Cell-penetrating peptide [WR]₅ has been previously shown to be an efficient molecular transporter for various hydrophilic and hydrophobic molecules. The peptide was synthesized using Fmoc/tBu solid-phase chemistry, and one arginine was replaced with one lysine to enable the conjugation with the anticancer drugs. Paclitaxel (PTX) was functionalized with an esterification reaction at the C2′ hydroxyl group of PTX with glutaric anhydride and conjugated with the cyclic peptide [W(WR)₄K(βAla)] in DMF to obtain the peptide-drug conjugate PTX1. Furthermore, camptothecin (CPT) was modified at the C(20)-hydroxyl group through the reaction with triphosgene. Then, it was conjugated with two functionalized cyclic peptides through a formyl linker affording two different conjugates, namely CPT1 and CPT2. All the conjugates showed better water solubility as compared to the parent drug. The cytotoxicity assay of the drugs and their conjugates with the peptides were evaluated in the human breast cancer MCF-7 cell line. PTX inhibited cell proliferation by 39% while the PTX-peptide conjugate inhibited the proliferation by ~18% after 72 h incubation. On the other hand, CPT, CPT1, and CPT2 reduced the cell proliferation by 68%, 39%, and 62%, respectively, in the MCF-7 cell lines at 5 µM concentration after 72 h incubation.

Linear (HR)_n and cyclic [HR]_n peptides (n = 4,5) containing alternate arginine and histidine residues were synthesized. The peptides showed 0–15% cytotoxicity at 5–100 µM in human ovarian adenocarcinoma (SK-OV-3) cells while they exhibited 0–12% toxicity in human leukemia cancer cell line (CCRF-CEM). Among all peptides, cyclic [HR]₄ peptide was able to improve the delivery of a cell impermeable fluorescence-labeled phosphopeptide by two-fold. Fatty acids of different alkyl chain length were attached at the N-terminal of the linear peptide (HR)₄ to improve the molecular transporter property. Addition of fatty acyl chains was expected to help with the permeation of the peptides through the cell membrane. Thus, we synthesized seven fatty acyl derivatives of the linear (HR)₄ peptide. The peptides were synthesized using Fmoc/tBu solid phase peptide chemistry, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption/ionization (MALDI) spectrometry. The fatty acyl peptides containing C₈, C₁₂, C₁₄, and C₁₈ alkyl chain did not show cytotoxicity on SK-OV-3 or CCRF-CEM cell lines up to 50 µM concentration; however, at higher concentration (100 µM), they showed mild cytotoxicity. For example, C₁₆-(HR)₄ was also found to reduce the proliferation of SK-OV-3 cells by 11% at 50 µM and C₂₀-(HR)₄ showed mild toxicity at 10 µM, reducing the proliferation of SK-OV-3 cells by 21%. Increase in the length of alkyl chain showed cytotoxicity to the cell lines. C₂₀-(HR)₄ peptide showed better efficiency in translocation of F′-GpYEEI to SK-OV-3 than the phosphopeptide alone. Further investigation of C₂₀-(HR)₄ peptide efficacy showed that the peptide could deliver doxorubicin and epirubicin into SK-OV-3 and also improved the drug antiproliferative ability. These studies provided insights into understanding the structural requirements for optimal cellular delivery of the fatty acyl-(HR)₄ peptide conjugates.

Prostate cancer (PCa) is the most common malignancy in men and is the leading cause of cancer-related male mortality. A disulfide cyclic peptide ligand [CTVRTSADC] 1 has been previously found to target extra domain B of fibronectin (EDB-FN) in the extracellular matrix that can differentiate aggressive PCa from benign prostatic hyperplasia. We synthesized and optimized the stability of ligand 1 by amide cyclization to obtain [KTVRTSADE] 8 using Fmoc/tBu solid-phase chemistry. Optimized targeting ligand 8 was found to be stable in phosphate buffered saline (PBS, pH 6.5, 7.0, and 7.5) and under redox conditions, with a half-life longer than 8 h. Confocal microscopy studies demonstrated increased binding of ligand 8 to EDB-FN compared to ligand 1. Therefore, we hypothesized that the EDB-FN targeted peptides (1 and 8) conjugated with an anticancer drug via a hydrolyzable linker would provide selective cytotoxicity to the cancer cells. To test our hypothesis, we selected both the normal prostate cell line, RWPE-1, and the cancerous prostate cell lines, PC3, DU-145, LNCaP, and C4-2, to evaluate the anticancer activity of synthesized peptide–drug conjugates. Docetaxel (Doce) and doxorubicin (Dox) were used as anticancer drugs. Dox conjugate 13 containing disulfide linkage showed comparable cytotoxicity versus Dox after 72 h incubation in all the cancer cell lines, whereas it was found to be less cytotoxic on RWPE-1, suggesting that it can act as a Dox prodrug. Doce conjugate 14 was found to be less cytotoxic in all the cell lines as compared to drug alone.

A cyclic peptide containing one cysteine and five alternating tryptophan and arginine amino acids [(WR)₅C] was synthesized using Fmoc/tBu solid-phase methodology. The ability of the synthesized cyclic peptide to produce gadolinium nanoparticles through an in situ one-pot mixing of an aqueous solution of GdCl₃ with [(WR)₅C] peptide solution was evaluated. Transmission electron microscopy showed the formed peptide-Gd nanoparticles in star-shape morphology with a size of ~250 nm. Flow cytometry investigation showed that the cellular uptake of a cell-impermeable fluorescence-labeled phosphopeptide (F′-GpYEEI, where F′ = fluorescein) was approximately six times higher in the presence of [(WR)₅C]-Gd nanoparticles than those of F′-GpYEEI alone in human leukemia adenocarcinoma (CCRF-CEM) cells after 2 h incubation. The antiproliferative activities of cisplatin and carboplatin (5 µM) were increased in the presence of [(WR)₅C]-GdNPs (50 μM) by 41% and 18%, respectively, after 72-h incubation in CCRF-CEM cells. The intracellular release of epirubicin, an anticancer drug, from the complex showed that 15% and 60% of the drug was released intracellularly within 12 and 48 h, respectively. This report provides insight about using a non-toxic MRI agent, gadolinium nanoparticles, for the delivery of various types of molecular cargos.