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Nanomedicine: Nanotechnology, Biology, and Medicinexx (2016) xxx–xxx

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HER2-positive breast cancer targeting and treatment by apeptide-conjugated mini nanodrug

Hui Ding, PhDa,1, Pallavi R. Gangalum, PhDa,1, Anna Galstyan, MD, PhDa, Irving Fox, BSa,Rameshwar Patil, PhDa, Paul Hubbard, PhDb, Ramachandran Murali, PhDb,2,

Julia Y. Ljubimova, MD, PhDa,2, Eggehard Holler, PhDa,⁎, 2aDepartment of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, United States

bDepartment of Biomedical Sciences, Research Division of Immunology, Cedars-Sinai Medical Center, Los Angeles, CA, United States

Received 6 May 2016; accepted 25 July 2016

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HER2+ breast cancer is one of the most aggressive forms of breast cancer. The new polymalic acid-based mini nanodrug copolymers aresynthesized and specifically characterized to inhibit growth of HER2+ breast cancer. These mini nanodrugs are highly effective and in theclinic may substitute for trastuzumab (the marketed therapeutic antibody) and antibody-targeted nanobiocongugates. Novel mini nanodrugsare designed to have slender shape and small size. HER2+ cells were recognized by the polymer-attached trastuzumab-mimetic 12-merpeptide. Synthesis of the nascent cell-transmembrane HER2/neu receptors by HER2+ cells was inhibited by antisense oligonucleotides thatprevented cancer cell proliferation and significantly reduced tumor size by more than 15 times vs. untreated control or PBS-treated group.We emphasize that the shape and size of mini nanodrugs can enhance penetration of multiple bio-barriers to facilitate highly effectivetreatment. Replacement of trastuzumab by the mimetic peptide favors reduced production costs and technical efforts, and a negligibleimmune response.© 2016 Published by Elsevier Inc.

Key words: HER2+ breast cancer; Polymer nanoconjugate; Trastuzumab mimetic peptide; 8-arm starPEG; Polymalic acid

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Abbreviations: AHNP, anti-HER2/neu peptide; Alexa, Alexa Fluor 680 (fluorescent dye); anti-TfR, anti-transferrin receptor; AON, antisenseoligonucleotide; ATCC, American Type Culture Collection; BT474, HER2 overexpressing human breast cancer cell line; DCC, N,N′-dicyclohexylcarbodiimide; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; EGFR, epidermal growth factor receptor; FBS, fetal bovineserum; HER2, neu, c-erbB2, members of the epidermal growth factor HER family of tyrosine kinase receptors; SEC-HPLC, size exclusion chromatography-highperformance liquid chromatography; IV, intravenous; LLL, leucyl-N-leucyl-N-leucine; Mal, maleimide; MEA, 2-mercapto-1-ethylamine; NHS,N-hydroxysuccinimide ester; No drug, P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2 (2%); PBS, phosphate-buffered saline; PEG200, PEG2000,PEG3400, polyethylene glycol (PEG) with molecular weights of 200, 2000, and 3400, respectively; PDP, 3-(2-pyridyldithio)-propionic acid; PMLA or P,poly(β-L-malic acid); SPR, surface plasmon resonance; starPEG, 8-arm PEG carboxylic acid (−COOH); TEM, transmission electron microscopy; TLC, thinlayer chromatography; RU, resonance unit.

Financial support: this work was supported by the Martz Translational Breast Cancer Research Discovery Fund (E.H.) and by the NIH/NCI grantR01CA136841 (J.Y.L.).

Author contributions: E.H. designed and coordinated the project together with J.Y.L. R.M. and P.H. carried out the SPR measurements. H.D. performed thechemical syntheses. R.P. contributed to the chemical synthesis. P.R.G., A.G., and I.F. conducted the biological experiments and contributed to the manuscriptpreparation.

Conflict of interest: J.Y.L. is co-founder and E.H. is director of chemical synthesis of Arrogene Inc.⁎Corresponding author at: Cedars-Sinai Medical Center, Nanomedicine Research Center, Department of Neurosurgery, Los Angeles, CA, USA.E-mail address: eggehard.holler@cshs.org (E. Holler).

1 Authors contributed equally.2 Senior authors.

http://dx.doi.org/10.1016/j.nano.2016.07.0131549-9634/© 2016 Published by Elsevier Inc.

Please cite this article as: Ding H., et al., HER2-positive breast cancer targeting and treatment by a peptide-conjugated mini nanodrug. Nanomedicine:NBM 2016;xx:1-9, http://dx.doi.org/10.1016/j.nano.2016.07.013

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The HER2 protein of the epidermal growth factor receptor(EGFR) family plays a key role in aggressive human breastcancer. The humanized anti-HER2/neu monoclonal antibodytrastuzumab (Herceptin; Genentech, Inc.) is used alone or incombination with other agents for the treatment of patients.1–3 Inmouse models, we have shown that the efficacy for the treatmentof human HER2-positive breast cancer with free trastuzumabalone could be significantly increased when the antibody wasco-liganded with poly(β-Lrange-malic acid and an antisenseoligonucleotide (AON) targeting HER2-mRNA.4,5 After thesuccessful use of trastuzumab with affinity in the low nanomolarrange,6,7 peptides with affinities in the high nanomolar region7–9

and affibodies with affinities in the picomolar to low nanomolarregions have been developed and successfully used for tumorimaging.10,11 Examples of targeted therapy using anti-HER2/neupeptide (AHNP) or affibodies alone or in combination with othermolecules have demonstrated variable degrees of tumor-specificgrowth inhibition.12–14

Although tissue penetration is a multifactorial issue in drugdelivery,14–15 an emerging concept suggests that targeted “mini”nanodrugs with a slim shape (i.e., polymeric nanoconjugateswith hydrodynamic diameter ~ 8 nm small but larger than~5 nm, the cut-off for fast renal clearance16) are desirable forachieving deep tissue penetration because they encounter thefewest diffusional barriers. Polymeric nanoconjugates based on70 to 100 kDa poly (β-L malic acid) (PMLA) have beensuccessfully used in delivery of drugs to brain and breast tumors.However, their sizes cannot be reduced below diameters of 15 to25 nm due to the sizes of the antibodies that are attached forspecific targeting.4,5 On the basis of hydrodynamic consider-ations, elongated nanomolecules would be superior in quickly“shuffling” their way through dense areas (e.g., intercellularmatrix) in comparison to spherically shaped molecules of similarsize. Small particles move faster than large ones, as can beroutinely observed under the microscope in the type ofmovement known as Brownian motion.

In addition to improving mobility, other aspects favor mininanodrugs. Examples include the replacement of antibodies thatcould result in reduced effort and cost for manufacturing,lyophilization for packaging, smaller infusion volume needed forcancer treatment, low if any immunogenicity, and stability. Thecombination of trastuzumab with PMLA has enabled a novel,unprecedentedly effective treatment of HER2-positive breastcancer.4,5 The biodegradable, nontoxic, and non-immunogenicplatform of PMLA offers a multitude of pendant carboxylicgroups that can be functionalized with (pro)drug(s), prodrug-ac-tivating groups, targeting peptide(s), protective polyethyleneglycol (PEG), membrane-destabilizing peptides, and dyes forimaging.4,5,18–20

Our design of a novel mini nanodrug follows the previousdesign involving a HER2-mRNA annealing Morpholino AON5′-CATGGTGCTCACTGCGGCTCCGGC-NH2–3′,

4 but re-duces the molecular weight of PMLA to 50 kDa and replacestrastuzumab by AHNP, the 12 amino acid exocyclic mimeticpeptide NH2-YCDGFYACYMDV-NH2 (analogue 3).

8 The AONis delivered as a prodrug while attached to PMLA. Drug activationoccurs in the recipient cancer cell when the disulfide linkage withthe polymer is reductively cleaved by cytoplasmic glutathione.

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Methods

Reagents

Polymalic acid with molecular weight 50,000 (SEC-HPLC/polystyrene sulfonate standards, polydispersity 1.2) was isolatedfrom the culture supernatant of Physarum polycephalumM3CVII and purified to homogeneity as previously described.19

A Morpholino AON that targeted HER2-mRNA (3′-aminemodified) was synthesized by Gene Tools (Philomath, OR,USA). AHNP targeting HER2, NH2-PEG200-AHNP, andNH2-PEG2000-AHNP were custom made by AnaSpec, Inc.(Fremont, CA, USA). Eight-arm PEG carboxylic acid (−COOH)(starPEG, 10 kDa) was obtained from Creative PEGWorks(Chapel Hill, NC, USA). NH2-PEG3400-Mal was obtained fromJenKem Technology USA Inc. (Plano, TX, USA). Alexa Fluor680 NHS ester and Alexa Fluor 680 C2 maleimide were obtainedfrom Thermo Fisher Scientific, Inc. (Waltham, MA, USA).PD-10 columns were obtained from GE Healthcare (Uppsala,Sweden).

Cell lines

HER2-positive BT474 human breast cancer cells wereobtained from American Type Culture Collection (ATCC;Manassas, VA, USA) and subcultured in Hybri-Care medium(ATCC) with 10% FBS at 37 °C in a humidified incubator, andpassaged every 4-5 days. The HER2-negative MDA-MB-468cell line was obtained from ATCC and used as a control.4

Synthesis of starPEG(PEG200-AHNP)2-8, P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%), and P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%)AON(1.5%)

Before conjugation with polymalic acid (P), starPEG(PEG200-AHNP)2-PEG-Mal was synthesized using theN-hydroxysuccinimide (NHS) ester dicyclohexylcarbodiimide(DCC) method for activation of the starPEG carboxylicgroups.19 Two of the 8 pendant carboxylic groups were amidatedwith NH2-PEG200-AHNP resulting in the simple conjugatesused to measure binding to the HER2 protein by surface plasmonresonance (SPR) and to demonstrate contraction by dynamiclight scattering. Then, after the synthesis of starPEG(PEG200-AHNP)2, Mal-PEG3400-NH2 was added in the samereaction mixture. It should be noted, because of conjugation ofthe Mal-PEG3400-NH2 linker, the number of sites available forthe attachment of NH2-PEG200-AHNP was 7 per starPEGmolecule. After stripping of the unreacted NHS (pH .8, 100 mMphosphate) and passage over a PD-10 column (pH .5, 100 mMphosphate), the purified Mal-PEG3400-starPEG(PEG200-AHNP)2 was thio-alkylated at the maleinyl residue of “pre-conjugate” P/LLL(40%)/MEA(10%) (Figure 1), prepared byamidation of NHS-activated polymalic acid (P) using(L-leucine)3 (LLL) and 2-mercapto-1-ethylamine (MEA).19

Note that the percentage indicates the amount of loaded ligandmolecules with regard to the number of malic acid residues perpolymer. Immediately after completion of the reaction, theremaining sulfhydryl groups of P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%) were conjugated with 3-(2-pyridyldithio)-propionyl-amido-AON (PDP-AON) to yield

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Figure 1. Synthesis of the lead mini nanodrug P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%)/AON(1.5%). The preconjugate andMal-PEG3400-starPEG(PEG200-AHNP)2 are synthesized independently before conjugation with PDP-AON and PDP to obtain the lead mini nanodrug.Star-PEG is an 8-arm branched PEG with terminal carboxylic acids. AHNP refers to the anti-HER2/neu peptide having an exocylic disulfide closed loop. Theindicated percentages refer to the fraction of malic acid residues attached with the indicated ligand. Abbreviations: P, poly(β-L-malic acid); LLL,leucyl-N-leucyl-N-leucine; Mal, maleimide; PEG200 or PEG3400, polyethylene glycol with molecular weights of 200 or 3400; starPEG, 8-arm PEG carboxylicacid (−COOH); AHNP, anti-HER2/neu peptide; NHS, N-hydroxysuccinimideester; PDP, 3-(2-pyridyldithio)-propionic acid; AON, antisense oligonucleotide.

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RP/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%)/AON(1.5%) or with free PDP to yield P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%) (no drug). Then,additional PDP reagent was added to block any remaining freesulfhydryl groups. The products were purified over SephadexG-75 (pH .4, PBS) and frozen in aliquots at −80 °C until use.Under the given conditions (i.e., pH, concentrations, reactiontime), maleinyl thiolation and MEA disulfide formation byreaction with 3(2-pyridyldithio)propionyl-amido-AON or thePDP reagent were smooth and complete, and sulfhydryl-inducedopening of the AHNP disulfide bridge was not observed. Table 1summarizes the results for chemical composition and hydrody-namic properties.

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starPEG conjugate tags with Alexa Fluor 680

Conjugate P/LLL(40%)/PEG3400-starPEG(2%)/Alexa(1%)was synthesized by thiolation of P/LLL(40%)/MEA withMal-PEG3400-starPEG and Alexa Fluor 680 C2-Mal.Conjugate P/PEG3400-star(PEG200-AHNP)2(2%)/Alexa(1%)

was synthesized by thio ether formation with Alexa Fluor680 C2-Mal with PMLA/MEA(10%) and then withMal-PEG3400-starPEG(PEG200-AHNP)2. In all syntheses in-volving thiolation, the unreacted sulfhydryls were blocked withPDP. All products were purified by sieving over a PD-10column.

Other conjugate tags with Alexa Fluor 680

Conjugates Alexa(1%)/star-PEG(PEG200-AHNP)2-8 wereobtained after NHS activation followed by amidation withappropriate amounts of NH2-PEG200-AHNP and amino-AlexaFluor 680. Excess NHS ester was hydrolyzed in phosphate buffer(pH .8, 100 mM). Conjugates Alexa(1%)/P/PEG(200)-AHNP(2%) or Alexa(1%)/P/PEG(2000)-AHNP(2%) andAlexa(1%)/P/LLL(40%)PEG(200)-AHNP(2%) or Alexa(1%)/P/LLL(40%)PEG(2000)-AHNP(2%) were similarly preparedby amidation of NHS-activated PMLA with NH2-PEG200-AHNP or NH2-PEG2000-AHNP, NH2-LLL, and NH2-AlexaFluor 680.

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Table 1t1:1

The composition and hydrodynamic properties of the nanoconjugates.t1:2

t1:3 Nanoconjugates

t1:4 #1† #2‡ #3§ #4||

t1:5 PMLA (kDa) 50 50 50 50t1:6 Malic acid per injection (μg) 21 12 50 50t1:7 Loading (%)t1:8 AHNP analogue 38 2 2 4.6 4.6t1:9 AON Not loaded 2 Not loaded 1.5t1:10 Dose (mg/kg)¶

t1:11 AHNP analogue 38 0.4 0.25 1.5 1.5t1:12 AON 0 1.5 0 2.4t1:13 Hydrodynamic diameter (nm) 3.3 3.7 7.8 7.8t1:14 Zetapotential (mV) −6.7 −15.8 −13.8 −14.4

Abbbreviations: PMLA, poly(β-L-malic acid); AHNP, anti-HER2/neupeptide; AON, antisense oligonucleotide; LLL, leucyl-N-leucyl-N-leucine;PEG200 or PEG3400, polyethylene glycol with molecular weights of 200 or3400; starPEG, 8-arm PEG carboxylic acid (−COOH); %, percent loading oftotal malic acid content.t1:15† #1 P/LLL(40%)/PEG200-AHNP(2%).t1:16‡ #2 P/LLL(40%)//PEG200-AHNP(2%)/AON(1.5%).t1:17§ #3 P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2(2%).t1:18|| #4 P/LLL(40%)/PEG3400-starPEG(PEG200)-AHNP)2(2%)/AON(1.5%).t1:19¶ Injected into tumor-bearing mice.t1:20

Table 2 t2:1

Kinetic parameters for AHNP (analogue 38) nanoconjugates binding toHER2. t2:2

t2:3kon(s−1 M−1) koff(10

−3 sec−1) Kd(10−6 M)⁎

t2:4AHNP 800 0.42 0.52t2:5starPEG(PEG200-AHNP)2 118 0.54 4.6t2:6starPEG(PEG200-AHNP)4 1.5 0.62 410t2:7starPEG(PEG200-AHNP)6 Binding not detectedt2:8starPEG(PEG200-AHNP)8 Binding not detected

Abbreviations: AHNP, anti-HER2/neu peptide; HER2, human epidermalgrowth factor receptor 2; starPEG, 8-arm PEG carboxylic acid (−COOH);PEG200, polyethylene glycol with molecular weight 200. t2:9⁎ Kd = koff/kon. t2:10

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Physico-chemical characterization of conjugates

Synthetic reactions were monitored by thin layer chromatog-raphy (TLC) using ninhydrin reagents for amidation. Ellman'sreagent was used to follow thiol consumption.21 Reactionproducts were tested by SEC-HPLC to verify purity. PMLAabsorbance was followed at a wavelength of 220 nm and wasclose to zero at 260 nm (maximum for AONs absorbance) and280 nm (maximum for peptides containing Tyr, Phe, Trp). Ahigh-resolu t ion SEC-HPLC column (PhenomenexPolysep-GFC-P 4000 column, Torrance, CA, USA) was usedin combination with the HPLC Elite LaChrom system L-2130equipped with a diode array detector L-2455 (Hitachi,Pleasanton, CA, USA).

In addition, nanoconjugate constituents were assessed byanalyzing malic acid and AON content as previouslydescribed.19 The AHNP peptide content was quantified asfollows: samples and standard peptide, each in 200 μL 6 M HClsolution, were hydrolyzed at 110 °C for 20 h in a sealedampoule. After HCl evaporation and reconstitution, the samplewas added to an aqueous mixture of potassium tetraborate,o-phthaldialdehyde, and mercaptopropinionic acid and kept for1 min. After addition of sodium acetate buffer to the sample,aliquots were injected into the reverse phase column LaChro-meUltra C18 column (Hitachi) using a gradient mobile phase:buffer A, 0.05 M sodium acetate (4.1 g/L) with 25 mL ofacetonitrile in 1 L of water (pH .0); buffer B, 0.05 M sodiumacetate in 300 mL of water and 700 mL of methanol. Theamount of peptide was calculated on the basis of the elutedamino acids of the standard peptide.

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Nanoconjugates were characterized with respect to hydrody-namic diameter and zeta potential using a Malvern ZetasizerNano-ZS90 (Malvern Instruments, UK). For the hydrodynamicdiameter measurements (25 °C), the solutions were prepared inPBS at a concentration of 2 mg/mL and filtered through a 0.2-mmpore membrane. For the zeta potential measurements, the sample(1-2 mg/mL) was dissolved in water containing 10 mMNaCl, anda voltage of 150 mV was applied. Means ± standard deviationsare reported for three independent measurements.

Dissociation constants of HER2-ligand complexes weremeasured with the SPR biosensor SensiQ Pioneer (SensiQ,Oklahoma City, OK, USA) similarly as previously described.8

Recombinant human HER2 (Thr 23 - Thr 652) fused to IgG1 FcTag was produced in human 293 cells (HEK293, catalog #HE2-H5253; ACROBiosystems, Atlanta, GA, USA). Forimmobilization, lyophilized HER2-Fc was reconstituted insterile water to a final concentration of 0.1 mg/mL. Immediatelyprior to immobilization, 150 μL (25 μg/mL diluted in 10 mMsodium acetate, pH .5) was flowed over a SensiQ COOH5 chip ata rate of 10 μL/min using a running buffer of 1× PBS and0.005% Tween 20. After deactivation using 1 M ethanolamine,pH .5, the final level of immobilization was determined to be~6500 resonance units (RUs). To test for binding activity,100 μL of a solution of trastuzumab (30 nM, 1× PBS, 0.005%Tween 20) was flowed over the surface at a rate of 20 μL/min.Immediately after immobilization, the antibody gave an RU levelof ~1500; while after 11 days, it still gave an RU level of ~1050.AHNP was initially solubilized in 10 mM glycine, pH .5. Themixture was vortexed and ultra-sonicated to maximize peptidesolubility. Then within 1 min during mixing, an equal volume of2× PBS and 0.01% Tween 20 was added to give a finalconcentration of 1 mg/mL (the resulting pH of the solution wasapproximately 8.5). Concentrations of peptides ranging from156 nM to 10 μM were made using running buffer (1× PBS,0.005% Tween, and 5 mM glycine, pH .5). Assays wereperformed using 700 μL of peptide at a fast flow rate of25 μL/min at a temperature of 25 °C. The kinetic parameterswere fit by rate constants using the calculation program providedby SensiQ. Experiments were performed in duplicate. Thesurface was regenerated by two 17-μL injections of 1 M NaCland 10 mM NaOH at a flow rate of 50 μL/min betweeneach cycle.

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Figure 2. Dynamic light scattering was used to measure the hydrodynamicdiameters of starPEG conjugates that contained from zero to eight AHNPsconjugated by amide bonds to the terminal carboxylic groups. (A)Compaction of the starPEG conjugates as a function of the number ofattached AHNPs is indicated by a shift toward smaller diameters with a largernumber of AHNPs. (B) Proposed hypothesis of intramolecular aggregationof hydrophobic AHNPs driving the compaction of the starPEG conjugate.Color codes in panels A and B indicate the number of attached AHNPs.Abbreviations: starPEG, 8-arm polyethylene glycol (PEG) carboxylic acid(−COOH); PEG200, polyethylene glycol with molecular weight 200; AHNP,anti-HER2/neu peptide; n, number of attached AHNPs.

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Mini nanodrug uptake in vitro

BT474 cells were trypsinized and maintained as single cells inHybri-Care medium. Cells were incubated with 0.1-10 μM AlexaFluor 680-stained nanoconjugates for 2 hours at 37 °C. Positivecontrols were obtained by incubation with Alexa Fluor 680conjugated to trastuzumab, while negative controls were notstained. Cells were washed, fixed with 2% paraformaldehyde, andrun on a Cyan (Beckman Coulter, Brea CA, USA) or BD LSR II(BD Biosciences, San Jose, CA, USA) flow cytometer. FlowingSoftware (version 2.3.3; flow cytometry data analysis softwareavailable for free on the internet) was used for the analysis.

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UNCAnimal experiments

Animal experiments were performed in accordance with theprotocols approved by the Cedars-Sinai Medical CenterInstitutional Animal Care and Use Committee (IACUC#4658). Athymic female NCr nude mice, age 8-10 weeks,(20 g body weight, CrTac:NCr-Foxn1nu homozygous; Taconic,Hudson, NY, USA) received estrogen pellets (0.72 mg, 90-daysustained release, Innovative Research of America, Sarasota, FL,USA) under the skin near the neck. One week after estrogenpellet placement, mice were inoculated in the flank with 107

BT474 cells re-suspended in Matrigel (Corning Life Sciences,Corning, NY, USA). Treatment started when tumors had reachedthe size of 120 mm3. Per injection, each mouse received 150-μLtest solution.

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HER2-positive breast cancer imaging in vivo

Mice with BT474 flank tumors that had reached the sizeof 120 mm3 were injected once with the dye-tagged nanoconju-gates. After 24 hours, mice were euthanized, and various organsand tumors were imaged using the Xenogen IVIS ImagingSystem (PerkinElmer, Waltham, MA, USA) with Living Imagesoftware (Symantec, Mountain View, CA, USA).

HER2-positive breast cancer treatment in vivo

Once BT474 flank tumors reached the size of 120 mm3, themice were given 150 μL each injection, twice a week with themini nanodrug and P/LLL(40%)/PEG3400-starPEG(PEG200-AHNP)2 (2%) (no drug). After a total of 8 injections, the animalswere kept under observation for one additional week. Tumor sizewas measured using calipers twice a week and calculated aslength × (width2)/2 in mm3.

Statistical analysis

Bars in the figures represent standard errors of the mean. TheP values were calculated using Student's t test, and P b 0.05was considered significant.

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Chemical synthesis of peptide nanoconjugates

The design of mini nanoconjugates was based on ourprevious success of inhibiting the growth of xenogeneicBT474 HER2-positive human breast cancer in nude mice.4

Instead of using trastuzumab for active tumor targeting and theanti-transferrin receptor (anti-TfR) antibody for extravasation,we designed a mini nanodrug for the same purpose but replacedtrastuzumab and the anti-TfR antibody by AHNP (analogue 38).Notably, AHNP (analogue 38) has a lower affinity for bindingthe HER2 receptor, as indicated by a dissociation constant ofKd = 0 .15 μM compa red wi t h Kd = 1 .04 nM fortrastuzumab.6,7 In addition, the mini nanodrug was subjectedto the low water solubility of the peptide and the tendency foraggregation. Other AHNP versions have been reported owinghigher solubility albeit at lower HER2-affinity.8 Kd values in themicromolar range and limited solubility are not unusual forrationally designed peptides and challenged us to presentevidence that our concept can circumvent these limitations. Wesynthesized AHNP-conjugates with straight linear linkers andwith branched PEG linkers and tested them for binding to theHER2 protein. While the linear linkers were not successful forbinding to the HER2 receptor, the branched 8-arm starPEGlinker yielded conjugates suitable for tumor targeting asevidenced by in vivo imaging and subsequent cancer treatment.

The nanoconjugates with or without attached Alexa Fluor 680were synthesized either with linear PEG-linkers of the type P/PEG(200-2000)-AHNP(2%-8%) or with branched PEG-linkersof the type starPEG(PEG200-AHNP)2-8. The percentage (%)refers to the fraction of ligand-attached malyl residues. Thecompounds were synthesized in 70%-80% yield and matched thedesigned compounds in composition. The conjugates were

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Figure 3. Mice with BT474 flank tumors that had reached the size of 120 mm3 received IV tail injections of Alexa Fluor 680-labeled P/LLL(40%)/PEG340-StarPEG(PEG200-AHNP)2(2%), the no drug without AHNP, or with labeled linear PEG conjugates. After 24 hours, imaging of the tumor and organswas performed. The doses (mg/kg) are marked in red type in both panels. (A) The amount of nanoagent in the tumor is reflected by the fluorescence intensity inthe region of interest. The fluorescence intensity after correction for the background intensity in the same region of an untreated mouse is depicted in thehistogram. (B) The distribution of the lead nanoagent, with and without the targeting AHNP, also indicates uptake in liver and kidneys but minimal uptake inspleen. Abbreviations: P, poly(β-L-malic acid); LLL, leucyl-N-leucyl-N-leucine; PEG200 or PEG3400, polyethylene glycol with molecular weights of 200 or3400; starPEG, 8-arm PEG carboxylic acid (−COOH); AHNP, anti-HER2/neu peptide.

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90%-95% pure by SEC-HPLC. The composition and hydrody-namic properties of representative compounds are summarized inTable 1. The small size indicated by a hydrodynamic diameterb10 nm is at the low end of the nanoscale range and justifies thename “mini nanoconjugates.” The molecular weight calculatedby the design is 210 kDa for the lead nanodrug. The ligation withAHNP and AON only moderately affected the diameter, butvariation of the linker size in the range of PEG200 to

PEG3400-starPEG(PEG200) increased the diameter from3.3 nm to 7.8 nm.

Flow cytometric measurement of HER2 binding with BT474cells was not possible because of the loss of bound,fluorescent-labeled nanoconjugates during cell washing. As analternate approach, we followed BT474 cell uptake of peptidenanoconjugates. Typically, when testing Alexa(1%)/P/LLL(40%)/PEG200-AHNPn=1, the peak of fluorescent cells

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Figure 4. Growth of HER2-positive BT474 human breast cancer tumors inmice that received IV tail injections of PBS (red line), the mini nanodrug(green line; 2.4 mg/kg AON, 1.5 mg/kg AHNP), or the “empty” drug carrier(blue line; 0 mg/kg AON, 1.5 mg/kg AHNP). Injections were given twice perweek, for a total of 8 injections, and were followed by one week ofobservation. Statistical significance was calculated with reference to the PBSgroup (P b 0.001 and marked as **). Abbreviations: PBS, phosphate-buf-fered saline; P, poly(β-L-malic acid); LLL, leucyl-N-leucyl-N-leucine;PEG200 or PEG3400, polyethylene glycol with molecular weights of 200or 3400; starPEG, 8-arm PEG carboxylic acid (−COOH); AHNP, anti-HER2/neu peptide; AON, antisense oligonucleotide.

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shifted by 8%-10% with reference to the shift for trastuzumab.By raising the number of conjugated AHNP or by increasing theconcentration from 0.025 to 1.0 μM, the shift increased slightly.However, at higher concentration, the shift reverted and wastentatively interpreted as AHNP self-aggregation that inactivatedreceptor binding and decreased uptake. Similar results wereobtained for the nanoconjugates in which linker PEG200 hadbeen replaced by the linker PEG2000, suggesting that thepresumed aggregation could not be influenced by an increase inlinker length. For Alexa(1%)/PLLL(40%)/starPEG(PEG200-AHNP2)2%, similar shifts were observed, however, withoutthe reversal. We surmised that in this nanoconjugate thebranched PEG-linker resisted aggregation and that it should beused in further experiments.

Binding of AHNP-nanoconjugates to the HER2 receptor

The affinity of the HER2 protein for binding free AHNP andAHNP-conjugates was measured by using SPR. Values of therate constants for complex formation (kon), complex dissociation(koff), and the calculated dissociation constant Kd = koff/kon aresummarized in Table 2. While starPEG with maximally 4conjugated AHNP ligands displayed binding, the conjugateswith 6 and 8 ligands were inactive. Further studies showed thatconjugates with linear PEG-AHNP linkers were all inactive.Next, we analyzed how the increase in ligation influenced thebinding kinetics. By comparing the binding of free AHNP andstarPEG conjugated with 2 or 4 AHNP ligands, we found that therate constant kon decreased, while koff remained essentiallyunchanged (Table 2). The decrease in kon resulted in the increaseof Kd, equivalent to a reduction in binding affinity. Considering

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the possibility of aggregation, the reduction in kon correspondedto an increased fraction of aggregated AHNP that was unable tobind HER2 protein. In fact, the AHNP contained 7 amino acidswith hydrophobic side chains out of 12 total amino acids andwas poorly water soluble.8 We proposed that the observeddecrease in binding affinity was the result of intramolecularaggregation and peptide inaccessibility, as schematicallydepicted in Figure 2.

This hypothesis of intramolecular aggregation was tested byusing dynamic light scattering to determine whether a decreaseof the hydrodynamic diameter occurred due to compaction.Experimental support for this hypothesis was obtained forstarPEG(PEG200-AHNP)n by varying the number of attachedAHNPs (i.e., n = 0, 2, 4, and 8). As shown in Figure 2, A, as thenumber of attached AHNPs was increased, the hydrodynamicdiameter was reduced from 7.5 nm (n = 0) to 3.5 nm (n = 8).

HER2-positive breast cancer imaging analysis

The targeting activity of AHNP was tested by imagingexperiments. The peptide-containing conjugates were attached tothe fluorescent dye Alexa Fluor 680 that reported the uptake andaccumulation in targeted tissue. After IV tail injection, nude micewith flank tumors derived from HER2-positive BT474 humanbreast cancer cells were imaged using the Xenogen IVIS ImagingSystem. To improve detection and minimize false–positiveresults, tumors were isolated 24 h after injection. The results areshown in Figure 3, A. The strong fluorescence signal obtainedfor P/PEG3400-star(PEG200-AHNP)2(2%)/Alexa(1%) indicat-ed tumor recognition and uptake and confirmed the binding tothe HER2 receptor seen by the SPR method. The tumor afterinjection of P/LLL(40%)/PEG3400-starPEG(2%)/Alexa(1%)did not accumulate fluorescence, in accord with the absence ofthe targeting peptide. Furthermore, the conjugates Alexa(1%)/P/PEG200 -AHNP(2%) and A l e x a ( 1%) /LLL ( 40%) /PEG200-AHNP(2%) with linear PEG-linkers were inactive, inagreement with the absence of HER2 binding noted above in theSPR experiments. The distribution of fluorescence in the organsof liver, kidney, spleen, lung, heart, brain, and skin wasmeasured and is shown in Figure 3, B (24 hours after injection).The high intensities in liver and kidney but low intensity inspleen are in agreement with the function of clearance pathways.Absence of fluorescence in lung, heart, brain, and skin suggestsan absence of major toxic side effects.

Growth inhibition of HER2-positive breast cancer in vivo

Nude mice inoculated with BT474 human breast cancer cells(HER2-positive) developed flank tumors with a size of120 mm3. The mice were given IV injections of AHNP(analogue 38) twice a week, for a total of 8 injections at thedoses indicated in Table 1. The last injection was followed by a1-week follow-up observation period. Tumor growth afterinjection of the no drug nanoconjugate (not containing theAON) was similar to the tumor growth in mice that received PBS(control), whereas significant inhibition of tumor growth wasobserved after injection of the mini nanodrug (Figure 4). Thetumor size when treated with the lead mini nanodrug remainedunchanged or reduced in size during the first to eight injections,

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Figure 5. Visual appearance of mice after treatment (upper panels) and tumors (lower panels). Mice were euthanized at the end of the experiment (i.e., aftertreatment and follow-up observation periods; time points shown in Figure 4). Results are shown for injected PBS (control), no drug nanoconjugate, and mininanodrug. Arrows indicate positions of tumors. Abbreviations: n.d., tumor not detectable; PBS, phosphate-buffered saline; P, poly(β-L-malic acid); LLL,leucyl-N-leucyl-N-leucine; PEG200 or PEG3400, polyethylene glycol with molecular weights of 200 or 3400; starPEG, 8-arm PEG carboxylic acid (−COOH);AHNP, anti-HER2/neu peptide; AON, antisense oligonucleotide.

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and one week after, treatment was finished in comparison withthe PBS group and the no drug group. The tumor size in thetreatment group was about 15 times smaller (medium size fortreatment group was 120 mm3 versus 1800 mm3 in control(PBS) group; P b 0.005). The resulting tumor growth in visualform is shown in Figure 5. Large tumors were seen in the PBScontrol group and the no drug group, whereas after treatmentwith the lead nanodrug, the tumor was small or not detectable.The amount of body weight lost during treatment with the mininanodrug or with the no drug nanoconjugate was 5%-6% of theweight for PBS-injected mice.

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UNCODiscussion

Targeted small nanoconstructs with an elongated shape havethe ability for deep tissue penetration due to hydrodynamicconsiderations15,17 and are, therefore, highly promising types ofnanodrugs. Our mini nanodrug consists of PMLA, attached toseveral HER2-specific AONs,4 several 12-mer mimetic peptidesthat specifically target the HER2 receptor,6–8 and a number oftrileucine residues that manage the escape into the cytoplasmafter endosome uptake into the recipient cell.19 We havepreviously shown that the apparent hydrodynamic diameter ofpolymalic acid nanoconjugates largely reflects the longitude ofthe polymer platform in spite of loading with peptides,antibodies, and oligonucleotides. 23 This finding has beenexplained by the assumption that the hydrodynamic radiusreflects the length of the negatively charged polymer backboneand that the peptide and oligonucleotide substituents along the

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chain are extended orthogonally to the polymer backbone, andthus, these substituents do not add significantly to the measuredradius. This feature has been directly visualized by transmissionelectron microscopy (TEM) for DOTA-gadolinium-polymalicacid conjugates.5 In our approach, we assumed that suchnanodrugs are ellipsoids with a given ratio of their long to shortaxis, where the long axis refers to the polymer backbone. Weestimated an axial ratio of 5:1 on the basis of bond lengths ofatoms in the polymer and substituents. On the basis of thesearguments, it seems appropriate to assume that our mininanodrug has an elongated shape.

The anti-HER2 mimetic peptide contains an exocyclicdisulfide bridge that formed spontaneously during sulfhydryloxidation in air.8 Furthermore, this exocyclic structure stabilizesthe peptide against degradation in the blood after IV injection.22

However, the relatively high proportion of hydrophobic sidechains of AHNP (analogue 38) posed the problem ofspontaneous self-aggregation and compaction of theAHNP-starPEG conjugate that became evident at higher thanmicromolar concentrations of the nanoconjugates. After aggre-gation, the AHNP residues did not bind HER2 protein. Results ofSPR-binding studies and in vivo imaging experiments suggestthat branched PEG linkers such as in starPEG, and in contrast tolinear PEG linkers, can improve the binding of polymalicacid-conjugated hydrophobic AHNP. This experimental ap-proach using branched PEG as linker for hydrophobic peptidescould be useful in general.

Although the binding for starPEG(PEG200-AHNP)n (n = 2)(Table 2) is moderate according to SRP experiments, targeteddelivery by AHNP conjugates could turn out to successfully

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block HER2 signaling in vivo if the dissociation rate of theHER2-AHNP conjugate complex is slow (i.e., koff for AHNP iscomparable to koff for trastuzumab).8 Kinetically, blocking couldbe achieved if the rate of dissociation was slower than the rate ofreceptor internalization (24 and references therein). Moreover, itcannot be ruled out that self-masking of AHNP by aggregationmight be partially reversed as a result of interaction, for examplewith serum constituents, allowing increased peptide access of thereceptor and binding affinity and even the possibility ofoligovalency for receptor binding.

The results of tumor growth inhibition compared favorablywith those of our previously reported results for nanodrugs of asimilar design that involved trastuzumab, which has high affinitybinding for the HER2 target.4,5 In those previous studies,trastuzumab itself caused a substantial degree of inhibition due toblocking of the HER2 signaling pathway.4 In our current study,however, the attached AHNP in the no drug nanoconjugate(Figure 4) did not give rise to detectable growth inhibition (doseof 1.5 mg/kg). Similarly, only a modest degree of inhibition wasreported for free AHNP (dose of 4 mg/kg).12 That the observed,significant inhibition could have been caused by AON itself andnot involve HER2-targeted cell uptake (dose of 1.5 mg/kg) isunlikely because AON in the absence of HER2 targeting at aneven higher dose of 2.5 mg/kg inhibited tumor growth by only30%-40%.4 The degree of inhibition by our lead mini nanodrug(containing starPEG-AHNP, Figure 4) was spectacular andcompared well with the previously reported degree of inhibitionby our nanoconjugates that contained trastuzumab.4,5 We thinkthat this increased inhibition resulted from the superior tumorpenetration of the mini nanodrug, because of a faster and deeperdiffusion. The favorable delivery profile, the biodegradability,and the demonstrated absence of toxicity among Polycefinnanodrugs4 are beneficial assets of the mini nanodrug, whichmight be successfully used in the future for treating HER2-positive breast cancer.

References

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13. Ding L, Tian C, Feng S, Fida G, Zhang C, Ma Y, et al. Small sizedEGFR1 and HER2 specific bifunctional antibody for targeted cancertherapy. Theranostics 2015;5:378-398.

14. Gujrati V, Lee M, Ko Y-J, Lee S, Kim D, Kim H, et al. Bioengineeredyeast-derived vacuoles with enhanced tissue-penetrating ability fortargeted cancer therapy. S A 2016;113:710-715.

15. Chan TC, Li HT, Li KY. Effects of shapes of solute molecules ondiffusion: a study of dependences on solute size, solvent, andtemperature. J Phys Chem B 2015;119:15718-15728.

16. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Ipe BI, et al. Renalclearance of nanoparticles. Nat Biotechnol 2007;25:1165-70.

17. Agarwal R, Jurney P, Raythatha M, Singh V, Sreenivasan V, Shi L.Effect of shape, size, and aspect ratio on nanoparticle penetration anddistribution inside solid tissues using 3D spheroid models. Adv HealthcMater 2015;4:2269-2280.

18. Lee B-S, Fujita M, Khazenon NM,Wawrowsky KA,Wachsmann-HogiuS, Dl F, et al. Polycefin, a new prototype of multifunctionalnanoconjugate based on poly(beta-L-malic acid) for drug delivery.Bioconjug Chem 2006;17:317-326.

19. DingH, Inoue S, LjubimovAV, Patil R, Portilla-Arias J,Hu J, et al. Inhibitionof brain tumor growth by intravenous poly(β-L-malic) acid nanobioconjugatewith pH-dependent drug release. S A 2010;107:18143-18148.

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1 Graphical Abstract

2 Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2016) xxx– xxx

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5 HER2-positive breast cancer targeting and treatment by a6 peptide-conjugated mini nanodrug

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8 Hui Ding, PhDa, Pallavi R. Gangalum, PhDa, Anna Galstyan, MD, PhDa, Irving Fox, BSa, Rameshwar Patil, PhDa,9 Paul Hubbard, PhDb, Ramachandran Murali, PhDb, Julia Y. Ljubimova, MD, PhDa, Eggehard Holler, PhDa,⁎

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aDepartment of Neurosurgery, Cedars-Sinai Medical Center, Los Angeles, CA, United States12

bDepartment of Biomedical Sciences, Research Division of Immunology, Cedars-Sinai Medical Center, Los Angeles, CA, United States

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14 Nanodrug trafficking efficacy through tissue and cellular bio-barriers depends on vehicle size and shape. Using slender15 polymers conjugated with minute-targeting molecules and drugs, we boosted delivery to and treatment efficacy of16 HER2+ breast cancer using a mini nanodrug with a specific mimetic peptide that replaced the HER2+ antibody.

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