Targeted Drug Delivery Systems Mediated by a Novel Peptide in Breast Cancer Therapy and Imaging

<div><p>Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. In this study, we utilized phage display to identify a new targeting peptide, SP90, which specifically binds to breast cancer cells, and recognizes tumor tissues from breast cancer patients. We used confocal and electron microscopy to reveal that conjugation of SP90 with liposomes enables efficient delivery of drugs into cancer cells through endocytosis. Furthermore, <i>in vivo</i> fluorescent imaging demonstrated that SP90-conjugated quantum dots possess tumor-targeting properties. In tumor xenograft and orthotopic models, SP90-conjugated liposomal doxorubicin was found to improve the therapeutic index of the chemotherapeutic drug by selectively increasing its accumulation in tumors. We conclude that the targeting peptide SP90 has significant potential in improving the clinical benefits of chemotherapy in the treatment and the diagnosis of breast cancer.</p></div

Identification of breast cancer cell-targeting peptides using <i>in vitro</i> phage display.

<p><b>a</b>, A phage-display random peptide library was used to identify peptides that bind to the breast cancer cell line, BT483. After four rounds of biopanning, the titer of phage eluted from the breast cancer cells had increased 60-fold relative to the first round of selection. PFU, plaque-forming units. <b>b</b>, Binding activity and specificity between individual phage clones and breast cancer cells were tested by cellular ELISA. Bar, mean; Error bar, s.d.; <i>n</i> = 4; OD490 nm, optical density at 490 nm. <b>c</b>, The binding activity of the PC90 phage to five breast cancer cell lines was analyzed by flow cytometry. Con-P, control phage. <b>d</b>, Verification of tumor-homing ability of phages <i>in vivo</i>. SCID mice bearing human breast cancer xenografts received intravenous injections of PC90 and control phage. After perfusion with PBS buffer, xenograft tumor masses were removed and phage titers were measured. <b>e</b>, The tumor-homing ability of the PC90 phage was competitively inhibited by its cognate peptide SP90. <b>f</b>, Immunohistochemical localization of PC90 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066128#pone-0066128-g001" target="_blank">figure 1d</a>. Scale bar: 50 µm. <b>g</b>, Immunohistochemical staining of human surgical specimens of breast infiltrating ductal carcinoma using PC90 phage. Tumor sections of surgical specimens incubated with PC90 or control phage. The phage signal was detected using HRP-conjugated anti-M13 phage antibody. Scale bar: 20 µm.</p

Generation of peptide-conjugated liposomal nanoparticles.

<p><b>a,</b> Schematic illustration of the targeting liposome, depicting the lipid bilayer membrane encapsulating a large amount of doxorubicin or sulforhodamine B, and the breast cancer targeting ligands that can be displayed on the surface of the liposome. Targeting peptide SP90 was chemically conjugated to NHS-PEG-DSPE in a 1.1∶1 molar ratio. There were about 500 peptide molecules per liposome. <b>b</b>, The percentage of conjugation between SP90 and NHS-PEG-DSPE was confirmed by subjecting the reaction product to electrophoresis on a Tricine-SDS gel, staining with coomassie blue for peptide, followed by barium chloride for PEG (SP90∶1.4 kDa, NHS-PEG-DSPE: 4.3 kDa, SP90-PEG-DSPE: 5.7 kDa). PEGylation efficiency for SP90 was 85% based on quantification of band intensity by densitometry. <b>c</b>, The SP90-PEG-DSPE conjugate was analyzed by MALDI-TOF mass spectrometry (bottom panel). A major peak appears at <i>m</i>/<i>z</i> 5702.8, which can be assigned as the PEGylated SP90 conjugate. Unconjugated SP90 (split peak at <i>m</i>/<i>z</i> 1452) and unreacted PEG (a broad peak around <i>m</i>/<i>z</i> 4250) were also visualized in the mass spectrum, which correspond to the peaks in the top and middle panels, respectively. <b>d</b>, Internalization of SP90-liposomal SRB (SP90-LS) and nontargeted LS (CP-LS and LS) by BT483 cells was studied by confocal microscopy (SRB, red). Nuclear staining was by DAPI (blue) (Scale bar: 10 µm). <b>e</b>, Time-course of SRB uptake by BT483 cells treated with SP90-LS, CP-LS and LS at the indicated times.</p

SP90-conjugated liposomes enhanced drug delivery to tumor.

<p><b>a,</b> BT483 breast cancer-bearing mice were treated with different formulations of liposomal (LD, CP-LD and SP90-LD) and free doxorubicin (FD). After perfusion with 50 ml PBS, doxorubicin concentration was determined in tumor tissue (<i>n</i> = 3 in each group; **<i>P</i><0.01, ***<i>P</i><0.005). <b>b</b>, Representative two-color images showing the distribution of doxorubicin (red) in relation to nuclei (blue) in tumor tissue sections. Accumulation of doxorubicin in tumor nuclei was examined at day 3 post-injection. Scale bar, 50 µm. Bar, mean; Error bar, s.d.</p

Treatment of SCID mice bearing human breast cancer xenografts with SP90-LD.

<p><b>a</b>, Mice bearing BT483-derived breast cancer xenografts with average tumor size of ∼75 mm³ were treated with 3 mg/kg at weekly intervals (for a total of three injections) of either FD, LD, SP90-LD, or an equal volume of PBS by intravenous injection. <i>n</i> = 6 each group. Points, mean tumor volumes. <b>b</b>, Mice bearing size-matched BT483-derived breast cancer xenografts with tumor size of ∼500 mm³ were treated with SP90-LD, LD, or FD, for a total doxorubicin dosage of 9 mg/kg (3 mg/kg/injection, twice a week). <i>n</i> = 8. The average tumor volumes at cessation of treatment in the LD, FD and control PBS groups were 1.5, 6.2 and 8.4 times larger than that in the SP90-LD-treated group, respectively. <b>c</b>, Mice bearing orthotopic breast cancer tumors with average tumor size of ∼500 mm³, MCF-7-Luc, were injected with either SP90-LD, FD or LD, for a total doxorubicin dosage of 9 mg/kg (3 mg/kg/injection, twice a week). The tumors were imaged and luminescence was quantified at the indicated days by IVIS200. <i>n</i> = 5. <b>d</b>, Volume of tumors depicted in (c). Error bar, s.d.; *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

<i>In vivo</i> tumor targeting and imaging with SP90-conjugated quantum dots.

<p><b>a</b>, SP90 was thiolated using Traut’s reagent to generate thiol-modified SP90 (SP90-SH). SP90-SH was subsequently conjugated with sulfo-SMCC-activated QD to produce SP90-QD. Mal, Maleimide. <b>b</b>, <i>In vivo</i> fluorescence imaging of SCID mice bearing BT483-derived tumors was performed after intravenous injection of QD (left) or SP90-QD (right). <i>n</i> = 3. Red arrows indicate the tumor loci. The NIR fluorescence images were captured at the indicated time points. <b>c</b>, Fluorescence signals within tumors were quantified using IVIS200 software. <i>n</i> = 3; Error bar, s.d; ***<i>P</i><0.001. <b>d</b>, Quantification and kinetics of <i>in vivo</i> targeting of SP90-QD. Fluorescence intensity was recorded as photons per second per square centimeter per steradian (p/s/cm²/sr). Representative images from three independent sets of studies all gave similar results. <i>n</i> = 3.</p

SP90-conjugated liposomes enhanced drug delivery and cytotoxicity towards cancer cells, through increased endocytosis.

<p><b>a,</b> Electron micrographs revealing liposomes in the endosomes of BT483 cells treated with SP90-LD (arrows) or CP-LD. Scale bar: 100 nm. <b>b,</b> The percentage of breast cancer cells harboring endocytosed liposomes following treatment with SP90-LD or CP-LD (<i>n</i> = 50 in each group; *<i>P</i><0.01). <b>c,</b> The average number of liposomes in each endosome following treatment with SP90-LD or CP-LD (n = 20 in each group; **<i>P</i><0.01). <b>d,</b> Cells were incubated with either SP90-LD or LD at 37°C. Doxorubicin uptake by the cells was quantified at the indicated times, following the removal of surface-bound liposomal drugs. <b>e,</b> Cells were treated with SP90-LD and LD at varying concentrations. Cell viability was determined by MTT assay, and calculated as a percentage of living cells. Each point represents the mean of four experiments. Error bar, s.d.</p

Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

<p>Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.</p