Cell-Penetrating and Enzyme-Responsive Peptides for Targeted Cancer Therapy: Role of Arginine Residue Length on Cell Penetration and In Vivo Systemic Toxicity

For the improved delivery of cancer therapeutics and imaging agents, the conjugation of cell-penetrating peptides (CPPs) increases the cellular uptake and water solubility of agents. Among the various CPPs, arginine-rich peptides have been the most widely used. Combining CPPs with enzyme-responsive peptides presents an innovative strategy to target specific intracellular enzymes in cancer cells and when combined with the appropriate click chemistry can enhance theranostic drug delivery through the formation of intracellular self-assembled nanostructures. However, one drawback of CPPs is their high positive charge which can cause nonspecific binding, leading to off-target accumulation and potential toxicity. Hence, balancing cell-specific penetration, toxicity, and biocompatibility is essential for future clinical efficacy. We synthesized six cancer-specific, legumain-responsive RnAANCK peptides containing one to six arginine residues, with legumain being an asparaginyl endopeptidase that is overexpressed in aggressive prostate tumors. When conjugated to Alexa Fluor 488, R1–R6AANCK peptides exhibited a concentration- and time-dependent cell penetration in prostate cancer cells, which was higher for peptides with higher R values, reaching a plateau after approximately 120 min. Highly aggressive DU145 prostate tumor cells, but not less aggressive LNCaP cells, self-assembled nanoparticles in the cytosol after the cleavage of the legumain-specific peptide. The in vivo biocompatibility was assessed in mice after the intravenous injection of R1–R6AANCK peptides, with concentrations ranging from 0.0125 to 0.4 mmol/kg. The higher arginine content in R4–6 peptides showed blood and urine indicators for the impairment of bone marrow, liver, and kidney function in a dose-dependent manner, with instant hemolysis and morbidity in extreme cases. These findings underscore the importance of designing peptides with the optimal arginine residue length for a proper balance of cell-specific penetration, toxicity, and in vivo biocompatibility

Single ¹⁹F Probe for Simultaneous Detection of Multiple Metal Ions Using miCEST MRI

The local presence and concentration of metal ions in biological systems has been extensively studied <i>ex vivo</i> using fluorescent dyes. However, the detection of multiple metal ions <i>in vivo</i> remains a major challenge. We present a magnetic resonance imaging (MRI)-based method for noninvasive detection of specific ions that may be coexisting, using the tetrafluorinated derivative of the BAPTA (TF-BAPTA) chelate as a ¹⁹F chelate analogue of existing optical dyes. Taking advantage of the difference in the ion-specific ¹⁹F nuclear magnetic resonance (NMR) chemical shift offset (Δω) values between the ion-bound and free TF-BAPTA, we exploited the dynamic exchange between ion-bound and free TF-BAPTA to obtain MRI contrast with multi-ion chemical exchange saturation transfer (miCEST). We demonstrate that TF-BAPTA as a prototype single ¹⁹F probe can be used to separately visualize mixed Zn²⁺ and Fe²⁺ ions in a specific and simultaneous fashion, without interference from potential competitive ions

Salicylic Acid Conjugated Dendrimers Are a Tunable, High Performance CEST MRI NanoPlatform

Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is well suited for imaging, however, existing small molecule CEST agents suffer from low sensitivity. We have developed salicylic acid conjugated dendrimers as a versatile, high performance nanoplatform. In particular, we have prepared nanocarriers based on generation 5-poly­(amidoamine) (PAMAM) dendrimers with salicylic acid covalently attached to their surface. The resulting conjugates produce strong CEST contrast 9.4 ppm from water with the proton exchange tunable from ∼1000 s^–1 to ∼4500 s^–1 making these dendrimers well suited for sensitive detection. Furthermore, we demonstrate that these conjugates can be used for monitoring convection enhanced delivery into U87 glioblastoma bearing mice, with the contrast produced by these nanoparticles persisting for over 1.5 h and distributed over ∼50% of the tumors. Our results demonstrate that SA modified dendrimers present a promising new nanoplatform for medical applications

Biophysical Characterization of Human Protamine‑1 as a Responsive CEST MR Contrast Agent

The protamines are a low-molecular-weight, arginine-rich family of nuclear proteins that protect chromosomal DNA in germ cells by packing it densely using electrostatic interactions. Human protamine-1 (hPRM1) has been developed as a magnetic resonance imaging (MRI) chemical exchange saturation transfer (CEST) reporter gene, based on a sequence that is approximately 50% arginine, which has a side chain with rapidly exchanging protons. In this study, we have synthesized hPRM1 and determined how its CEST MRI contrast varies as a function of pH, phosphorylation state, and upon noncovalent interaction with nucleic acids and heparin (as antagonist). CEST contrast was found to be highly sensitive to phosphorylation on serine residues, intra- and intermolecular disulfide bridge formation, and the binding of negatively charged nucleotides and heparin. In addition, the nucleotide binding constants (<i>K</i>_eq) for the protamines were determined through plotting the molar concentration of heparin versus CEST contrast and compared between hPRM1 and salmon protamine. Taken together, these findings are important for explaining the CEST contrast of existing arginine-rich probes as well as serving as a guideline for designing new genetic or synthetic probes

Metal Ion Sensing Using Ion Chemical Exchange Saturation Transfer ¹⁹F Magnetic Resonance Imaging

Although metal ions are involved in a myriad of biological processes, noninvasive detection of free metal ions in deep tissue remains a formidable challenge. We present an approach for specific sensing of the presence of Ca²⁺ in which the amplification strategy of chemical exchange saturation transfer (CEST) is combined with the broad range of chemical shifts found in ¹⁹F NMR spectroscopy to obtain magnetic resonance images of Ca²⁺. We exploited the chemical shift change (Δω) of ¹⁹F upon binding of Ca²⁺ to the 5,5′-difluoro derivative of 1,2-bis­(<i>o</i>-amino­phenoxy)­ethane-<i>N</i>,<i>N</i>,<i>N</i>′,<i>N</i>′-tetra­acetic acid (5F-BAPTA) by radiofrequency labeling at the Ca²⁺-bound ¹⁹F frequency and detection of the label transfer to the Ca²⁺-free ¹⁹F frequency. Through the substrate binding kinetics we were able to amplify the signal of Ca²⁺ onto free 5F-BAPTA and thus indirectly detect low Ca²⁺ concentrations with high sensitivity

Mesoporous Silica-Coated Hollow Manganese Oxide Nanoparticles as Positive <i>T</i>₁ Contrast Agents for Labeling and MRI Tracking of Adipose-Derived Mesenchymal Stem Cells

Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO₂) nanoparticles were developed as a novel <i>T</i>₁ magnetic resonance imaging (MRI) contrast agent. We hypothesized that the mesoporous structure of the nanoparticle shell enables optimal access of water molecules to the magnetic core, and consequently, an effective longitudinal (<i>R</i>₁) relaxation enhancement of water protons, which value was measured to be 0.99 (mM⁻¹s⁻¹) at 11.7 T. Adipose-derived mesenchymal stem cells (MSCs) were efficiently labeled using electroporation, with much shorter <i>T</i>₁ values as compared to direct incubation without electroporation, which was also evidenced by signal enhancement on <i>T</i>₁-weighted MR images in vitro. Intracranial grafting of HMnO@mSiO₂-labeled MSCs enabled serial MR monitoring of cell transplants over 14 days. These novel nanoparticles may extend the arsenal of currently available nanoparticle MR contrast agents by providing positive contrast on <i>T</i>₁-weighted images at high magnetic field strengths