supramolecular nanoparticles that target phosphoinositide...

Therapeutics, Targets, and Chemical Biology

Supramolecular Nanoparticles That TargetPhosphoinositide-3-Kinase Overcome Insulin Resistanceand Exert Pronounced Antitumor Efficacy

Ashish A. Kulkarni1,2,3,5, Bhaskar Roy1, Poornima S. Rao1, Gregory A. Wyant4, Ayaat Mahmoud1,Madhumitha Ramachandran1, Poulomi Sengupta1,10, Aaron Goldman1,2,5, Venkata Ramana Kotamraju7,8,Sudipta Basu1,3,9, Raghunath A. Mashelkar1,10, Erkki Ruoslahti7,8, Daniela M. Dinulescu4,5, andShiladitya Sengupta1,2,3,5,6

AbstractThe centrality of phosphoinositide-3-kinase (PI3K) in cancer etiology is well established, but clinical

translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intratumoral concentration,and an insulin resistance "class effect." This study was designed to explore the use of supramolecularnanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations.PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supra-molecular nanoassembly with L-a-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phos-phoethanolamine-N-[amino(polythylene glycol)]. The supramolecular nanoparticles (SNP) that were assem-bled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy wasquantified in vivo using 4T1 breast cancer and K-RasLSL/þ/Ptenfl/fl ovarian cancer models, with effects onglucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogatemolecules to engineer the SNPs highlighted the need to keep design principles in perspective; specifically,potency of the active molecule and the linker chemistry were critical principles for efficacy, similar toantibody–drug conjugates. We found that the SNPs exerted a temporally sustained inhibition of phosphor-ylation of Akt, mTOR, S6K, and 4EBP in vivo. These effects were associated with increased antitumor efficacyand survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticlesurface with tumor-homing peptides. Notably, the use of SNPs abrogated the insulin resistance that has beenassociated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use ofsupramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering aparadigm for extension to other molecularly targeted therapeutics being explored for cancer treatment.Cancer Res; 73(23); 6987–97. �2013 AACR.

IntroductionAccording to the World Health Organization, mortality due

to cancer is expected to increase from 7.6 million in 2008 to 12million deaths in 2030 (1). To address this growing problem,two emerging paradigms driving the evolution of newer treat-ment strategies are as follows: (i) better understanding ofoncogenic drivers, leading to the development of molecularly"targeted" therapeutics (2, 3), and (ii) the use of nanotechnol-ogy to deliver cytotoxic drugs specifically to the tumor, result-ing in improved therapeutic index (4, 5). However, the interfacebetween these two paradigms, which can offer unique oppor-tunities for improving chemotherapeutic outcomes, currentlyremains largely underexplored. In this study, we demonstratethe potential advantages of bringing these two paradigmstogether through the rational design of supramolecular nano-particles (SNP) that target the phosphoinositide-3-kinase(PI3K) pathway.

The centrality of the PI3K family of lipid kinases in theetiology of cancer is well established (6). Of the 3 classes of

Authors' Affiliations: 1Laboratory for Nanomedicine, Division of Biomed-ical Engineering, Department ofMedicine, Brigham andWomen's Hospital;2Harvard–MIT Division of Health Sciences and Technology; 3Indo-US JointCenter for Nanobiotechnology, Cambridge; 4Department of Pathology,Brigham and Women's Hospital; 5Harvard Medical School, Boston; 6DanaFarber Cancer Institute, Brookline, Massachusetts; 7Cancer ResearchCenter, Sanford-BurnhamMedical Research Institute, La Jolla, San Diego;8Center for Nanomedicine, Department of Cell, Molecular and Develop-mental Biology, University of California, Santa Barbara, California; 9IndianInstitute for Science Education Research (IISER); and 10National ChemicalLaboratories, Pune, India

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

B. Roy, P.S. Rao, and G.A. Wyant contributed equally to this work.

Corresponding Authors: Shiladitya Sengupta and Ashish Kulkarni,Brigham and Women's Hospital, 65 Landsdowne Street, Room 317,Cambridge, MA 02139. Phone: 617-768-8994; Fax: 617-768-8595; E-mail:[email protected] and [email protected]; Sudipta Basu,Indian Institute of Science Education and Research (IISER), GarwareCircle, Sutarwadi, Pashan, Pune, Maharashtra, India, 411021. Phone:08888842206; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4477

�2013 American Association for Cancer Research.

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PI3K, class IA PI3K is most implicated in driving humancancers (7). PIK3CA and PIK3R1, which encode the p110acatalytic subunit and the regulatory p85a subunit of PI3K,respectively, are somatically mutated or amplified in multipleprimary cancers, including of breast and ovarian origins(7). Similarly, the lipid phosphatase PTEN, an inhibitor ofPI3K signaling, is a commonly inactivated tumor suppressor(8). Activation of this pathway can also occur upstream at thelevel of mutated or amplified tyrosine receptor kinase ordownstream through mutations of AKT and RAS (7). Con-sequently, small-molecule inhibitors that target PI3K path-way have emerged as an exciting area of research, and severalmolecules that either inhibit specific catalytic subunits (a, b,d, and g) of p110 or act as pan-PI3K inhibitors are currentlyin development (9). However, recent studies have implicatedp110a as also playing a predominant role in glucose homeo-stasis (10). Indeed, recent data from a phase I clinical studywith a pan-class I selective PI3K inhibitor (NVP-BKM120)showed dose-dependent hyperglycemia, possibly an exampleof a class effect consistent with PI3K inhibition (11). Fur-thermore, studies have reported that approximately 10-foldhigher concentration of PI3K inhibitors might be requiredto block phosphorylation of downstream pathway proteins(such as ribosomal protein S6) than that needed for inhibit-ing more proximal AKT phosphorylation (12). We rational-ized that a natural approach to overcome these challengesassociated with targeting the PI3K pathway is through theuse of nanotechnology.

Nanovectors capitalize on the unique leaky angioge-nic tumor vasculature to preferentially home to tumors,which coupled with impaired lymphatic drainage resultsin increased intratumoral drug concentrations (termed asthe enhanced permeation and retention or EPR effect;ref. 13). However, traditional processes for nanoformula-tion are often incompatible with physicochemical proper-ties of many chemotherapeutic agents, which can limitthe entrapment efficiency or introduce suboptimal re-lease kinetics. Indeed, our early attempts in entrappingLY294002, one of the earliest PI3K inhibitors, resulted insuboptimal loading efficiency that prevented translation toin vivo tumor efficacy studies (14). Similarly, in a recentstudy, wortmannin-encapsulated polymeric nanoparticleswere shown to act as a radiosensitizer (15), but suchformulations are limited by burst release, which compli-cate clinical translation. We rationalized that this can beaddressed using supramolecular nanochemistry (16), i.e.,evolution of complex nanostructures from molecularbuilding blocks interacting via noncovalent intermolecularforce (17, 18). Indeed, supramolecular nanochemistry is anemerging concept in cancer theranostics; for example, in arecent study, gandolinium (III)-encapsulated SNPs wereused in diagnosis of cancer metastasis (19). Here, we reportthat rational modification of PI3K inhibitors facilitatessupramolecular assembly in the nanoscale dimension.Such PI3K-targeting SNPs exhibit the desired pharmaco-dynamic profile with enhanced antitumor efficacy, and canemerge as a new paradigm in targeted molecular thera-peutics development.

Materials and MethodsDichloromethane (DCM), anhydrous DCM, methanol, cho-

lesterol, dimethylamino pyridine (DMAP), succinic anhydride,sodium sulfate, pyridine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), L-a-phosphatidylcholine, and sephadexG-25were purchased fromSigma-Aldrich (all analytical grades).PI103 and PI828 were obtained from Selleckchem and TocrisBiosciences, respectively. 1,2-distearoyl-sn-glycero-3 phospho-ethanolamine-N-[amino(polythylene glycol)2000], mini hand-held Extruder kit was purchased from Avanti Polar Lipids Inc.1H spectra were recorded on Bruker DPX 400 MHz spectro-meter. Chemical shifts are reported in d (ppm) units usingresidual 1H signals from deuterated solvents as references.Spectra were analyzed with Mest-Re-C Lite (MestrelabResearch) and/or XWinPlot (Bruker Biospin) softwares. Elec-trospray ionization mass spectra were recorded on a Micro-mass Q Tof 2 (Waters) and data were analyzed withMassLynx4.0 software (Waters). The, 4T1 and MDA-MB-231s cell lineswere obtained from American Type Culture Collection andused within 6 months of resuscitation of frozen stock.

Synthesis of PI103–cholesterol conjugateCholesterol (500 mg, 1.29 mmol) was dissolved in 5 mL of

anhydrous pyridine. Succinic anhydride (645 mg, 6.45 mmol)and catalytic amount of DMAP was added to the reactionmixture to form a clear solution. The reaction mixture wasstirred under argon atmosphere for 12 hours. Pyridinewas thenremoved under vacuum and the crude residue was diluted in30 mL DCM. It was washed with 1N HCl (30 mL) and water(30 mL), and the organic layer was separated and dried overanhydrous sodium sulfate, filtered, and concentrated in vacuo.Completion of the reaction was confirmed by thin layer chro-matography (TLC) in 1:99 methanol:DCM solvent mixtures.PI103 (25 mg, 0.072 mmol) was dissolved in 3 mL anhydrousDCM followed by addition of cholesterol–succinic acid (0.216mmol, 105 mg), EDC (0.216 mmol, 41.4 mg), and DMAP (0.216mmol, 26 mg). The reaction mixture was stirred at roomtemperature for 12 hours under argon. Upon completion ofreaction asmonitoredbyTLC, the reactionmixturewas dilutedwith 10 mL DCM and washed with dilute HCl and water. Theorganic layers were separated, combined, and dried overanhydrous sodium sulfate. The solvent was evaporated undervacuum and the crude product was purified by using columnchromatography, eluting with methanol:methylene chloridegradient, to give PI103–cholesterol conjugate as a light yellowsolid (52mg, 90%). 1H-NMR (CDCl3, 400MHz): d 8.65 to 8.53 (m,1H), 8.36 (d, J¼ 8.3 Hz, 1H), 8.19 (d, J¼ 1.7 Hz, 1H), 7.56 to 7.41(m, 1H), 5.29 (s, 1H), 4.28 to 4.15 (m, 2H), 3.97 to 3.86 (m, 2H),3.64 (s, 1H), 2.93 (d, J¼ 7.0 Hz, 1H), 2.76 (d, J¼ 7.0 Hz, 1H), 2.35(s, 1H), 2.17 (s, 1H), 1.59 (s, 4H), 1.29 (d, J ¼ 34.2 Hz, 3H), 1.25to 1.23 (m, 6H), 1.13 to 0.80 (m, 13H), 0.66 (s, 2H), and 0.03(m, 12H). High resolution mass spectrometry calculated for[C50H64N4O6þH]þ:817.4899 found: 817.4883.

Synthesis of PI828–cholesterol conjugatePI828 [28 mg (0.088 mmol) dissolved in 2.0 mL of dry

DCM] was added to 20.0 mg (0.044 mmol) of cholesteryl

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chloroformate (dissolved in 2.0 mL dry DCM). Finally, 15.5 mL(0.088mmol) of dryN,N-Diisopropylethylaminewas added to itdrop-wise at room temperature in an inert condition. Progressof the reaction was monitored by TLC. After 24 hours, it wasquenched with 100 mL 0.1N HCl and the compound wasextracted in DCM. The desired product was separated bycolumn chromatography using a solvent gradient of 0% to5% MeOH in DCM. 1H-NMR (300 MHz) d (ppm) ¼ 8.165–8.13(m); 7.59 to 7.40 (m, aromatic); 6.72 (s); 5.98 to 5.93 (m); 5.42to 5.40 (m); 4.67 to 4.59 (m); 3.75 to 3.74 (m); 3.44 to 3.40 (m);2.43 to 2.34 (m); 2.04 to 1.93 (m); 1.86 to 1.77 (m); 1.65 to 1.43 (m);1.35 to 1.43 (m); and 1.32 to 0.85 (m).

Synthesis and characterization of SNPsDrug–cholesterol conjugates, L-a-phosphatidylcholine, and

DSPE-PEG2000 (at optimized weight ratios) were dissolved in1.0 mL DCM. Resulting solutions were evaporated in a round-bottomed flask with the help of a rotary evaporator andthoroughly dried. The resulting thin films were hydrated withPBS with constant rotation at 55�C for 2 hours. Nanoparticleswere eluted through a Sephadex column and extruded through200 nm pores. The size was checked using dynamic lightscattering (DLS), and drug loading was determined by spec-troscopy. For release kinetics studies, the drug-loaded nano-particles (1 mg drug/mL; 5 mL) were suspended in PBS buffer(pH 7.4), 4T1 or 4306 cell lysates within a dialysis tube (MWCO¼ 3,500 Da; Spectrum Lab). The dialysis tube was suspended in1 L PBS pH 7.4 with gentle stirring to simulate infinite sink tankcondition. A 100 mL portion of the aliquot was collected fromthe sample at predetermined time intervals and replaced byequal volume of PBS buffer, and the released drug was quan-tified by spectrometry. More details are available in Supple-mentary Data.

In vitro assaysThe 4T1 and MDA MB 468 breast cancer cells were cultur-

ed in RPMI, whereas 4306 ovarian cancer cells were culturedin Dulbecco's Modified Eagle Medium (supplemented with10% FBS and 1% of antibiotic–antimycotic 100� solution).Cells (4 � 103) were seeded into 96-well flat-bottomed plates,and incubated with free drug or drug-loaded nanoparticles(normalized to equivalent amounts of free drug) for desiredtime periods. Cell viability was quantified using the CellTiter96 Aqueous One Solution assay (Promega). To study druginternalization, 4T1 breast cancer cells were incubated withfree PI103 or PI103-SNPs (with equivalent amount of PI103) for4 hours, then washed and incubated in fresh media. Afterdesired time of incubation, cells were lysed, and drug con-centrations quantified using spectroscopy.

Murine 4T1 breast cancer modelThe 4T1 breast cancer cells (1 � 106) were implanted sub-

cutaneously in the flanks of 4-week-old BALB/c mice. The drugtherapy was started on day 9. Animals were randomized intothe following treatment groups: (i) vehicle, (ii) free drug(5 mg/kg), and (iii) SNPs (at dose equivalent to 5 mg/kg ofthe PI3K inhibitor). In a separate experiment, we includedan additional group treated with iRGD-PI103-SNPs to test for

effect of active targeting on efficacy. Animals were dosed every48 hours. The tumors were measured regularly, tumor volume(Vt) was calculated using the formula, L� B2=2, and relativetumor volume was calculated using the formula Vt=V0 (whereV0 was tumor volume at the time of first injection). In a separatestudy, we monitored survival of 4T1 tumor-bearing mice andtreatmentswereperformed asdescribed above. Animals (n¼ 10in eachgroup)were sacrificed as soonas they reachedmoribundstate, which is attained before reaching tumor volume cutoff inthismodel. All animal procedureswere approved by theHarvardInstitutional Use and Care of Animals Committee.

Insulin tolerance test using PI103-SNPsRandom fed mice (with 4T1 breast cancer) were injected

with a single dose of empty nanoparticles (control), free PI103or PI828 and PI828-SNP or PI103-SNPs (at doses equivalent to5 mg/kg of the parent inhibitor molecule) via the tail vein. Themice were injected with freshly prepared insulin solution (0.75U/kg) in 0.1 mL 0.9% NaCl at defined time points after drugadministration. Blood glucose levelsweremeasured before and45 minutes after insulin injections using a glucometer.

Efficacy study of PI103-SNPs in murine ovarian cancertumor model

Ovarian adenocarcinomas were induced in genetically engi-neered K-rasLSL/þ/Ptenfl/fl mice via intrabursal delivery ofadenovirus-carrying Cre recombinase (Adeno-Cre). Tumorcells were engineered to express luciferase activated byAdeno-Cre. Once mice developed medium to large tumors,they were placed into one of four groups, and treated withvehicle, free PI103, PI103-SNP, or iRGD-PI103-SNP at dosesequivalent to 5mg/kg of PI103. Tumor imaging was performedusing an IVIS Lumina II Imaging System. Quantification ofbioluminescence was achieved by using Living Image Software3.1 (Caliper Life Sciences). Images were taken a day beforeinitial treatment (day 0, baseline image), and the day after threeor five cycles of treatments. The 4306-cell line was establishedfrom these Cre-induced K-rasþ/Pten murine tumors in theDinulescu Laboratory, and genotyping was done periodicallyby PCR analysis of DNA for characterization.

Western blot analysisFor in vitro studies, 5� 104 cells were seeded in each well of a

6-well plate and incubated with free drug or SNPs (with equiv-alent amount of drug) for 24 hours. For in vivo studies, tumorstored in �80�C was pulverized in a mortar and pestle usingliquid nitrogen. Proteins were extracted with radioimmunopre-cipitation assay buffer. Protein lysates were fractionated byelectrophoresis, transferred to membranes, which were incubat-ed with antibodies against phosphorylated proteins, and probedwith horseradish peroxidase–conjugated secondary antibody.Detection was done using a G-box (Syngene), and densitometricquantification was done by ImageJ software. Expression wasnormalized to total expression of the specific protein or b-actin.

Tumor histocytochemistryTumor cryosections were directly imaged using a

Nikon TE2000 epifluorescence microscope for studying the

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localization of fluorescein amidite (FAM)-labeled iRGD-SNPs.Blood vessels were delineated using vonWillebrand factor(vWF) immunostaining. For studying apoptosis, formalin-fixedtumor sections were stained with a standard TMR-red fluo-rescent terminal deoxynucleotidyl tranferase–mediated dUTPnick end labeling (TUNEL) kit following the manufacturer'sprotocol (the In Situ Cell Death Detection Kit; TMR-Red;Roche).

StatisticsThe statistical analysis was done using a two-tailed Stu-

dent t test or one-way ANOVA followed by Newman–Keulspost hoc test, with P < 0.05 as the threshold for significance.

ResultsSynthesis and characterization of PI3K-inhibiting SNPs

We used two different PI3K inhibitors, the pyridofuropyr-imidine PI103, and PI828 (8-bromo-2-morpholin-4-yl-chro-men-4-one) to engineer the SNPs. PI828 is a derivative of theearlier generation and widely used PI3K inhibitor LY294002, inwhich an amine linker has been inserted in 4-position hydro-gen of the exocyclic phenyl substituent, enabling conjugationto cholesterol via a carbamate bond (Fig. 1A). Previous studieshave demonstrated that conjugation via this linker maintainsaffinity for the catalytic site of PI3K class I isoforms (20).However, PI828, like LY294002, is a weak inhibitor (20). Wetherefore included, PI103, which has been reported to exhibitexcellent potency in the low nanomolar range and selectivityfor class IA PI3Ks aswell asmTOR (12). However, PI103was notfound suitable for clinical development as the planar tricyclicstructure resulted in limited aqueous solubility and the phe-nolic hydroxyl group is rapidly glucoronidated (12). Theselimitations, however, made PI103 a suitable molecule to engi-neer the SNPs. As shown in Fig. 1B, the phenolic hydroxyl groupwas conjugated via an ester linkage to cholesterol–succinatecomplex. The intermediate and products were characterizedby 1H-NMR spectroscopy andmass spectrometry (Supplemen-tary Figs. S1–S3).

We engineered the SNPs from the cholesterol–PI828 orcholesterol–PI103 conjugates, phosphatidylcholine andDSPE-PEG2000 at optimized weight ratios using a lipid-filmhydration self-assembly method (Fig. 1C; ref. 21). The incor-poration efficiency for the cholesterol-PI828 SNPs was 43%,and 60% � 5% for PI103–cholesterol conjugate SNPs. Asshown in Fig. 1D, cholesterol–PI828 conjugates resultedin the formation of SNPs with hydrodynamic diameter of108 � 8.9 nm as determined by DLS (Fig. 1D). PI103 SNPsshowed a mean particle diameter of 172 � 1.8 nm (Fig. 1E).Ultrastructure analysis using cryo-transmission electronmicroscopy (cryo-TEM; Fig. 1F) revealed the formation ofpredominantly unilamellar structures 100 nm or less in diam-eter. The size difference between TEM and DLS measure-ments can be attributed to the hydration sphere arising fromthe PEG coating, which can facilitate the masking from thereticuloendothelial system (22). In addition, aliquots of thePI103-SNPs stored for a period of more than a month exhib-ited no changes in size and z potential, indicating that the

formulations were stable (Fig. 1G). Temporal release kineticsrevealed a sustained release of active drug in cell lysate(Fig. 1H and I), consistent with the cleavage of the linkers inacidic and enzymatic (esterase) conditions. Interestingly, therate of release of PI828 was significantly lower, consistent withthe more stable carbamate linker (cleaved by carboxyes-terases). As a control experiment, we tried engineering nano-particles using traditional approaches of nanoformulation,in which we entrapped PI103 in the lipid bilayer (Supplemen-tary Fig. S4A). Using the lipid ratio employed with the SNPsresulted in minimal incorporation efficiency of 2% PI103,which could be optimized by changing the compositionalratio (Supplementary Fig. S4B). A sustained release of PI103was observed from the formulation (Supplementary Fig. S4C),resulting in similar effects on cell viability (SupplementaryFig. S4D), and inhibition of Akt phosphorylation (Supplemen-tary Fig. S4E). However, light scattering studies revealed atemporal increase in the size of the nanoparticles (Supple-mentary Fig. S4F), leading to precipitation (instability; Sup-plementary Fig. S4G), consistent with the fact that currentlyavailable strategies for nanoformulation are not be compatiblewith many molecules, which has limited the repertoire ofnanomedicines.

In vitro efficacy of SNPsWe evaluated the efficacy of the SNPs in vitro using 4T1

(murine breast cancer), MDA-MB-468 (human breast cancer),and a PI3K-overexpressing 4306 (ovarian cancer) cell lines (Fig.2A–F). Temporal effect on cell viability and IC50 values areshown in Fig. 2I. Although we observed a decrease in thepotency following conjugation of the active molecules tocholesterol, this is consistent with a prodrug approach, inwhich the construct requires activation to the parent moleculefor efficacy. Western blot analysis showed that continuousincubation with both the free drug as well the SNP (at equi-molar concentrations of PI103) induced a sustained inhibitionof basal phosphorylation of Akt (Fig. 3A). Interestingly, on theother hand, a transient exposure more than 4 hours resulted ina rebound increase in phosphorylation of AKT in the case offree PI103, whereas SNP-PI103 inhibited Akt phosphorylationin a more sustained manner (Fig. 3B and C). Indeed, thetransient treatment resulted in an initial higher intracellularconcentration of PI103 in the cells treated with the free drugcompared with SNP-PI103. However, although the concentra-tion remained elevated in the PI103-SNP–treated cells, onlytraces of the drug were detected in the cells treated with thefree drug by 18 hours (Fig. 3D) in line with the need forconversion to active drug in the case of SNPs. Consistent withthe above observations, PI828-SNPs and free PI828 exhibitedsimilar cytotoxic effect on the 4T1 cells (Fig. 2G) and 4306 cells(Fig. 2H). The cells treated with PI103-SNPs and PI828-SNPsexhibited similar inhibition of Akt phosphorylation after36 hours of treatment (Fig. 3E).

Efficacy of SNP in an in vivo 4T1 breast cancer modelWenext investigated the antitumor efficacy of PI103-SNPs in

the 4T1-cell line, which is negative for estrogen receptor andprogesterone receptor, and expresses a low level of the mouse

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Her2/neu equivalent (23). Transplanted into syngeneic mice,the 4T1s form aggressive, highly metastatic breast cancers.Mutations in genes that constitute the PI3K pathway occur inmore than 70% of breast cancers (24). We have previouslydemonstrated that the 4T1 cells mount a survival response to

standard chemotherapy via an upregulation of PI3K signaling(25). As shown in Fig. 4A, treatment with PI103 resulted intumor growth inhibition relative to PBS-treated controls, but atumor rebound was observed after the treatment was stopped.In contrast, treatment with PI103-SNP resulted in a sustained

Figure 1. Synthesis and characterization of SNP. Synthetic scheme showing conjugation of PI828 (A) and PI103 (B) to cholesterol via carbamateand ester linkages, respectively. C, schematic representation shows assembly of SNPs from phosphatidylcholine, PI103-/PI828–cholesterolconjugates, and DSPE-PEG2000. D and E, the graphs show the distribution of hydrodynamic diameter of PI828-SNPs (D) and PI103-SNPs (E),measured using dynamic light scattering. F, high-resolution cryo-TEM image of PI103-SNPs (scale bar, 100 nm). G, the physical stability of PI103-SNPsduring storage condition at 4�C as measured by changes in size and z potential of nanoparticles. H and I, graphs show release kinetics of PI103from SNPs in PBS, pH 7.4, and 4T1 breast cancer cell lysate (H) and release kinetics of PI828 from SNPs in PBS, pH 7.4, 4T1 breast cancer cell lysate,and PI3K-overexpressing 4306 ovarian cancer cell line (I). Data represent mean � SEM (at least triplicates at each condition).






tumor growth inhibition over the study period. To test whethertargeting the nanoparticles to the tumor using "homing" pep-tides increases antitumor efficacy, a separate group of tumor-bearing mice were treated with PI103-SNPs that were surface-decorated with iRGD peptide. As shown in Fig. 4A, such atreatment resulted in greater tumor inhibition compared withSNPs that accumulated via passive uptake. Indeed, previousobservations have shown that iRGD-coated nanostructuresexhibit increased extravasation and tissue penetration in atumor-specific and neuropilin-1–dependent manner (26). Toelucidate the mechanism underlying the increased in vivoefficacy, the tumors were excised after treatment, and process-ed for TUNEL as a marker for apoptosis. As shown in Fig. 4BandC, treatmentwith PI103-SNPs resulted in greater apoptosisthan treatment with free PI103. Although we did observe anenhanced antitumor efficacy with iRGD-coated PI103-SNPs,stereologic analysis ofmultiple tumor sections revealed a stati-stically insignificant increase in the level of apoptosis com-pared with PI103-SNP. Epifluorescence imaging of tumorcross-sections did reveal intratumoral localization of FAM-labeled iRGD-coated PI103-SNPs (Fig. 4D).We next studied theeffect of treatment on survival of 4T1-bearing mice. The 4T1model is an aggressive form of breast tumor, and the animalsbecome moribund and have to be sacrificed before the tumorreaching maximum tumor cutoffs. Hence, the 4T1 syngeneicimplants serves as an excellent model for generating Kaplan–Meier survival curves. As shown in Fig. 4E, three cycles of PI103-SNP significantly increased the median survival by 2 days ascompared with free PI103 treatment (P < 0.05). Pharmacody-namic monitoring revealed that phosphorylation of Akt anddownstream signaling molecules mTOR and 4EBP were sig-nificantly inhibited in the PI103-SNP–treated tumors thanin the PI103-treated tumors (P < 0.05, Student t test; Fig. 4 F

and G). Treatment with PI828-SNPs (5mg/kg PI828 equivalent,3 doses) also exerted an inhibitory effect on Akt phosphory-lation in vivo translating into superior tumor growth inhibitionas compared with free PI828 (Supplementary Fig S5). However,consistent with its low potency, the antitumor efficacy of PI828or PI828-SNP was significantly lower than seen with PI103-SNPs. However, given that the both PI103-SNPs and PI828-SNPs did inhibit PI3K signaling, it is possible that the releasekinetics (rate of release) of the active agent plays a critical rolein efficacy and needs to be considered in the design of SNPs.

Efficacy of PI103-SNPs in an in vivo K-RasLSL/þ/Ptenfl/fl

ovarian cancer modelWe further evaluated the effect of PI103-SNP in a K-RasLSL/þ/

Ptenfl/fl ovarian cancer model (27). We selected this modelbecause tumors that lack Pten have been reported to beaddicted to PI3K signaling (7). On the other hand, tumorsthat present a mutated or activated Ras have been reportedto be less responsive to PI3K inhibitors (7). As shown in Fig.5A–C, bioluminescence quantification of tumor luciferasesignal indicates that free PI103, PI103-SNP, and iRGD-PI103-SNP resulted in significant tumor regression as comparedwith vehicle control. The antitumor response to iRGD-PI103-SNP was statistically significantly superior to free PI103 afterthree cycles of treatments as quantified by the biolumines-cence signal. This distinction between free drug and PI103-SNPs was attained only after five cycles of treatment, con-sistent with previous observations that iRGD facilitatesintratumoral penetrance and accumulation. No change inbody weight was observed in any treatment group (Fig. 5D).The expression levels of PI3K/mTOR pathway markers, asassessed by Western blot analysis of tumor samples fromdifferent groups, showed a significant decrease in the

Figure 2. Cell viability assays of PI3K-inhibiting SNPs. A and B,MTS assay showing the effect of free or PI103-SNPs at different concentrations on 4T1 cells at48hours (A) and72hours (B).C–F,MDA-MB-468cells at 48hours (C) and72hours (D), and4306cells at 48hours (E) and72hours (F).GandH, graphs show theeffect of treatment with PI828 or PI828-SNPs on viability of 4T1 breast cancer cells (G) or 4306 cells (H). I, the table shows IC50 of PI103 and PI103-SNP indifferent cell lines at 48 and 72 hours. Data represent mean � SEM (n ¼ 3; with at least triplicates in each independent experiment).

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expression of phospho-mTOR, phospho-AKT, phospho-S6,and phospho4EBP1 in the PI103-SNP and the iRGD-PI103-SNP–treated groups as compared with the free PI103-treatedtumors (Fig. 5E).

Effect of PI103-SNP on glucose homeostasisPI3K plays a central role in mediating insulin signaling

that is conserved throughout eukaryotic evolution. Wetherefore investigated the effect of PI103-SNPs on insulintolerance in a 4T1 breast cancer model. Consistent withprevious studies (28), mice injected with free PI103 exhibiteda transient tolerance to insulin compared with the signifi-cant insulin-induced decrease in blood glucose levelobserved in mice pretreated with PI103-SNP or with emptynanoparticles as a control (Fig. 6A). The insulin responsewas restored at later time points, consistent with previousstudies with PI103 (28), and can arise from the short half-lifeof PI103. Interestingly, we did not observe any change inglucose levels following PI103-SNP treatment comparedwith vehicle-treated controls over the study period, suggest-ing that the sustained release of PI103 from the nanopar-ticles and the increased efficacy did not manifest in adelayed onset of insulin resistance. Treatment with freePI828 or PI828-SNP did not affect glucose response to insulinat the doses used (Fig. 6B), consistent with the low potencyof the active agent as compared with PI103. Furthermore, we

analyzed tissue sections from liver, kidney, and spleen, threeorgans that are implicated in clearance of nanoparticles. Asshown in Fig. 6C, TUNEL study revealed no distinctionsbetween the different treatments. This was validated usingWestern blotting for monitoring the expression of phospho-mTOR as well as cleaved caspase-3 and PARP (as markers ofinduction of apoptosis), which revealed no differencesbetween free PI103- or PI103-SNP–treated animals (Fig. 6D).

DiscussionThe challenges faced in clinical translation of PI3K inhibitors

have highlighted the need to overcome feedback signaling andinsulin resistance "class effect," and to achieve high intratu-moral concentration of the drug (29). Here, we demonstratethat the SNPs can induce a sustained inhibition of the PI3K,resulting in potentially overcoming feedback signaling. Fur-thermore, this sustained inhibition of PI3K signaling results inincreased antitumor efficacy without insulin resistance, indi-cating that the use of supramolecular nanochemistry canemerge as a powerful strategy for overcoming the challengesfaced during clinical translation of PI3K inhibitors.

Indeed, our study demonstrates that an acute exposure tothe free drug (PI103) results in an increase in the phospho-Aktlevels at later time points. Such a rebound activation of thepathway is consistent with previous reports (29, 30), arising

Figure 3. In vitro characterizationof PI3K-inhibitingSNPs. A, representativeWestern blot analysis showsexpressionof phospho-AKTand total AKT in 4T1cellsat 3, 9, 24, and 48 hours after treatment with either 5 mmol/L of free PI103 or PI103-SNP. B and C, effect of acute treatment (4 hours incubation) withPI103 or PI103-SNP on PI3K activity over time. After 4 hours of exposure to drug, the cells were washed three times with cold PBS to remove additionaldrug outside cells and then incubated with fresh media with 1% FBS. Cells were collected at 0, 12, 24, 36, and 48 hours. PI103-SNPs inducedsustained inhibition of phosphorylation of Akt. D, graph showing internalization of free PI103 and PI103-SNP at 4 and 18 hours. The amount of druginternalizedwas quantifiedbyUV -vis spectroscopy. Data representmean�SEM fromat least three replicates; �,P <0.05; ��,P <0.01 (Student t test). E, effectof treatment with PI828 or PI828-SNP (5.0 and 7.0 mmol/L) on phospho-Akt levels at 36 hours after treatment.






from a homeostatic feedback loop via the upregulation ofreceptor tyrosine kinases (31). Interestingly, treatment withthe PI103-SNPs could potentially overcome this feedback loopas evident from a sustained inhibition of the phospho-Aktsignal. This could arise from an increase in the intracellularconcentration of PI103 with time, achieved with SNPs. Indeed,Western blot analysis of the in vivo tumor samples (extracted72 hours of administration of dose) revealed a robust PI3Ksignaling in tumors from animals that were treated with freePI103. In contrast, treatment with PI103-SNP resulted incomplete shutdown of the pathway, as evident from decreasedlevels of phosphorylated forms of Akt, S6K, 4E-BP1, or mTORin both tumor models (indeed PI828-NPs exerted a similarsustained inhibition on phosphorylation of Akt in vitro and

in vivo as compared with free PI828, but it is unlikely thatPI828-SNP will emerge as a potential drug candidate forreasons described later). Indeed, in a recent commentary,Courtney and colleagues had posed the question whetherlack of efficacy of PI3K inhibitors is due to inadequate inhi-bition of the target or because complete inhibition of thetarget is not sufficient to produce antitumor activity (31). Thecurrent results indicate that in addition to the level of inhibi-tion of the pathway, the temporality or kinetics of inhibitionmay be a critical element in determining antitumor outcome.Interestingly, a similar observation was made during theevolution of a current clinical candidate GDC-0941 from PI103,in which approximately 90% inhibition of Akt phosphoryla-tion for several hours was seen as a requirement for antitumor

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Figure 4. In vivo efficacy of PI103 or PI103-SNP in a syngenic 4T1 breast cancer BALB/c mice model. A, growth curves show effect of different multidosetreatments on tumor volume. Each animal was injected with three doses of either PBS (for control group), 5 mg/kg of free PI013, 5 mg/kg of PI103-SNP, oriRGD-tagged PI103-SNPs (at dose equivalent to PI103) on every alternate day. First day of treatment was considered as day 1. End point for each animalwas tumor size more than 2,000 cm3 or tumor ulceration or necrosis or animal death. Treatment with PI103-SNPs or iRGD-tagged PI103-SNP wasstatistically more effective than treatment with free PI103. Data represent mean� SEM; P < 0.01; ANOVA. Top, representative tumors from each group. B,representative epifluorescent images of tumor sections from animals treated as above were labeled for apoptosis using TUNEL (red) and counterstainedwith DAPI (blue). C, the quantification of apoptosis from the labeled tumor sections as a percentage of TUNELþ cells as a function of total nuclei. D,immunoflurescence image of the frozen section of tumor from either control or iRGD-tagged PI103-SNP, showing effective targeting with iRGD (iRGDpeptide was attached with FAM)-coated PI103-SNP; the section was counter-labeled for vWF (Texas Red), which delineates the vasculature. E,Kaplan–Meier survival curves show that treatment with PI103-SNP increases survival (P < 0.05) as compared with free PI103 (n ¼ 10 in each treatmentgroup). F, representative Western blot analysis shows the expression of phospho and total forms of mTOR, Akt, and 4EBP (normalized to actin) in tumors72 hours after a single dose injection of either free PI103 or PI103-SNP (at 5 mg/kg PI103 dose equivalent) in a syngenic 4T1 breast model. G,densitometric quantification from the above study. Data represent mean � SEM from n ¼ 3; �, P < 0.05; ��, P < 0.01; ��, P < 0.01.

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activity, establishing a link between pharmacokinetic expo-sure and pharmacodynamic biomarker changes (9). Indeed,this is validated by the fact that free inhibitors were found tobe more potent that the SNPs in vitro under conditions ofsustained exposure (as the latter has to be activated into theactive moiety), whereas SNPs were more effective in vivo,potentially arising from sustained intratumoral concentra-tions as compared with the free drug. An additional point tonote was that we did see a disconnect between the expressionlevels of phosphorylated Akt and downstream phosphorylatedproteins, such as S6K, 4E-BP1, in which although p-Akt wasobserved in some SNP-treated tumor tissues the downstream

proteins were still inhibited. This captures the kinetics offlow of information through a signaling cascade, potentiallydissected because of the temporal inhibition attained with theSNPs as opposed to an "all or none" inhibition seen withfree inhibitors. This also highlights the necessity to includethe distinct levels of PI3K signal transduction pathway, asopposed to monitoring only phosphorylation of Akt, as bio-markers for efficacy (32).

Clearly, the transient resistance to insulin observed withPI103, together with its short half-life and its ability to inhibittumor growth, suggests that insulin resistance could possibly beaCmax-driven effect, whereas the antitumor efficacy is a function

Figure 5. PI103-SNP inhibits tumor growth in a K-rasLSL/þ/Ptenfl/fl ovarian cancer model. A, representative pictures from the four treatment groups before andafter three and five treatments (n¼ 3). Tumor images were obtained using an IVIS Lumina II Imaging System. Quantification of bioluminescencewas achievedby using the Living Image Software 3.1. Mice received 150 mg/kg of D-luciferin firefly potassium salt via intraperitoneal injection before imaging. B,bioluminescence quantification indicates a significantly decreased tumor luciferase signal in mice treated with free PI103, PI103-SNP, and iRGDPI103-SNPcompared with vehicle (P < 0.05; one-way ANOVA analysis) after three treatments. Following five treatments, bioluminescence quantification indicatedresponse to PI103-SNP was significantly higher compared with free PI103 (P < 0.01; one-way ANOVA analysis). C, representative K-rasLSL/þ/Ptenfl/fl tumorsexcised from animals treated with free PI103, PI103-SNP, and iRGD-PI103-SNP. D, drug toxicity assessed by measurements in overall body weight.Daily recordings indicated no difference in body weight after five treatments of all treatment groups. E, PI3K/mTOR pathway markers were assessed inK-rasLSL/þ/Ptenfl/fl tumors treated with vehicle, 5 mg/kg free PI103, 5 mg/kg PI103-SNP, and 5 mg/kg iRGDPI103-SNP by Western blotting as described inMaterials and Methods. Inhibition of mTOR substrates by PI103-SNP and iRGD-PI103-SNP treatment was much greater in comparison with free drugin K-rasLSL/þ/Ptenfl/fl tumors. �, P < 0.05; ��, P < 0.01.






of sustained exposure (area under curve or AUC-driven effect).This is supported by the results with PI828, which showed noresistance to insulin at the dose used but did exert an inhibitoryeffect on tumor growth. This could also explain why PI103-SNPexerted a significant intratumoral PI3K inhibition, resulting inincreased antitumor efficacy (arising from a sustained release ofactive drug), but did not induce insulin tolerance, indicating thatthe SNPs approach can indeed overcome the current challengesassociated with PI3K inhibitors.

Indeed, the integration of supramolecular nanochemistrywith targeted therapeutics can open up new opportunities toharness the full potential of targeted therapeutics byenhancing the therapeutic index. As seen in the case ofPI103, it can potentially overcome limitations that hadprevented potent molecules from advancing further, andcan enable the rescue of "failed" drugs (12). Furthermore,this study reveals that the supramolecular nanochemistry–based approach can potentially enable the fabrication ofnanomedicines from drugs that are not compatible withcurrently used techniques of nanoformulation. Indeed, in arecent study, we had demonstrated that such SNPs ofcisplatin resulted in stable formulations, increased drugloading efficiency and enhanced delivery to the tumors asopposed to free cisplatin (16). However, the contrasts in theoutcomes with PI103- and PI828-SNPs also highlight theneed for keeping design principles in perspective whenengineering the SNPs. In some manner, this is similar tothe design principles for a successful antibody–drug conju-gate (33), in which selection of a potent active agent and the

optimal linker chemistry is critical to efficacy, with thenanoparticle enabling a preferential delivery to the tumor.

In summary, several components of the current approachcan facilitate future therapy in humans. First, the ability ofnanoparticles to accumulate in tumors can lead to increasedefficacy. Second, the sustained release resulting in prolongedinhibition of the PI3K pathway and absence of the "feedbackloop" could be a critical determinant in clinical success.Third, the absence of insulin resistance with the PI103-SNPindicates that supramolecular nanochemistry can significantlyimpact the therapeutic index. We also demonstrate that theefficacy can be further improved by placing active targetingmoieties such as iRGD peptides on the nanoparticle surface.Indeed, the fact that the nanoparticle assembles from unitmolecules offers an exquisite control over the stoichiometryin terms of active agents as well as the valency of targetingagents. This stoichiometric control also means that supramo-lecular nanochemistry platform can potentially be extendedto additional cytotoxics andmolecularly targeted therapeuticsincluding enabling combination therapy from a single nano-particle, thereby facilitating an integrative approach towardcancer chemotherapy.

Disclosure of Potential Conflicts of InterestR.A. Mashelkar has ownership interest (including patents) in Invictus Oncol-

ogy. E. Ruoslahti has received commercial research grant support and is aconsultant/advisory board member in EnduRx Pharmaceuticals, also has hon-oraria from speakers' bureau from Zealand Pharma, and has ownership interest(including patents) in CendR Therapeutics, Inc. S. Sengupta has ownershipinterest (including patents) in Cerulean Pharmaceuticals and Invictus Oncology

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Figure 6. Testing off-tumor effectsof PI3K inhibition. A and B, graphsshow the effect of PI103-SNP (A)and PI828-SNP (B) on insulintolerance. Each animal wasinjectedwith a singledoseof emptynanoparticles, free PI3K inhibitor(5 mg/kg), or SNP (equivalent to5 mg/kg of the corresponding PI3Kinhibitor). At defined time points,animals were injected with insulin(0.75U/kg). Glucose levels in bloodwere measured in blood samplesbefore insulin injections and 45minutes after insulin injection.Results are mean � SEM (n ¼ 5);��, P < 0.01. C, representativeepifluorescence images of cross-sections of different organs fromanimals treated with PI103 orPI103-SNP. D, Western blotanalysis of kidney and spleen cellsisolated from animals treated withblank SNPs (lanes 1 and 5), freePI103 (lanes 2 and 6), PI103-SNP(lanes 3 and 7), and iRGD-coatedPI103-SNP (lanes 4 and 8) showsexpression levels of phospho- andtotal mTOR (downstream of Akt)and state of apoptosis markers,caspase-3 and PARP (cleavedand total).

Kulkarni et al.





as well as is a consultant/advisory board member of Cerulean Pharmaceuticals.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConceptionanddesign:A.A. Kulkarni, B. Roy, P. Sengupta, S. Basu, E. Ruoslahti,D.M. Dinulescu, S. SenguptaDevelopment ofmethodology:A.A. Kulkarni, B. Roy, P.S. Rao, P. Sengupta, V.R.Kotamraju, S. Basu, E. RuoslahtiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.A. Kulkarni, B. Roy, P.S. Rao, G.A. Wyant,M. Ramachandran, A. Goldman, D.M. DinulescuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.A. Kulkarni, B. Roy, P.S. Rao, G.A. Wyant,M. Ramachandran, P. Sengupta, S. Basu, D.M. Dinulescu, S. SenguptaWriting, review, and/or revision of the manuscript: A.A. Kulkarni, B. Roy,G.A. Wyant, P. Sengupta, S. Basu, R.A Mashelkar, D.M. Dinulescu, S. SenguptaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G.A. Wyant, E. Ruoslahti, D.M.Dinulescu

Study supervision: A.A. Kulkarni, B. Roy, S. Basu, R.A Mashelkar, E. Ruoslahti,D.M. Dinulescu, S. Sengupta

Grant SupportThisworkwas supported byU.S. Department of Defense (DOD) Breast Cancer

Research Program (BCRP) Era of Hope Scholar Award W81XWH-07-1-0482, aDOD Collaborative Innovator Grant W81XWH-09-0698/700, NIH grant R011R01CA135242-01A2, and an American Lung Association Discovery Award(LCD-259932-N; S. Sengupta), Department of Defense Ovarian Cancer ResearchProgramAwardW81XWH-10-1-0263, a V Foundation Scholar Award, anOvarianCancer Research Fund Liz Tilberis Award, by the BurroughsWellcome,Mary KayAsh, and Rivkin Foundations, and by a generous contribution from the MildredMoorman Ovarian Cancer Research Fund (D.M. Dinulescu).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 11, 2012; revised September 18, 2013; accepted September20, 2013; published OnlineFirst October 11, 2013.

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2013;73:6987-6997. Published OnlineFirst October 11, 2013.Cancer Res Ashish A. Kulkarni, Bhaskar Roy, Poornima S. Rao, et al. Pronounced Antitumor EfficacyPhosphoinositide-3-Kinase Overcome Insulin Resistance and Exert Supramolecular Nanoparticles That Target

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