The specific targeting ability of novel breast cancer targeting peptides as ligands coupled to polymeric micelles was evaluated in the present study. In this context, engineered breast cancer cell targeting peptides, denoted as peptide 11 (RGDPAYQGRFL) and peptide 18 (WXEAAYQRFL), were compared with the lead 12-mer p160 peptide and cyclic RGDfK peptide. All four peptides were conjugated individually to poly(ethylene oxide)-b-poly(caprolactone) (PEO-b-PCL) diblock polymeric micelles to obtain targeted carrier systems PM11, PM18, PM 160, and PM c-RGD. Physical blending of the peptides 11 and 18 with PEO-b-PCL was also done to yield combination micelles, comPM11 and comPM18. The structural confirmation of polymer was carried out using 1H NMR and MALDI-TOF, and the size distribution and zeta potential of the micelles were determined using dynamic light scattering. Lipophilic cyanine fluorescent probe DiI was physically incorporated in the polymeric micelles to imitate the hydrophobic drug loaded in the micellar core. The cellular uptake of DiI-loaded peptide-containing polymeric micelles by MDA-MB-435, MDA-MB-231, and MCF7 breast cancer cell lines, as well as HUVEC and MCF10A noncancerous cells, were analyzed using flow cytometry and confocal microscopy techniques. Modification of polymeric micelles with peptide 11 or 18 led to an increase in micellar uptake specifically in breast cancer cells compared to p160, c-RGD modified, or naked micelles. The peptide–micelle combinations (comPM11 and comPM18) displayed better uptake by the cells compared to the covalently conjugated PM11 and PM18 micelles; however, the combinations were less selective toward cancer cells. The results point to a potential for peptides 11- and 18-micelle conjugates as attractive platforms for improved performance of a wide range of chemotherapeutic drugs and/or imaging agents in cancer therapy and diagnosis.

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β-Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β-peptides, β3- and β2-peptides derived from l-aspartic acid and l-diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β-peptidic compounds and a mixed α /β-peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α-peptide. Peptides containing β-linkages were resistant to all conditions. The mixed α /β-peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β-peptide was slower than that would be expected for an α-peptide. In addition, these β-peptides were not toxic to HeLa and COS-1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β-peptides containing β-amino acids or small β-peptide fragments as therapeutic peptides.

Peptides containing the Asn-Gly-Arg (NGR) motif are known to bind CD13 isoforms expressed in tumor vessels and have been widely used for tumor targeting. Residues flanking the NGR sequence play an important role in modulating the binding affinity and specificity of NGR for the CD13 receptor. Herein, we have used a rapid, easy, and reliable peptide array–whole cell binding assay for screening a library of NGR peptides with different flanking residues. A peptide array consisting of forty-five NGR containing peptides was synthesized on a cellulose membrane, followed by screening against CD13 positive (HUVEC and HT-1080) and CD13 negative cell lines (MDA-MB-435 and MDA-MB-231). The library screening led to the identification of five cyclic and acyclic NGR peptides that display higher binding (up to 5-fold) to CD13 positive cells with negligible binding to CD13 negative cell lines when compared to the lead sequence cyclic CVLNGRMEC. Peptides with high binding affinity for the CD13 positive cells also showed improved in vitro cellular uptake and specificity using flow cytometry and fluorescence microscopy. Interestingly, the identified peptides resemble the NGR sequences present in the human fibronectin protein. These NGR peptides are promising new ligands for developing tumor vasculature targeted drugs, delivery systems and imaging agents with reduced systemic toxicity.

In this paper, we describe a novel method to screen peptides for specific recognition by cancer cells. Seventy peptides were synthesized on a cellulose membrane in an array format, and a direct method to study the peptide−whole cell interaction was developed. The relative binding affinity of the cells for different peptides with respect to a lead 12-mer p160 peptide, identified by phage display, was evaluated using the CyQUANT fluorescence of the bound cells. Screening allowed identification of at least five new peptides that displayed higher affinity (up to 3-fold) for MDA-MB-435 and MCF-7 human cancer cells compared to the p160 peptide. These peptides showed very little binding to the control (noncancerous) human umbilical vein endothelial cells (HUVECs). Three of these peptides were synthesized separately and labeled with fluorescein isothiocyanate (FITC) to study their uptake and interaction with the cancer and control cells using confocal laser scanning microscopy and flow cytometry. The results confirmed the high and specific affinity of an 11-mer peptide 11 (RGDPAYQGRFL) and a 10-mer peptide 18 (WXEAAYQRFL) for the cancer cells versus HUVECs. Peptide 11 binds different receptors on target cancer cells as its sequence contains multiple recognition motifs, whereas peptide 18 binds mainly to the putative p160 receptor. The peptide array−whole cell binding assay reported here is a complementary method to phage display for further screening and optimization of cancer targeting peptides for cancer therapy and diagnosis.

Polymeric micelles based on poly(ethylene oxide)-b-poly(ε-caprolactone) PEO-b-PCL or poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) PEO-b-PBCL block copolymers were prepared and decorated with either c(RGDfK) or p160, a cancer cell-specific peptide ligand, on their surface. The cellular uptake of p160-decorated PEO-b-PBCL micelles containing DiI fluorescent label by MDA-MB-435 cancer cells was assessed and compared to that for c(RGDfK)-decorated micelles. The hydrophobic anticancer drug paclitaxel (PTX) was physically encapsulated into PEO-b-PCL or PEO-b-PBCL micelles (with and without peptide ligands) using a dialysis technique. The effect of the micellar formulation on the specificity of encapsulated PTX against cancer cells was assessed by investigating the in vitro cytotoxicity of free and encapsulated PTX against MDA-MB-435 cancer cell line versus two normal cells, Human Umbilical Vein Endothelial Cells (HUVEC) and MCF10A cells, using the MTT assay. Our results showed both peptide ligands to facilitate the association of micelles with MDA-MB-435 cells. The p160-micelles, however showed better binding and internalizing in MDA-MB-435 cells than c(RGDfK)-micelles. In general, peptide decoration enhanced the selective cytotoxicity of encapsulated PTX against MDA-MB-435 cells over normal HUVEC and MCF10A cells. The extent of this increase in cancer cell specificity for encapsulated PTX was more for p160-decorated micelles than c(RGDfK)-decorated ones.

Formation of stable secondary structures by oligomers that mimic natural peptides is a key asset for enhanced biological response. β(3) -Peptides with an additional methylene group in their backbone are proteolytically stable and fold into stable 14-helical structures in solution. Here we show that oligomeric β(3) -hexapeptides synthesized from L-aspartic acid monomers (β(3) -peptides 1, 5a and 6) or homologated β(3) -amino acids (β(3) -peptide 2), fold into similar stable 14-helical secondary structures in solution, except that the former form right-handed 14-helix and the later form left-handed 14-helix. β(3) -Peptides from L-Asp monomers contain an additional amide bond in the side chains that provides opportunities for more hydrogen bonding. However based on the NMR solution structures, we found that β(3) -peptide from L-Asp monomers (1) and from homologated amino acids (2) form similar structures with no additional side chain interactions. These results suggest that the β(3) -peptides derived from L-Asp are promising peptide-mimetics that can be readily synthesized using L-Asp monomers as well as the right-handed 14-helical conformation of these β(3) -peptides (such as 1 and 6) may prove beneficial in the design of mimics for right-handed α -helix of α -peptides.

Naphthenic acids (NAs) are water-soluble components of petroleum. The characterization and quantification of NAs by analytical methods have proved quite challenging, whilst the toxic effects of these water-soluble compounds on a variety of organisms adversely affecting reproduction and steroid production is becoming apparent. In this study, we report a fluorescence-based competitive binding method for rapid sensing of the presence of NAs using cellulosic peptide array strips as sensors. The peptide array was designed from sequences derived from the estrogen receptor (ER). Several of these peptides were able to detect the presence of NAs at low micromolar (∼5 mg L−1) levels in different oil sands process affected water samples. The specific binding of one of the peptides, peptide 17 (EGXVEIFDXLLATS) with NAs was evaluated using isothermal titration calorimetry. The results show that peptide 17 interacts strongly with NAs with an apparent binding constant (Ka) of 96 × 106 M−1, and may bind NAs in a similar fashion as ER interacts with estrogen. Finally, the data support that the peptides displaying high affinity for NAs can be used for developing disposable peptide-based sensor arrays for NA detection in oil sands process affected water samples

Abstract The identification of cancer cell-specific ligands is a key requirement for the targeted delivery of chemotherapeutic agents. Usually phage display system is employed to discover cancer-specific peptides through a biopanning process. Synthetic peptide array libraries can be used as a complementary method to phage display for screening and identifying cancer cell-specific ligands. Here, we describe a peptide array-whole cell binding assay to identify cancer cell-specific peptides. A peptide array library based on a lead dodecapeptide, p160, is synthesized on a functionalized cellulose membrane using solid phase chemistry and a robotic synthesizer. The relative binding affinity of the peptide library is evaluated by incubating the library with fluorescently labeled cancerous or non-cancerous cells. Thereby the assay allows picking peptides that show selective and high binding to cancerous cells. These peptides represent potential candidates for use in cancer-targeted drug delivery, imaging, and diagnosis.

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