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A novel approach to deliver anticancer drugs to key cell types in tumors using a PDGFreceptor-binding cyclic peptide containing carrier
Jai Prakash a,b,⁎, Edwin de Jong a,b, Eduard Post a, Annette S.H. Gouw c, Leonie Beljaars a, Klaas Poelstra a,b
a Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute for Pharmacy, University of Groningen, The Netherlandsb BiOrion Technologies BV, Groningen, The Netherlandsc Department of Pathology and Laboratory Medicine, University Medical Centre Groningen, The Netherlands
Abbreviations: HSA, human serum albumin; pPB, PDGβ, platelet derived growth factor receptor beta; Dox, do⁎ Corresponding author. Department of Pharmacokine
Groningen Research Institute for Pharmacy, UniverDeusinglaan 1, 9713 AV, Groningen, The Netherlands+31 50 3633247.
Tumor stromal cells have been recently recognized to contribute to tumor growth. Therefore, wehypothesized that delivery of anticancer drugs to these cells in addition to the tumor cells might treatcancer more effectively. Stromal cells abundantly expressed Platelet-Derived Growth Factor Receptor-beta(PDGFR-β) in different human tumors as shown with immunohistochemistry. To achieve targeting throughPDGFR-β, we developed a carrier by modifying albumin with a PDGFR-β recognizing cyclic peptide (pPB-HSA). pPB-HSA specifically bound to PDGFR-β-expressing 3T3 fibroblasts, C26 and A2780 cancer cells invitro. Subsequently, doxorubicin was conjugated to pPB-HSA through an acid-sensitive hydrazone linkage. Invitro, Dox-HSA-pPB was taken up by fibroblasts and tumor cells and a short exposure of the conjugateinduced cell death in these cells. In vivo, the conjugate rapidly accumulated into PDGFR-β expressing cells inC26 tumors. Treatment with Dox-HSA-pPB significantly reduced the C26 tumor growth in mice while freedoxorubicin treated mice had lower response to the therapy. Furthermore, in contrast to free doxorubicin theconjugate did not induce loss in body weight. In conclusion, the present study reveals a novel approach totarget key cell types in tumors through PDGFR-β, which can be applied to enhance the therapeutic efficacy ofanticancer drugs.
FR-β binding peptide; PDGFR-xorubicin.tics, Toxicology and Targeting,sity of Groningen, Antonius. Tel.: +31 50 3636414; fax:
Tumor targeting of anticancer drugs has become a vital approachin anticancer therapies as conventional chemotherapies lack thera-peutic efficacy in many patient groups and usually exhibit side-effects[1–5]. For a long time, tumor growth was supposed to be mainlydependent on malignant cancer cells and angiogenesis [6]. In therecent years, however, tumor stromal cells including tumor-associat-ed fibroblasts, endothelial cells, vascular pericytes and infiltratinginflammatory cells have been recognized as key cell types in theinduction of tumor growth and progression [7,8]. So far, major tumordelivery strategies were based on passive targeting through theenhanced permeability and retention (EPR) effect and on the tumorvasculature targeting to the endothelial cell receptors such as alpha(v)beta(3) integrin and vascular endothelial growth factor receptors[9,10]. However, these approaches offer targeting of therapeutic
agents only to a limited number of cell types in a tumor. To deal withtumor variability, more drug carriers are needed.
Most of the human tumors possess a large stroma which isenriched with tumor fibroblasts and other stromal cell types [8].Therefore, we hypothesized that targeting to tumor stromal cells inaddition to tumor cells might be a highly interesting new approach.We have recently shown that tumor cells and tumor stromalfibroblasts could be targeted through the mannose-6-phosphate/insulin-like growth factor II receptor, which is highly expressed onthese cell types [11]. In continuation of our investigation on thetargeting to tumor stromal cells, we now propose to target stromalcells through the Platelet-Derived Growth Factor (PDGF) receptor.PDGFs are a family of dimeric forms of growth factors and exert theiraction through tyrosine kinase receptors, the PDGF-α and -β receptors[12]. PDGFR-β is normally present in the cells from mesenchymalorigin such as vascular smooth muscle cells, fibroblasts, bone marrowand monocytes [13]. However, its expression gets upregulated duringvarious disorders such as atherosclerosis, fibrosis and cancer [14,15].Particularly in tumors, PDGFR-β is abundantly expressed in stromalcells and pericytes of several human cancers e.g. colorectal, pancre-atic, ovarian, lung and breast cancer and it is in several cases alsopresent on the tumor cells of some of the cancer types [16].
In earlier studies, we have designed a cyclic peptide (pPB) againstPDGFR-β and conjugated it to human serum albumin (HSA) in order
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to develop the PDGFR-β-specific drug carrier (pPB-HSA) [17]. Thiscarrier was shown to bind specifically to the PDGFR-β and wasemployed to target the activated hepatic stellate cells in fibrotic liverswhich express this receptor during fibrogenesis [17]. In addition, pPBpeptide has also been applied for gene transfer to stellate cells usingadenovirus as a carrier [18]. In the present study, we applied pPB-HSAfor delivery of anticancer drugs to tumors. First, we determined thePDGFR-β expression in many human tumors and in C26 coloncarcinoma tumor in mice to identify the receptor cellular localization.We further examined the binding and uptake of pPB-HSA in PDGFR-βexpressing fibroblast and tumor cells. To examine whether pPB-HSAcan be used as a drug carrier, we conjugated doxorubicin to it andinvestigated uptake and anti-tumor effects in vitro in different celltypes. Subsequently, we evaluated the conjugate in vivo for itsbiodistribution and therapeutic efficacy in C26 tumor-bearing mice.
2. Materials and methods
2.1. Materials
Mouse NIH3T3 fibroblasts were purchased from ATCC (Manassas,VA). Mouse colon carcinoma cells (C26) were obtained from theDepartment of Medical Biology, University Medical Centre Groningen,The Netherlands and human A2780 ovarian carcinoma cells werereceived from the Department of Pharmaceutical Gene Modulation,University of Groningen. Polyclonal anti-PDGF-β receptor IgG origi-nated from rabbit or goat species was purchased from Santa CruzBiotechnologies (Santa Cruz, CA) and monoclonal rat anti-mousePECAM-1 (CD31) from BD PharMingen. Rabbit anti-human serumalbumin (HSA) was purchased from ICN Biomedics (Eschwege,Germany). Goat anti-HSA antibody was bought from Sigma. Rabbitanti-pPB antibody was developed by Harlan (Zeist, The Netherlands).The human serum albumin (HSA, Cealb®) was purchased fromSanquin (Amsterdam, The Netherlands).
2.2. Immunohistochemistry and immunofluorescence
Immunohistochemical staining on human cancer tissues wasperformed on the 4-μm thick paraffin-embedded section whereasthe rest of the immunohistochemical and immunofluorescencestainings were performed on 4-µm thick cryostat sections. Forstaining on paraffin-embedded tissues, sections were deparaffinizedin xylene and rehydrated in alcohol and distilled water. Antigen-retrieval was done by overnight heating at 80 °C in citrate buffer (pH6.0). For frozen tissues, sections were fixed in acetone for 20 min andthen rehydrated in PBS. Thereafter, common steps were followed for
Fig. 1. Representative microscopic pictures showing the PDGFR-β receptor expression in dwere stained with anti-PDGFR-β antibody. A) normal human liver B) liver metastasis of coloncarcinoma in 1 patient sample and E) pancreatic carcinoma in 11 patient samples. Tumor stralso found in the tumor nest (T) in all tumors.
both paraffin and frozen sections. Sections were incubated withprimary antibody for 1 h. Endogenous peroxidase activity was blockedby incubating with 0.05% hydrogen peroxide/PBS for 20 min. After 3washings with PBS, sections were incubated with horseradishperoxidase-labeled secondary antibodies for 30 min. In case offluorescent staining, no endogenous peroxidase blocking step wasperformed and sections were directly incubated with secondaryantibodies labeled with either FITC (green) or TRITC (red). Subse-quently, sections were washed and incubated with hematoxyllin orDAPI (fluorescent staining) for nuclear staining. Sections weremounted with glycerol/kieselguhr or glycerol/PBS (for fluorescentstaining) solution. For paraffin-embedded sections, sections weredehydrated with alcohol and xylol and finally mounted with Depex™mounting medium. Microscopic pictures were taken with a light orfluorescent microscope.
2.3. Synthesis and characterization of pPB-HSA and Dox-HSA-pPB
The PDGFR-β recognizing cyclic peptide (pPB; CSRNLIDC with C–Cdisulfide bond)was custom-made andmodifiedwith N-Succinimidyl-S-acetylthioacetate by Ansynth Service BV., Roosendaal, TheNetherlands. pPB-HSA was synthesized as described elsewhere [17].Briefly, HSA (1.5 μmol, dissolved in PBS) was reacted with Gamma-MaleimidoButyryloxy-Succinimide ester (GMBS, 30 μmol, dissolvedin DMF; Sigma) for 2 h and then dialyzed against PBS in 10 KDa cut-offdialysis membrane cassette (Thermo Scientific, Rockford, IL). Then,SATA-modified pPB (pPB-ATA; 34.5 μmol, dissolved in DMF) wasadded to the GMBS-modified HSA and reacted for overnight anddialyzed against PBS and then further dialyzed against ultrapurewater. The final product was freeze-dried for long-term storage at−20 °C. pPB-HSAwas characterized withMass spectrometry analyses(MALDI-TOF) and Western blot analyses using anti-HSA and anti-pPBantibodies. For the Western blot analyses, 100 ng HSA and pPB-HSAwere applied on the 10% SDS-PAGE gel and the proteins weretransferred to polyvinylidene fluoride membrane electrophoretically.Membranes were blocked with 5% non-fat milk in Tris-buffered salinecontaining 0.05% Tween-20 and then incubated with anti-pPB or anti-HSA antibody at 4 °C overnight. Then, after 3 times washinghorseradish peroxidase-conjugated secondary antibody was appliedfor 1 h. Protein bands were developed with ECL detection reagent andthe blots were visualized by enhanced chemiluminescence camera(Syngene, Cambridge, UK).
Doxorubicin was conjugated to pPB-HSA through an acid-sensitivehydrazone linkage (Fig. 7A). Doxorubicin (16 µmol) was reacted with3,3′-N-[ε-Maleimidocaproic acid] hydrazide (48 µmol, ThermoScientific)in methanol for 24 h and then the doxorubicin-maleimide product was
ifferent human carcinoma. Paraffin-embedded sections of different human carcinomascarcinoma in 8 patient samples C) breast carcinoma in 11 patient samples D) prostrate
oma (S) had a strong staining (brown color) for PDGFR-βwhereas a weak staining was
Fig. 2. Distribution and localization of the PDGFR-β in C26 tumors and major organs in mice. (A) Microscopic pictures show the PDGFR-β staining (red color) in tumor tissue (a) andother organs such as heart (b), kidneys (c), liver (d), spleen (e) and lungs (f). Note that tumor possesses a high expression of the receptor compared to other organs. (B) Fluorescentmicroscopic pictures show the localization of the PDGFR-β in tumor blood vessels through double staining with the endothelial cell marker CD31. Arrows indicate the co-localizationof the receptor with tumor endothelium. Light microscopic pictures show the expression of the alpha-smooth muscle actin (α-SMA), a marker for fibroblasts, in tumor stroma (S).PDGFR-β was highly expressed in the same area.
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isolated by the crystallization process. To conjugate doxorubicin-maleimide to pPB-HSA, thiol (–SH) groups were introduced in pPB-HSA(1.33 µmol) using N-Succinimidyl-S-acetylthioacetate (20 µmol, Sigma)cross-linker. Then, doxorubicin-maleimide (16 µmol) was reacted withpPB-HSA-SH (1.33 µmol) for 3 h to obtain Dox-HSA-pPB conjugate. Thefinal product was dialyzed in 10 KDa cut-off membrane against PBSovernight and further purified using gel-filtration chromatography withAKTA™ system (Amersham Biosciences, Uppsala, Sweden) to collect themonomeric fraction of the conjugates. The molar ratio of doxorubicincoupled to pPB-HSA in Dox-HSA-pPB conjugate was determined byanalyzing doxorubicin concentration with UV-spectrophotometer at480 nm and by analyzing protein concentration using Lowry's assay(Bio-Rad). Furthermore, we determined whether doxorubicin could bereleased from the Dox-HSA-pPB conjugate at lysosomal pH and itsstability at pH 7.0. Therefore, we incubated the conjugate (equivalent to5 μg/ml doxorubicin) at 37 °C in phosphate buffers of pH 5.0 and 7.0 andcollected samples up to 6 h and determined the released doxorubicinusing a high-performance liquid chromatography method as describedearlier [11].
Fig. 3. Structure and characterization of pPB-HSA. (A) Schematic representation of pPB-HSA.(B) Western blots of HSA and pPB-HSA stained with anti-HSA and anti-pPB antibodies. Theshift in the bands of pPB-HSA indicates that the observed shift of HSA is due to pPB coupling.
2.4. Cell experiments
NIH3T3, C26, A2780 and human hepatocellular carcinoma HepG2cells were maintained on Dulbecco's Modified Eagle's Medium(DMEM, BioWhittaker, Verviers, Belgium) supplemented with 10%fetal calf serum (FCS, BioWhittaker) and antibiotics (penicillin, 50units/ml plus streptomycin, 50 ng/ml for 3T3, A2780 and HepG2 cellsand gentamicin for C26 cells, 10 µg/ml) at 37 °C in a humidifiedincubator containing 5% CO2. Medium for 3T3 cells was added with2 mM L-glutamine.
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2.4.1. Cell viability studiesThe cytotoxic effects of doxorubicin and Dox-HSA-pPB conjugate
on 3T3, C26 and A2780 cells were determined using the Alamar Blueassay. Cells were seeded into the 96-well plate as 1×104 cells per well.After 24 h, cells were incubated with different compounds for 2 h toallow internalization of the conjugate and thenwashed twicewith thecell culture medium and cultured for another 46 h. Thereafter, cellswere incubated with Alamar Blue dye (Serotec, Oxford, UK) for 4 h atthe end of the experiment, and measurements were performedaccording to the instructions of the manufacturer.
2.4.2. In vitro cell binding and uptake studiesWe examined the binding of pPB-HSA on 3T3 fibroblasts and
HepG2. Cells (2×104 cell/well) were seeded in eight-chamberedslides (Nunc Lab-Tek™, Naperville, IL) and grown for 48 h. Then, cellswere incubated with pPB-HSA or HSA (100 µg/ml in 0.2% bovineserum albumin) at room temperature for 1 h and washed withmedium 2 to 3 times. Cells were fixed with acetone:methanol (1:1) at−20 °C for 1 h. Subsequently, anti-HSA and anti-pPB immunofluo-rescent staining was performed as described above. For the targetedconjugate, it is highly important to internalize into the cells andtherefore we examined the uptake of the Dox-HSA-pPB in 3T3fibroblasts, A2780 and C26 tumor cells. Cells (2×104 cells/well for 3T3
Fig. 4. PDGFR-β specific binding of pPB-HSA in vitro. (A) Microscopic pictures showing the PDC26 and A2780 all show strong staining of PDGFR-β while HepG2 cells have no staining at afluorescent) and anti-HSA (green fluorescent) immunofluorescent staining in 3T3 cells (B) anspecifically bound to pPB-HSA as unmodified HSA did not bind to the cells. In contrast, PDG
cells and 2.5×104 cells/well for A2780 and C26 cells) were grown onLab-Tek for 48 h and then incubated with Dox-HSA-pPB at 37 °C for2 h. Thereafter, cells were washed 2–3 times, fixed and stained withanti-HSA and anti-pPB antibodies.
For quantitative analyses of binding and uptake of pPB-HSA intumor cells, we radiolabeled pPB-HSA with 125I using N-bromosucci-nimide. Briefly, an equal volume of HCl was added to the sodium saltof 125I for neutralization and then 50 µl Tris/HCl buffer (1 M) wasadded. Subsequently, pPB-HSA (0.13 nmol) and N-bromosuccinimide(14 nmol) were added and vortexed for 30 s. 125I-pPB-HSA waspurified through PD-10 desalting column (GE Healthcare, Piscataway,NJ) to receive N98% purity. The binding and uptake experiments wereperformed both in C26 and A2780 tumor cells. 1×105 cells/well wereseeded in 12-well plate and allowed to attach overnight. Cells wereincubated with 1% BSA for 15 min and subsequently with differentcompetitors (pPB-HSA, 1 mg/ml; HSA, 1 mg/ml; monensin, 4 μM) in0.2% BSA in DMEM for 15 min and subsequently incubated with1×105 cpm/well of 125I-pPB-HSA at 4 °C, room temperature or 37 °C.After 2 h, cells were washed 3 times with cold medium and thenlysed with 1 M sodium hydroxide. Thereafter, the lysed cells wereremoved by centrifugation, and the cell-associated radioactivity wasmeasured using a gamma-counter (Riastar; Packard Instruments, PaloAlto, CA).
GFR-β immunofluorescent staining in 3T3, C26, A2780 and HepG2 cells. Pictures of 3T3,ll. Panels B and C illustrate representative microscopic pictures showing anti-pPB (redd HepG2 cells (C) after incubating with pPB-HSA for 1 h at room temperature. 3T3 cellsFR-β negative HepG2 cells did not have any binding of pPB-HSA.
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2.5. Biodistribution of Dox-HSA-pPB in C26 tumor-bearing mice
Normal male balb/c mice (20–25 g) were obtained from Harlan(Zeist, The Netherlands). Animals were kept in cages and received adlibitum normal diet, at a 12 h light and 12 h dark cycle. Allexperimental protocols for animal studies were approved by theAnimal Ethics Committee of the University of Groningen. Subcutane-ous tumors of C26 cells were induced by injecting (1×106 cells/mousesuspended in 100 μl of PBS) in the flank of balb/c mice. Tumor growthwas followed by measuring tumor size using a digital Vernier Caliper,and tumor volume was established using the following formula(a×b2/2) where a denotes to the tumor length and b denotes to thetumor width. Tumors were allowed to grow up to the size ofapproximately 550 mm3. Then, animals were injected with Dox-HSA-pPB (1.6 mg per mouse) intravenously through the penile vein. After30 and 120 min, tumors, heart, kidneys, liver, lungs and spleen wereisolated and immunohistochemical staining with anti-HSA IgG wasperformed on cryosections to localize Dox-HSA-pPB in these tissues.
2.6. Effect of Dox-HSA-pPB on C26 tumor growth
C26 tumors were grown in balb/c mice as described above. Afterattaining a tumor size of approximately 100 mm3, mice were injectedintravenously with either vehicle (PBS), pPB-HSA, doxorubicin(50 µg/mouse/day) or Dox-HSA-pPB (equivalent to 50 µg doxorubi-cin/mouse/day) on days 5, 7, 9, 10, 11, and 12. Tumor volumes weremeasured at the time of dosing under anesthesia. At the end of theexperiment, animals were sacrificed under anesthesia and blood wascollected to perform liver and kidney function tests on serum.
2.7. Statistical analyses
The statistical analyses were performed using Student's t-test withpb0.05 as the minimal level of significance. Results are presented asmean±SEM. Cell viability datawere fitted for sigmoidal dose–responsecurve to calculate IC50 using Graphpad Prism 4 software (La Jolla, CA).
Fig. 5. In vitro binding and uptake of radiolabeled (125I)-pPB-HSA in tumor cells.(A) Binding of pPB-HSA to A2780, C26 and HepG2 cells after incubation of 2 h at roomtemperature. Data represent the mean±SEM of at least n=3. *pb0.05. Panel Brepresents the cell-associated radioactivity as the binding of 125I-pPB-HSA to C26 cellsand A2780 cells at 4 °C and room temperature, respectively whereas Panel C representsthe cell-associated radioactivity of 125I-pPB-HSA at 37 °C. pPB-HSA and HSAconcentrations were 1 mg/ml and the concentration of endocytosis inhibitor,monensin, was 4 μM. Data represent the mean±SEM of at least n=3. *pb0.01.
3. Results
3.1. PDGFR-β expression in different human and mouse tumors and invarious mouse organs
We determined the PDGFR-β expression in different humancarcinoma tissues such as metastasis of colon carcinoma in liver andbreast, prostate and pancreatic cancers using immunohistochemicalstaining.We found that all tumors had a strong staining in their stroma(S) in addition to a weak staining in the tumor nest (T) (Fig. 1A to E).
Furthermore, we examined the PDGFR-β expression in subcuta-neously induced C26 colon carcinoma tumors and normal organs inmice using immunohistochemistry (Fig. 2). We found that PDGFR-βwas abundantly expressed in subcutaneous solid C26 tumors whereasorgans such as heart, kidneys, liver, spleen and lung only displayed aweak expression in smooth muscle cells around the blood vessels(Fig. 2A). Using immunofluorescent staining, we found that thereceptor was localized in the endothelial cells of tumor blood vesselsas indicated by the co-localization (yellow color) of the stainings forCD31 and PDGFR-β. In addition, there was a high expression of thereceptor in tumor stroma which is likely to be localized in tumor-associated fibroblasts as these tumor stromal cells also were positivefor alpha-smooth muscle actin (α-SMA), a marker for the (myo)fibroblasts (Fig. 2B). These data clearly demonstrate that PDGFR-β isstrongly present in tumors compared to the other organs and withinthe tumors the receptor is present on key cell types that supporttumor growth.
3.2. Synthesis and characterization of the PDGFR-β targeting construct(pPB-HSA)
To target anticancer drugs to PDGFR-β in tumors, we developed adrug carrier, pPB-HSA, by modifying HSA with a cyclic peptide againstPDGFR-β (Fig. 3A). The pPB-HSA was successfully synthesized, andthe successful conjugation of the peptides to HSA was confirmed byMass spectrometry and Western blot analyses (Fig. 3B and C).
The shift in the mass spectra of pPB-HSA indicates that many pPBpeptides (on average 7 to 8) were randomly conjugated to HSA. Inaddition,Western blot analyses with anti-HSA antibody also showed a
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complete shift in the HSA band after pPB conjugation, while the blotwith anti-pPB antibody confirmed that the shift was due to thecoupling of the peptides. Many batches of pPB-HSA were synthesizedduring these experiments and theywere found to be highly consistent(data not shown).
3.3. Binding and uptake of pPB-HSA in PDGFR-β expressing fibroblastsand tumor cells
In earlier studies, we have shown the specificity of pPB-HSA toPDGFR-β in hepatic stellate cells [17,18]. In the present study, weexamined whether pPB-HSA binds to stromal fibroblasts and tumorcells using immunofluorescent staining and radioactive studies(Figs. 4 and 5).
We first determined the PDGFR-β expression in stromal and tumorcells. We found that 3T3 cells representing stromal fibroblasts, C26and A2780 tumor cells expressed PDGFR-β whereas HepG2 cells didnot express this receptor at all (Fig. 4A). Using anti-pPB and anti-HSAimmunofluorescent staining, we showed that pPB-HSA bound to the3T3 cells after incubation at room temperature whereas unmodifiedHSA did not show any binding to these cells (Fig. 4B). Similar resultswere obtained with C26 tumor cells (pictures not shown). However,in HepG2 cells, which do not have PDGFR-β (Fig. 4C) and no binding ofpPB-HSA to the PDGFR-β negative cells indicates the specificity ofpPB-HSA to the PDGFR-β. Through radioactive studies, we alsodemonstrated that radiolabeled (125I)-pPB-HSA was able to bind toPDGFR-β expressing A2780 and C26 cells but not to the PDGFR-β
Fig. 6. Synthesis and characterization of Dox-HSA-M6P. (A) Schematic illustration of the conspectrometry analyses showing the shift in the spectra of HSA after conjugation with pPchromatograms of size-exclusion chromatography demonstrating that doxorubicin was suabsorption wavelength for doxorubicin. In addition, the purified conjugate contained monowas released from the Dox-HSA-pPB construct at the lysosomal pH (5.0) but remained ratsymbol as mean±SEM.
negative HepG2 cells (Fig. 5A). Furthermore, we showed that thebinding of 125I-pPB-HSAwas specific for the PDGFR-β binding peptideas the binding was only blocked by cold pPB-HSA but not by HSA(Fig. 5B). Furthermore, we showed that 125I-pPB-HSA is taken up bythe cells when incubated at 37 °C as the uptake was significantlyblockedwith the endocytosis inhibitormonensin (Fig. 5C). The uptakewas also blocked by the addition of cold pPB-HSA but not byunmodified HSA, again demonstrating the involvement of the peptidein the cellular uptake.
3.4. Conjugation of doxorubicin to pPB-HSA
To demonstrate that pPB-HSA can be applied as a drug carrier fortumor targeting, we conjugated a well-known anticancer agent,doxorubicin, to the core protein using an acid-sensitive hydrazonelinkage (Fig. 6A). The coupling of doxorubicin to pPB-HSA wasconfirmed by gel-filtration chromatography through which weshowed that the doxorubicin conjugated pPB-HSA (Dox-HSA-pPB)had an extra peak at 480 nm, a specific wavelength for doxorubicin, inaddition to the peak of the carrier at 280 nm. A peak of freedoxorubicin was not detectable in the carrier preparation (Fig. 6B).The basic characteristics of the conjugate are described in Table 1. Theconjugates had N95% bound doxorubicin as determined with HPLCmethods. Furthermore, we examined whether doxorubicin could bereleased from the conjugate at the lysosomal pH (5.0) as this issupposed to occur after the receptor-mediated endocytosis of theconjugate into lysosomal compartments of the target cells. Using
jugation of doxorubicin to pPB-HSA using an acid-sensitive hydrazone linkage. (B) MassB and a further shift in the spectra of pPB-HSA after coupling doxorubicin. (C) Theccessfully conjugated to pPB-HSA as the peak of Dox-HSA-pPB is at 480 nm, a specificmeric form. (D) pH-dependent release of doxorubicin from Dox-HSA-pPB. Doxorubicinher stable at pH 7.0. The experiments were performed in triplicate representing each
Table 1Characteristics of the conjugates.
Compounds Coupling molar ratio (wt.%) Average molecular weight
HSA NA 66,430pPB-HSA Peptide:HSA=8:1
(10.5%)75,800
Dox-HSA-pPB Dox:pPB-HSA=4.2:1(3.0%)
78,000
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HPLC analyses, we demonstrated that doxorubicin was released fromthe conjugate with time after incubation in a buffer at pH 5.0 withN70% release at t=6 h while the conjugate remained stable at pH 7.0with b5% release at t=6 h indicating its stability in plasma (Fig. 6C).
3.5. Intracellular uptake of Dox-HSA-pPB in fibroblasts and tumor cells
Since the conjugation of doxorubicin could affect the binding ofpPB-HSA to its target receptor, we investigated the uptake of the Dox-HSA-pPB conjugate in fibroblasts and tumor cells. We found that Dox-HSA-pPB was taken up by these cells after incubation at 37 °C asdemonstrated with the anti-pPB and anti-HSA immunofluorescentstainings (Fig. 7). The dotted pattern of the staining adjacent to nucleiclearly indicates the intracellular localization of the construct, mostprobably in the lysosomes. In addition, this uptake of the conjugate ina variety of cells was specifically induced by the peptide asunmodified HSA did not show any uptake.
3.6. In vitro effects of Dox-HSA-pPB
To investigate whether Dox-HSA-pPB is pharmacologically activeafter internalization into the cells, we incubated the cells with the
Fig. 7. In vitro uptake of the Dox-HSA-pPB conjugate. Dox-HSA-pPB and HSA were incubatedimmunofluorescent stainings were performed to detect the uptake of the conjugate. Arrointernalization of the conjugate. In contrast, HSA-incubated cells did not show any staining
conjugate only for 2 h to allow its uptake and then removed the excessof conjugate from the medium and determined the effect on the cellviability after another 46 h. We found that the conjugate induced celldeath in 3T3 fibroblasts (IC50 of doxorubicin is 0.65 µM and Dox-HSA-pPB is 0.41 µM), C26 (IC50 of doxorubicin is 0.56 µM and Dox-HSA-pPB is 1.98 µM) and A2780 (IC50 of doxorubicin is 0.07 µM and Dox-HSA-pPB is 0.27 µM) tumor cells with the increasing doses (Fig. 8A toC). In addition, the effects of the conjugate were not due to the carrieras pPB-HSA did not induce any effect at the maximum equivalentdoses (Fig. 8D). To demonstrate that the effects of Dox-HSA-pPB werenot due to passive uptake, we examined the effects of Dox-HSA(without the targeting peptide) in C26 cells and found that Dox-HSAdid not induce any cell death up to 10 µM whereas Dox-HSA-pPBinhibited the cell viability by 75% at the same concentration(Supplementary Fig. 1). These results demonstrate that Dox-HSA-pPB is able to deliver the active drug intracellularly through thePDGFR-β which subsequently induces cell death in stromal cell-likefibroblasts and tumor cells in vitro.
3.7. In vivo tumor distribution of Dox-HSA-pPB
Next, we determined the tumor distribution of the Dox-HSA-pPBconjugate in C26 colon carcinoma tumor-bearing mice after a singleintravenous injection.We found that the conjugate distributed rapidlyto the solid tumors within 30 min after the injection as indicated bythe strong anti-HSA immunostaining and doxorubicin fluorescence intumors (Fig. 9A). 2 h after the injection, both stainings could still bevisualized in tumors by anti-HSA staining but the intensity wasreduced probably due to degradation of the albumin. Interestingly, wefound that the conjugate was localized with the PDGFR-β expressingcells as demonstrated by the co-localization of the immunofluores-cent stainings for HSA and the PDGFR-β (Fig. 9B). We also examined
with 3T3 fibroblasts, C26 and A2780 tumor cells for 2 h at 37 °C. Anti-HSA and anti-pPBws indicate the dotted pattern of the staining adjacent to nuclei demonstrating the.
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the biodistribution of the conjugate in normal organs such as heart,kidneys, liver, lungs and spleen using anti-HSA immunostaining(Fig. 9C).We found that anti-HSA stainingwas present in these organsat t=30 min. Notably, this was not cell-associated but mainly presentin the blood capillaries as can be seen in the interstitial space of heartand kidneys, sinusoids of liver, blood vessels of lungs and red pulp ofspleen, which are enriched with blood capillaries. After 2 h, theconjugate was completely disappeared from these organs. Of note,there was still the staining present in tumors at t=2 h so that theconjugate does not accumulate in these organs but in tumors. Theseresults are in line with the biodistribution data of pPB-HSA as shownin Supplementary Fig. 2. pPB-HSA also accumulated in tumors but notin other organs. In order to determine the pharmacokinetics of pPB-HSA, we used radiolabeled pPB-HSA in C26 tumor-bearing mice.However, we found that the iodine radiolabel was not stable in vivo asonly 6% of the injected dose was found in blood after 2 h and highradioactivity was found in stomach which is known to accumulatefree iodine (Supplementary Fig. 3). These data indicate that radiolabelwith pPB-HSA is not stable in vivo, probably due to access to de-iodinizing enzymes in liver. To demonstrate the biodistribution ofDox-HSA-pPB is highly different and better than a passive targetedconstruct (Dox-HSA), we also performed the biodistribution of Dox-HSA in C26 tumors and found that Dox-HSA distributed throughoutthe body as anti-HSA staining was also present at t=2 h in all organsand tissues examined (Supplementary Fig. 2).
3.8. In vivo anti-tumor effects of Dox-HSA-pPB
To obtain proof-of-principle that delivery of an anticancer drug tokey cell types in a tumor using pPB-HSA could exhibit anti-tumoreffects, we evaluated our Dox-HSA-pPB conjugate in C26 tumor-bearing mice. We found that treatment with the conjugate signifi-cantly (pb0.01) reduced the tumor growth with high response rate asall the treated animals had similar reduction in tumor size. In contrast,animals treated with free doxorubicin had a poor response rate asonly 50% animals had reduction in tumor size which resulted in aninsignificant effect (pb0.08 at day 14) (Fig. 10A).
Importantly, we found that after the 2nd dosing, free doxorubicininduced significant side-effects as indicated by the continuous loss inbodyweight (Fig. 10B). Interestingly, the targeted doxorubicin did notshow such a side-effect even after multiple dosing. In addition, weexamined the liver function by measuring plasma ALT, AST andalkaline phosphatase levels and kidney function by analysis of serumcreatinine levels at the end of the experiment and found no significantchanges in these parameters in any of the groups (SupplementaryFig. 4).
4. Discussion
In the last decade, a better understanding of the tumormicroenvironment has revealed that tumor-associated cells such asstromal fibroblasts, vascular endothelial cells and pericytes signifi-cantly support the tumor growth [8]. Therefore, it is crucial to deliveranticancer agents to these cells in addition to tumor cells. In thepresent study, we demonstrate for the first time that tumor cells andtumor-associated stromal cells can be targeted through a PDGFR-βrecognizing cyclic peptide-based drug carrier (pPB-HSA) as these cellsstrongly express the PDGFR-β during tumor growth. By conjugatingdoxorubicin as a model anticancer drug to the carrier, we show thatthe Dox-HSA-pPB conjugate is taken up by stromal fibroblasts and
Fig. 8. In vitro effects of Dox-HSA-pPB in fibroblasts and tumor cells. Cells were incubatedwith Dox-HSA-pPB conjugate or free doxorubicin only for 2 h and then cells werewashedand the cell viability was determined after 46 h using Alamar blue dye. (A) 3T3 fibroblasts,(B) C26 and (C) A2780 tumor cells. (D) pPB-HSA alone (equivalent to the highestconcentration of the conjugate) did not show any cytotoxic effects. Mean±SEM, n=3.
Fig. 9. In vivo tumor and organ distribution of Dox-HSA-pPB. (A) Representative microscopic pictures of the anti-HSA (red color) and doxorubicin staining (red fluorescent) 30 minand 120 min after a single i.v. injection of the Dox-HSA-pPB conjugate in C26 tumor-bearing mice. (B) Double immunofluorescent staining demonstrates the co-localization of theconjugate with PDGFR-β in tumor. (C) Anti-HSA immunostaining illustrates the distribution of the conjugate in different organs at 30 min and 120 min after the injection of Dox-HSA-pPB.
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tumor cells and induces cell death in vitro. Furthermore, the conjugateaccumulates into the PDGFR-β expressing cells in C26 tumors in vivoand exhibits improved therapeutic efficacy and reduced side-effectscompared to the free doxorubicin.
Other than the commonly used targets for tumor delivery such astumor cells and tumor vasculature, the tumor stroma may also serveas an interesting target as it occupies a large area of the whole tumor(Fig. 1). In addition, mutation frequency and cell heterogeneity instromal tissue are much lower than in tumor cell population which isan important benefit of stromal cell targeting, especially in therapy-resistant tumors. Many studies have shown that tumor stromal cellsand vasculature abundantly express PDGFR-β [19,20], which is alsoconfirmed in the present study in many human tumors andsubcutaneous tumors in mice. In addition, tumor cells itself alsoexpress the PDGFR-β receptor although the expression level may varysignificantly among different tumors [16,19]. PDGFR-β was found tobe more expressed in tumors than in any other organ, as demon-strated in the present study, which favors the tumor selectivity.
We employed a cyclic peptide-based drug carrier to target thePDGFR-β because cyclic peptides are known for their higher affinityand selectivity compared to linear peptides [21]. The specificity of thepPB peptide (CSRNLIDC) to the target receptor was confirmed inearlier studies by simultaneous testing of a scrambled peptide(CIDNLSRC) that did not show any efficacy in adenovirus-mediatedgene transfer studies [18]. Furthermore, we used HSA as a coremolecule to introduce many peptides on the surface to enhance thereceptor-binding affinity of the peptides as shown earlier [17,18] andto allow therapeutic drug incorporation. Albumin has been used as a
tumor delivery vehicle because it penetrates into the tumor throughthe EPR effect [22]. However, in this situation no active cellular uptakeof albumin takes place (Fig. 7), which is an essential process for cell-specific delivery as well as intracellular release of a drug. Efficientbinding and intracellular uptake of our pPB peptide-modified albumin(pPB-HSA) in different PDGFR-β expressing cells signifies itsapplication as an active tumor targeting vehicle. Since the bindingof pPB-HSA only occurred in PDGFR-β expressing cells but not inPDGFR-β negative cells (Figs. 4C and 5A), a cell-specific targeting tokey cell types in a tumor can be anticipated. In addition, pPB-HSAbound and internalized into both stromal fibroblasts and tumor celltypes and of note, these processes occurred both in human andmousecells. The PDGFR-β binding peptide we used incorporates a bindingsequence which is common for mouse as well as human PDGFR-β.
In order to establish the application of pPB-HSA as a drug carrier,we conjugated doxorubicin to it with an acid-sensitive hydrazonelinker. The conjugate released approximately 70% doxorubicin at pH5.0 in 6 h, which is in line with another study in which doxorubicinwas conjugated to HPMA polymer via hydrazone linkage [23]. Theconjugation process did not affect the binding and uptake of thewhole construct as the Dox-HSA-pPB conjugate was taken up byfibroblasts and tumor cells. In addition, only a short exposure of theconjugate to the target cells in order to allow its internalizationinduced significant cell death in stromal and tumor cells. Compared tothe free drug, a lower or similar effectivity of the conjugate in vitro isexpected since the free drug enters the cells through passive diffusionwith no rate limiting steps whereas the conjugate enters through atime-consuming and rate limiting receptor-mediated endocytosis
Fig. 10. In vivo efficacy of Dox-HSA-pPB in C26 subcutaneous tumor. (A) Tumor growthcurve after the treatment with PBS, Dox-HSA-pPB, pPB-HSA and free doxorubicin atdifferent days. Treatment with Dox-HSA-pPB significantly (*pb0.05, **pb0.01 vs.vehicle group) reduced the tumor growth while free doxorubicin showed onlymoderate effects (non-significant vs. vehicle). (B) Percentage change in the animalbody weight after different treatments. Changes in the body weight were calculatedrelative to the body weight at the start of the treatment on day 5. In can be seen thatanimals treated with free doxorubicin lost weight continuously after the second dosingwhereas other treatments did not cause any substantial loss in the body weight. Dataare represented asmean±SEM, n=6. *pb0.05, **pb0.01 vs. vehicle group and #pb0.05vs. free doxorubicin.
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process. The ineffectiveness of Dox-HSA, which lacks the peptide, intumor cells in vitro confirms that the cytotoxic effects of Dox-HSA-pPBwere obtained through active targeting.
In vivo, Dox-HSA-pPB efficiently accumulated into the subcutane-ous C26 tumors in comparison to the other organs examined. Theaccumulation of the conjugate was strongly associatedwith PDGFR-β-expressing cells (Fig. 9). The passively targeted conjugate Dox-HSAaccumulated in tumors but in addition to this, in many other organs aswell, which was clearly evident 2 h after injection. In addition,unmodified HSA was not endocytosed by any cell type in vitro and invivo. Earlier it has been shown that treatment with Dox-HSA did notshow any improvement in the therapy compared to the freedoxorubicin [24], which demonstrates that passive delivery is notsuitable to enhance therapeutic efficacy. Treatment with our targetedDox-HSA-pPB conjugate induced strong anti-tumor effects and lowerside-effects compared to the free drug although subcutaneous tumorshave little stroma compared to human tumors (Fig. 1 vs. Fig. 2) andC26 tumors have poor vascularization and therefore do not allowefficient penetration of albumin [25]. It can therefore be anticipated
that a better effectiveness might be obtained in a tumor containinghigher PDGFR-β expression and possessing more stroma or vascular-ization. These data demonstrate that the conjugate has an overallbenefit over free doxorubicin as there was higher response rate andbetter tolerance.
In summary, the present study demonstrates a novel strategy totarget both stromal cells and tumor cells through PDGFR-β using ourcyclic peptide-based drug carrier pPB-HSA. pPB-HSA was able todeliver doxorubicin in these cells in vitro and in vivo. In addition,delivery of anticancer agents using pPB-HSA to tumors in vivo resultedin higher anti-tumor effects and reduced side-effects compared to thefree drug. It can be concluded that this approach offers an innovativeapproach for the cell-selective delivery of therapeutic agents to thesetumor-associated cells for the treatment of cancer.
Acknowledgements
Authors thank Alie de Jager-Krikken, Catharina Reker-Smit andMieke Zeinstra-Smith for their excellent technical assistance. Ara K.Mohammad is acknowledged for her participation in this study. J.H.Pol from the Department of the Nuclear Medicine performedradiolabeling of the proteins. This study was supported by the STWValorisation Grant and the Innovative Action Program Groningen(IAG2), The Netherlands.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jconrel.2010.03.018.
References
[1] Y. Singh, M. Palombo, P.J. Sinko, Recent trends in targeted anticancer prodrug andconjugate design, Curr. Med. Chem. 15 (2008) 1802–1826.
[2] R. Duncan, Polymer conjugates as anticancer nanomedicines, Nat. Rev. Cancer 6(2006) 688–701.
[3] D. Peer, J.M. Karp, S. Hong, O.C. Farokhzad, R. Margalit, R. Langer, Nanocarriers asan emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751–760.
[4] T. Lammers, W.E. Hennink, G. Storm, Tumour-targeted nanomedicines: principlesand practice, Br. J. Cancer 99 (2008) 392–397.
[5] M.E. Davis, Z.G. Chen, D.M. Shin, Nanoparticle therapeutics: an emergingtreatment modality for cancer, Nat. Rev. Drug Discov. 7 (2008) 771–782.
[6] J. Folkman, Role of angiogenesis in tumor growth andmetastasis, Semin. Oncol. 29(2002) 15–18.
[7] F. Angeli, G. Koumakis, M.C. Chen, S. Kumar, J.G. Delinassios, Role of stromalfibroblasts in cancer: promoting or impeding? Tumour Biol. 30 (2009) 109–120.
[8] M.M. Mueller, N.E. Fusenig, Friends or foes — bipolar effects of the tumour stromain cancer, Nat. Rev. Cancer 4 (2004) 839–849.
[9] K. Temming, R.M. Schiffelers, G. Molema, R.J. Kok, RGD-based strategies forselective delivery of therapeutics and imaging agents to the tumour vasculature,Drug Resist. Updat. 8 (2005) 381–402.
[10] S. Modi, J.J. Prakash, A.J. Domb, N. Kumar, Exploiting EPR in polymer drugconjugate delivery for tumor targeting, Curr. Pharm. Des. 12 (2006) 4785–4796.
[11] J. Prakash, L. Beljaars, A.K. harapanahalli, M. Zeinstra-Smith, A. de Jager-Krikken,M. Hessing, H. Steen, K. Poelstra, Tumor-targeted intracellular delivery ofanticancer drugs through the mannose-6-phosphate/insulin-like growth factorII receptor, Int. J. Cancer 26 (2009) 1966–1981.
[12] R.H. Alvarez, H.M. Kantarjian, J.E. Cortes, Biology of platelet-derived growth factorand its involvement in disease, Mayo Clin. Proc. 81 (2006) 1241–1257.
[13] R. Board, G.C. Jayson, Platelet-derived growth factor receptor (PDGFR): a target foranticancer therapeutics, Drug Resist. Updat. 8 (2005) 75–83.
[14] J. Andrae, R. Gallini, C. Betsholtz, Role of platelet-derived growth factors inphysiology and medicine, Genes Dev. 22 (2008) 1276–1312.
[15] J.C. Bonner, Regulation of PDGF and its receptors in fibrotic diseases, CytokineGrowth Factor Rev. 15 (2004) 255–273.
[16] C.H. Heldin, B. Westermark, Mechanism of action and in vivo role of platelet-derived growth factor, Physiol. Rev. 79 (1999) 1283–1316.
[17] L. Beljaars, B. Weert, A. Geerts, D.K. Meijer, K. Poelstra, The preferential homing ofa platelet derived growth factor receptor-recognizing macromolecule to fibro-blast-like cells in fibrotic tissue, Biochem. Pharmacol. 66 (2003) 1307–1317.
[18] M.H. Schoemaker, M.G. Rots, L. Beljaars, A.Y. Ypma, P.L. Jansen, K. Poelstra, H.Moshage, H.J. Haisma, PDGF-receptor beta-targeted adenovirus redirects genetransfer from hepatocytes to activated stellate cells, Mol. Pharm. 5 (2008)399–406.
[19] Y. Kitadai, T. Sasaki, T. Kuwai, T. Nakamura, C.D. Bucana, S.R. Hamilton, I.J. Fidler,Expression of activated platelet-derived growth factor receptor in stromal cells of
101J. Prakash et al. / Journal of Controlled Release 145 (2010) 91–101
human colon carcinomas is associated with metastatic potential, Int. J. Cancer 119(2006) 2567–2574.
[20] G. Bergers, S. Song, N. Meyer-Morse, E. Bergsland, D. Hanahan, Benefits oftargeting both pericytes and endothelial cells in the tumor vasculature withkinase inhibitors, J Clin. Investig. 111 (2003) 1287–1295.
[21] M. Katsara, T. Tselios, S. Deraos, G. Deraos, M.T. Matsoukas, E. Lazoura, J.Matsoukas, V. Apostolopoulos, Round and round we go: cyclic peptides in disease,Curr. Med. Chem. 13 (2006) 2221–2232.
[22] F. Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates andnanoparticles, J. Control Release 132 (2008) 171–183.
[23] P. Chytil, T. Etrych, C. Konak, M. Sirova, T. Mrkvan, B. Rihova, K. Ulbrich, Propertiesof HPMA copolymer-doxorubicin conjugates with pH-controlled activation: effectof polymer chain modification, J. Control Release 115 (2006) 26–36.
[24] B. Schmid, D.E. Chung, A. Warnecke, I. Fichtner, F. Kratz, Albumin-bindingprodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker thatare cleaved by cathepsin B: synthesis and antitumor efficacy, Bioconjug. Chem. 18(2007) 702–716.
[25] J. Prakash, R. Bansal, E. Post, A. de Jager-Krikken, M.H. de Hooge, K. Poelstra,Albumin-binding and tumor vasculature determine the anti-tumor effect of 15-deoxy-delta12,14-Prostaglandin J2 in vivo, Neoplasia 11 (2009) 1348–1358.
