human), U266 (Human) and 5TGM1 (Mouse) cells at 24 h. (B, C) Experimental protocol for the in vivo tumor growth assay in C57BL/KaLwRij. The groups were as follows: 1) ND/NT 200; 2) T/ND 200; 3) T/D 200; 4) NT/ND 20; 5) T/ND 20 and 6) T/D 20 all injected on days 3,5,7,10,12 and 14 following the i.v. injections of 5TGM1 cells (Day 0). (B & C) Kaplan-Meier survival curves of the treated mice with 20 and 200nm NPs. (ND=no drug, NT=non-targeted). Reproduced with permission from reference (80). Copyright (2015) American Association for Cancer Research.

Conclusion Sn2 lipase labile phospholipid prodrugs in conjunction with contact facilitated drug delivery offer an important advancement in nano-based therapy. For lipid-encapsulated nanoemulsions Sn2 phospholipid prodrugs inactivate the drug and minimize premature drug escape until bound to target cells and there reactivated by intracellular lipases. They increase intracellular target cell bioavailability by circumventing endosomal clearance pathways. This technology reflects one approach to nano-based drug delivery, and its success reflects the potential for nanomedicines in general to address intractable medical issues and unmet needs.

202 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

References

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

1.

Muthu, M. S.; Leong, D. T.; Mei, L.; Feng, S. S. Nanotheranostics application and further development of nanomedicine strategies for advanced theranostics. Theranostics 2014, 4, 660–77. 2. Gabizon, A.; Peretz, T.; Sulkes, A.; Amselem, S.; Ben-Yosef, R.; Ben-Baruch, N.; Catane, R.; Biran, S.; Barenholz, Y. Systemic administration of doxorubicin-containing liposomes in cancer patients: A phase I study. Eur. J. Cancer Clin. Oncol. 1989, 25, 1795–803. 3. Panahadjopoulos, D.; Allen, T.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S.; Lee, K.; Woodle, M.; Lasic, D.; Redemann, C.; et al. Sterically stabilized liposomes: Improvements in pharmacokinetics and anti-tumor therapy efficacy. Proc. Natl. Acad. Sci. 1991, 88, 11460–11464. 4. Gabizon, A.; Catane, R.; Uziely, B.; Kaufman, B.; Safra, T.; Cohen, R.; Martin, F.; Huang, A.; Barenholz, Y. Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes. Cancer Res. 1994, 54, 987–992. 5. Cabanes, A.; Tzemach, D.; Goren, D.; Horowitz, A.; Gabizon, A. Comparative study of the antitumor activity of free doxorubicin and polyethylene glycol-coated liposomal doxorubicin in a mouse lymphoma mode. Clin. Cancer Res. 1998, 4, 499–505. 6. Suzuki, S.; Watanabe, S.; Uno, S.; Tanaka, M.; Masuko, T.; Hashimoto, Y. Endocytosis does not necessarily augment the cytotoxicity of adriamycin encapsulated in immunoliposomes. Biochim. Biophys. Acta 1994, 1224, 445–453. 7. Abes, R.; Arzumanov, A. A.; Moulton, H. M.; Abes, S.; Ivanova, G. D.; Iversen, P. L.; Gait, M. J.; Lebleu, B. Cell-penetrating-peptide-based delivery of oligonucleotides: An overview. Biochem. Soc. Trans. 2007, 35, 775–779. 8. Wiethoff, C. M.; Middaugh, C. R. Barriers to nonviral gene delivery. J. Pharm. Sci. 2003, 92, 203–217. 9. El-Sayed, A.; Khalil, I. A.; Kogure, K.; Futaki, S.; Harashima, H. Octaarginine- and octalysine-modified nanoparticles have different modes of endosomal escape. J. Biol. Chem. 2008, 283, 23450–23461. 10. Kobayashi, S.; Nakase, I.; Kawabata, N.; Yu, H. H.; Pujals, S.; Imanishi, M.; Giralt, E.; Futaki, S. Cytosolic targeting of macromolecules using a pH-dependent fusogenic peptide in combination with cationic liposomes. Bioconjugate Chem. 2009, 20, 953–959. 11. Hatakeyama, H.; Ito, E.; Akita, H.; Oishi, M.; Nagasaki, Y.; Futaki, S.; Harashima, H. A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo. J. Controlled Release 2009, 139, 127–132. 12. Rosenholm, J. M.; Peuhu, E.; Eriksson, J. E.; Sahlgren, C.; Linden, M. Targeted intracellular delivery of hydrophobic agents using mesoporous hybrid silica nanoparticles as carrier systems. Nano Lett. 2009, 9, 3308–3311.

203 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

13. Wang, X. L.; Xu, R.; Lu, Z. R. A peptide-targeted delivery system with pH-sensitive amphiphilic cell membrane disruption for efficient receptormediated siRNA delivery. J. Controlled Release 2009, 134, 207–213. 14. Adler, A. F.; Leong, K. W. Emerging links between surface nanotechnology and endocytosis: Impact on nonviral gene delivery. Nano Today 2010, 5, 553–569. 15. Dehousse, V.; Garbacki, N.; Colige, A.; Evrard, B. Development of pH-responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for sirna delivery. Biomaterials 2010, 31, 1839–1849. 16. Shim, M. S.; Kwon, Y. J. Acid-transforming polypeptide micelles for targeted nonviral gene delivery. Biomaterials 2010, 31, 3404–3413. 17. Lehto, T.; Simonson, O. E.; Mager, I.; Ezzat, K.; Sork, H.; Copolovici, D. M.; Viola, J. R.; Zaghloul, E. M.; Lundin, P.; Moreno, P. M.; et al. A peptidebased vector for efficient gene transfer in vitro and in vivo. Mol. Ther. 2011, 19, 1457–1467. 18. Liu, J.; Jiang, Z.; Zhou, J.; Zhang, S.; Saltzman, W. M. Enzyme-synthesized poly(amine-co-esters) as nonviral vectors for gene delivery. J. Biomed. Mater. Res. A 2011, 96, 456–65. 19. Curcio, A.; Marotta, R.; Riedinger, A.; Palumberi, D.; Falqui, A.; Pellegrino, T. Magnetic pH-responsive nanogels as multifunctional delivery tools for small interfering rna (sirna) molecules and iron oxide nanoparticles (ionps). Chem. Commun. (Camb.) 2012, 48, 2400–2402. 20. Shrestha, R.; Elsabahy, M.; Florez-Malaver, S.; Samarajeewa, S.; Wooley, K. L. Endosomal escape and sirna delivery with cationic shell crosslinked knedel-like nanoparticles with tunable buffering capacities. Biomaterials 2012, 33, 8557–8568. 21. Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoter, M.; et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638–646. 22. Peetla, C.; Jin, S.; Weimer, J.; Elegbede, A.; Labhasetwar, V. Biomechanics and thermodynamics of nanoparticle interactions with plasma and endosomal membrane lipids in cellular uptake and endosomal escape. Langmuir 2014, 30, 7522–7532. 23. Ahmad, A.; Ranjan, S.; Zhang, W.; Zou, J.; Pyykko, I.; Kinnunen, P. K. Novel endosomolytic peptides for enhancing gene delivery in nanoparticles. Biochim. Biophys. Acta 2015, 1848, 544–553. 24. Ortega, R. A.; Barham, W. J.; Kumar, B.; Tikhomirov, O.; McFadden, I. D.; Yull, F. E.; Giorgio, T. D. Biocompatible mannosylated endosomal-escape nanoparticles enhance selective delivery of short nucleotide sequences to tumor associated macrophages. Nanoscale 2015, 7, 500–510. 25. Li, J.; Chen, Y. C.; Tseng, Y. C.; Mozumdar, S.; Huang, L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J. Controlled Release 2010, 142, 416–421.

204 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

26. Li, J.; Yang, Y.; Huang, L. Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor. J. Controlled Release 2012, 158, 108–114. 27. Lanza, G. M.; Yu, X.; Winter, P. M.; Abendschein, D. R.; Karukstis, K. K.; Scott, M. J.; Chinen, L. K.; Fuhrhop, R. W.; Scherrer, D. E.; Wickline, S. A. Targeted antiproliferative drug delivery to vascular smooth muscle cells with a magnetic resonance imaging nanoparticle contrast agent: Implications for rational therapy of restenosis. Circulation 2002, 106, 2842–2847. 28. Partlow, K.; Lanza, G.; Wickline, S. Exploiting lipid raft transport with membrane targeted nanoparticles: A strategy for cytosolic drug delivery. Biomaterials 2008, 29, 3367–3375. 29. Soman, N. R.; Lanza, G. M.; Heuser, J. M.; Schlesinger, P. H.; Wickline, S. A. Synthesis and characterization of stable fluorocarbon nanostructures as drug delivery vehicles for cytolytic peptides. Nano Lett. 2008, 8, 1131–1136. 30. Offodile, R.; Walton, T.; Lee, M.; Stiles, A.; Nguyen, M. Regression of metastatic breast cancer in a patient treated with the anti-angiogenic drug TNP-470. Tumori 1999, 85, 51–53. 31. Herbst, R. S.; Madden, T. L.; Tran, H. T.; Blumenschein, G. R., Jr.; Meyers, C. A.; Seabrooke, L. F.; Khuri, F. R.; Puduvalli, V. K.; Allgood, V.; Fritsche, H. A., Jr.; et al. Safety and pharmacokinetic effects of TNP-470, an angiogenesis inhibitor, combined with paclitaxel in patients with solid tumors: Evidence for activity in non-small-cell lung cancer. J. Clin. Oncol. 2002, 20, 4440–4447. 32. Logothetis, C. J.; Wu, K. K.; Finn, L. D.; Daliani, D.; Figg, W.; Ghaddar, H.; Gutterman, J. U. Phase i trial of the angiogenesis inhibitor TNP-470 for progressive androgen-independent prostate cancer. Clin. Cancer Res. 2001, 7, 1198–203. 33. Winter, P. M.; Schmieder, A. H.; Caruthers, S. D.; Keene, J. L.; Zhang, H.; Wickline, S. A.; Lanza, G. M. Minute dosages of alpha(nu)beta3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits. FASEB J. 2008, 22, 2758–2767. 34. Winter, P.; Neubauer, A.; Caruthers, S.; Harris, T.; Robertson, J.; Williams, T.; Schmieder, A.; Hu, G.; Allen, J.; Lacy, E.; et al. Endothelial alpha(nu)beta(3)-integrin targeted fumagillin nanoparticles inhibit angiogenesis in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2006, 26, 2103–2109. 35. Zhou, H. F.; Chan, H. W.; Wickline, S. A.; Lanza, G. M.; Pham, C. T. Alphavbeta3-targeted nanotherapy suppresses inflammatory arthritis in mice. FASEB J. 2009, 23, 2978–2985. 36. Bhargava, P.; Marshall, J. L.; Rizvi, N.; Dahut, W.; Yoe, J.; Figuera, M.; Phipps, K.; Ong, V. S.; Kato, A.; Hawkins, M. J. A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin Cancer Res 1999, 5, 1989–1995. 37. Kudelka, A. P.; Verschraegen, C. F.; Loyer, E. Complete remission of metastatic cervical cancer with the angiogenesis inhibitor TNP-470. N. Engl. J. Med. 1998, 338, 991–992. 205 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

38. Lanza, G. M.; Yu, X.; Winter, P. M.; Abendschein, D. R.; Karukstis, K. K.; Scott, M. J.; Chinen, L. K.; Fuhrhop, R. W.; Scherrer, D. E.; Wickline, S. A. Targeted antiproliferative drug delivery to vascular smooth muscle cells with a magnetic resonance imaging nanoparticle contrast agent: Implications for rational therapy of restenosis. Circulation 2002, 106, 2842–2847. 39. Pan, D.; Sanyal, N.; Schmieder, A. H.; Senpan, A.; Kim, B.; Yang, X.; Hu, G.; Allen, J. S.; Gross, R. W.; Wickline, S. A.; et al. Antiangiogenic nanotherapy with lipase-labile Sn-2 fumagillin prodrug. Nanomedicine (Lond.) 2012, 7, 1507–1519. 40. Pan, D.; Schmieder, A. H.; Wang, K.; Yang, X.; Senpan, A.; Cui, G.; Killgore, K.; Kim, B.; Allen, J. S.; Zhang, H.; et al. Anti-angiogenesis therapy in the Vx2 rabbit cancer model with a lipase-cleavable sn 2 taxane phospholipid prodrug using alpha(v)beta(3)-targeted theranostic nanoparticles. Theranostics 2014, 4, 565–578. 41. Wymer, N.; Gerasimov, O.; Thompson, D. Cascade liposomal triggering: Light-induced Ca2+ release from diplasmenylcholine liposomes triggers PLA2-catalyzed hydrolysis and contents leakage from DPPC liposomes. Bioconjugate Chem. 1998, 9, 305–308. 42. Davidsen, J.; Jøgensen, K.; Andresen, T. L.; Mouritsen, O. G. Secreted phospholipase A2 as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue. Biochim. Biophys. Acta, Biomembr. 2003, 1609, 95–101. 43. Andresen, T. L.; Davidsen, J.; Begtrup, M.; Mouritsen, O. G.; Jørgensen, K. Enzymatic release of antitumor ether lipids by specific phospholipase A2 activation of liposome-forming prodrugs. J. Med. Chem. 2004, 47, 1694–1703. 44. Jensen, S. S.; Andresen, T. L.; Davidsen, J.; Høyrup, P.; Shnyder, S. D.; Bibby, M. C.; Gill, J. H.; Jørgensen, K. Secretory phospholipase a2 as tumorspecific trigger for targeted delivery of a novel class of liposomal prodrug anticancer etherlipids. Mol. Cancer Ther. 2004, 3, 1451–1458. 45. Andresen, T. L.; Jørgensen, K. Synthesis and membrane behavior of a new class of unnatural phospholipid analogs useful as phospholipase A2 degradable liposomal drug carriers. Biochim. Biophys. Acta, Biomembr. 2005, 1669, 1–7. 46. Andresen, T. L.; Jensen, S. S.; Madsen, R.; Jørgensen, K. Synthesis and biological activity of anticancer ether lipids that are specifically released by phospholipase a2 in tumor tissue. J. Med. Chem. 2005, 48, 7305–7314. 47. Peters, G.; Møller, M.; Jørgensen, K.; Rönnholm, P.; Mikkelsen, M.; Andresen, T. Secretory phospholipase A2 hydrolysis of phospholipid analogues is dependent on water accessibility to the active site. J. Am. Chem. Soc. 2007, 129, 5451–5461. 48. Kaasgaard, T.; Andresen, T. L.; Jensen, S. S.; Holte, R. O.; Jensen, L. T.; Jørgensen, K. Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery. Chem. Phys. Lipids 2009, 157, 94–103.

206 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

49. Linderoth, L.; Peters, G.; Madsen, R.; Andresen, T. Drug delivery by an enzyme-mediated cyclization of a lipid prodrug with unique bilayer-formation properties. Angew. Chem., Int, Ed. 2009, 48, 1823–1826. 50. Pedersen, P.; Christensen, M.; Ruysschaert, T.; Linderoth, L.; Andresen, T. L.; Melander, F.; Mouritsen, O.; Madsen, R.; Clausen, M. Synthesis and biophysical characterization of chlorambucil anticancer ether lipid prodrugs. J. Med. Chem. 2009, 52, 3408–3415. 51. Pedersen, P.; Adolph, S.; Subramanian, A.; Arouri, A.; Andresen, T.; Mouritsen, O.; Madsen, R.; Madsen, M.; Peters, G.; Clausen, M. Liposomal formulation of retinoids designed for enzyme triggered release. J. Med. Chem. 2010, 53, 3782–3792. 52. Madsen, J.; Linderoth, L.; Subramanian, A.; Andresen, T.; Peters, G. Secretory phospholipase A2 activity toward diverse substrates. J. Phys. Chem. B 2011, 115, 6853–6861. 53. Winter, P. M.; Caruthers, S. D.; Kassner, A.; Harris, T. D.; Chinen, L. K.; Allen, J. S.; Lacy, E. K.; Zhang, H.; Robertson, J. D.; Wickline, S. A.; et al. Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel alpha(nu)beta3-targeted nanoparticle and 1.5 Tesla magnetic resonance imaging. Cancer Res. 2003, 63, 5838–5843. 54. Schmieder, A. H.; Winter, P. M.; Williams, T. A.; Allen, J. S.; Hu, G.; Zhang, H.; Caruthers, S. D.; Wickline, S. A.; Lanza, G. M. Molecular MR imaging of neovascular progression in the Vx2 tumor with alphavbeta3-targeted paramagnetic nanoparticles. Radiology 2013, 268, 470–480. 55. Zhang, R.; Pan, D.; Cai, X.; Yang, X.; Senpan, A.; Allen, J. S.; Lanza, G. M.; Wang, L. V. Alphanubeta(3)-targeted copper nanoparticles incorporating an sn 2 lipase-labile fumagillin prodrug for photoacoustic neovascular imaging and treatment. Theranostics 2015, 5, 124–133. 56. Pan, D.; Pramanik, M.; Senpan, A.; Allen, J. S.; Zhang, H.; Wickline, S. A.; Wang, L. V.; Lanza, G. M. Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons. FASEB J. 2011, 25, 875–882. 57. Jain, R. Normalizing tumor vasculature with anti-angiogenic therapy: A new paradigm for combination therapy. Nat Med 2001, 7, 987–989. 58. Jain, R. K.; Finn, A. V.; Kolodgie, F. D.; Gold, H. K.; Virmani, R. Antiangiogenic therapy for normalization of atherosclerotic plaque vasculature: A potential strategy for plaque stabilization. Nat. Clin. Pract. Cardiovasc. Med. 2007, 4, 491–502. 59. Jain, R. K. Taming vessels to treat cancer. Sci. Am. 2008, 298, 56–63. 60. Monach, P.; Hattori, K.; Huang, H.; Hyatt, E.; Morse, J.; Nguyen, L.; OrtizLopez, A.; Wu, H. J.; Mathis, D.; Benoist, C. The K/BXN mouse model of inflammatory arthritis: Theory and practice. Methods Mol. Med. 2007, 136, 269–282. 61. Zhou, H. F.; Yan, H.; Senpan, A.; Wickline, S. A.; Pan, D.; Lanza, G. M.; Pham, C. T. Suppression of inflammation in a mouse model of rheumatoid arthritis using targeted lipase-labile fumagillin prodrug nanoparticles. Biomaterials 2012, 33, 8632–8640. 207 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

62. Bernier, S. G.; Lazarus, D. D.; Clark, E.; Doyle, B.; Labenski, M. T.; Thompson, C. D.; Westlin, W. F.; Hannig, G. A methionine aminopeptidase-2 inhibitor, PPI-2458, for the treatment of rheumatoid arthritis. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 10768–10773. 63. Bainbridge, J.; Madden, L.; Essex, D.; Binks, M.; Malhotra, R.; Paleolog, E. M. Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: Potential role for angiogenesis inhibition. Arthritis Res. Ther. 2007, 9. 64. Zhou, H. F.; Yan, H.; Hu, Y.; Springer, L. E.; Yang, X.; Wickline, S. A.; Pan, D.; Lanza, G. M.; Pham, C. T. Fumagillin prodrug nanotherapy suppresses macrophage inflammatory response via endothelial nitric oxide. ACS Nano 2014, 8, 7305–7317. 65. Edwards, B.; Ward, E.; Kohler, B.; Eheman, C.; Zauber, A.; Anderson, R.; Jemal, A.; Schymura, M.; Lansdorp-Vogelaar, I.; Seeff, L.; et al. Annual report to the nation on the status of cancer, 1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer 2010, 116, 544–573. 66. Kuehl, W. M. Mouse models can predict cancer therapy. Blood 2012, 120, 238–40. 67. Kuehl, W. M.; Bergsagel, P. L. Myc addiction: A potential therapeutic target in mm. Blood 2012, 120, 2351–2352. 68. Murre, C.; Mccaw, P. S.; Baltimore, D. A new DNA-binding and dimerization motif in immunoglobulin enhancer binding, daughterless, myod, and myc proteins. Cell 1989, 56, 777–783. 69. Amati, B.; Brooks, M. W.; Levy, N.; Littlewood, T. D.; Evan, G. I.; Land, H. Oncogenic activity of the c-MYC protein requires dimerization with MAX. Cell 1993, 72, 233–245. 70. Freytag, S. O.; Dang, C. V.; Lee, W. M. Definition of the activities and properties of c-MYC required to inhibit cell differentiation. Cell Growth Differ. 1990, 1, 339–343. 71. Smith, M. J.; Charron-Prochownik, D. C.; Prochownik, E. V. The leucine zipper of c-MYC is required for full inhibition of erythroleukemia differentiation. Mol. Cell. Biol. 1990, 10, 5333–5339. 72. Chen, J. P.; Lin, C.; Xu, C. P.; Zhang, X. Y.; Fu, M.; Deng, Y. P.; Wei, Y.; Wu, M. Molecular therapy with recombinant antisense c-MYC adenovirus for human gastric carcinoma cells in vitro and in vivo. J. Gastroenterol. Hepatol. 2001, 16, 22–28. 73. Lewis, D. L.; Hagstrom, J. E.; Loomis, A. G.; Wolff, J. A.; Herweijer, H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat .Genet. 2002, 32, 107–108. 74. Berg, T.; Cohen, S. B.; Desharnais, J.; Sonderegger, C.; Maslyar, D. J.; Goldberg, J.; Boger, D. L.; Vogt, P. K. Small-molecule antagonists of myc/ max dimerization inhibit MYC-induced transformation of chicken embryo fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 3830–3835. 75. Kiessling, A.; Sperl, B.; Hollis, A.; Eick, D.; Berg, T. Selective inhibition of c-MYC/MAX dimerization and DNA binding by small molecules. Chem. Biol. 2006, 13, 745–751. 208 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by NORTH CAROLINA STATE UNIV on December 30, 2017 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch008

76. Bagnasco, L.; Tortolina, L.; Biasotti, B.; Castagnino, N.; Ponassi, R.; Tomati, V.; Nieddu, E.; Stier, G.; Malacarne, D.; Parodi, S. Inhibition of a protein-protein interaction between ini1 and c-MYC by small peptidomimetic molecules inspired by helix-1 of c-MYC: Identification of a new target of potential antineoplastic interest. FASEB J. 2007, 21, 1256–1263. 77. Clausen, D. M.; Guo, J.; Parise, R. A.; Beumer, J. H.; Egorin, M. J.; Lazo, J. S.; Prochownik, E. V.; Eiseman, J. L. In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of 10074-G5, a novel small-molecule inhibitor of c-MYC/MAX dimerization. J. Pharmacol. Exp. Ther. 2010, 335, 715–727. 78. Yin, X.; Giap, C.; Lazo, J. S.; Prochownik, E. V. Low molecular weight inhibitors of MYC-MAX interaction and function. Oncogene 2003, 22, 6151–6159. 79. Pan, D.; Kim, B.; Hu, G.; Gupta, D. S.; Senpan, A.; Yang, X.; Schmieder, A.; Swain, C.; Wickline, S. A.; Tomasson, M. H.; et al. A strategy for combating melanoma with oncogenic c-MYC inhibitors and targeted nanotherapy. Nanomedicine (Lond.) 2015, 10, 241–251. 80. Soodgupta, D.; Pan, D.; Cui, G.; Senpan, A.; Yang, X.; Lu, L.; Weilbaecher, K. N.; Prochownik, E. V.; Lanza, G. M.; Tomasson, M. H. Small molecule MYC inhibitor conjugated to integrin-targeted nanoparticles extends survival in a mouse model of disseminated multiple myeloma. Mol. Cancer Ther. 2015, 14, 1286–1294. 81. Fowler, J. A.; Mundy, G. R.; Lwin, S. T.; Lynch, C. C.; Edwards, C. M. A murine model of myeloma that allows genetic manipulation of the host microenvironment. Dis. Models Mech. 2009, 2, 604–611. 82. Radl, J.; Croese, J. W.; Zurcher, C.; Van den Enden-Vieveen, M. H.; de Leeuw, A. M. Animal model of human disease. Multiple myeloma. Am. J. Pathol. 1988, 132, 593–597. 83. Radl, J.; De Glopper, E. D.; Schuit, H. R.; Zurcher, C. Idiopathic paraproteinemia. Ii. Transplantation of the paraprotein-producing clone from old to young C57BL/Kalwrij mice. J. Immunol. 1979, 122, 609–613.

209 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.