Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors.

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine-leucine-leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

Targeted imaging of urothelium carcinoma in human bladders by an ICG pHLIP peptide ex vivo.

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with nonbuffered saline and instilled with a solution of the ICG pHLIP construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80% if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. The ICG pHLIP imaging agent marked high-grade urothelial carcinomas, both muscle invasive and nonmuscle invasive. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four cases were seen using white light, so imaging with the ICG pHLIP peptide offers improved early diagnosis of bladder cancers and may also enable new treatment alternatives.

pHLIP Peptides Target Acidity in Activated Macrophages.

PURPOSE: Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity. PROCEDURES: The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed. RESULTS: We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45+, CD11b+, F4/80+, and CD206+ tumor-associated macrophages with no significant targeting of other immune cells. Also, fluorescent pHLIPs target CD206-positive cells found in the inguinal lymph nodes of animals inoculated with breast cancer cells in mammary fat pads. CONCLUSIONS: pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery.

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration.

PARaDIM: A PHITS-Based Monte Carlo Tool for Internal Dosimetry with Tetrahedral Mesh Computational Phantoms.

Mesh-type and voxel-based computational phantoms comprise the current state of the art for internal dose assessment via Monte Carlo simulations but excel in different aspects, with mesh-type phantoms offering advantages over their voxel counterparts in terms of their flexibility and realistic representation of detailed patient- or subject-specific anatomy. We have developed PARaDIM (pronounced "paradigm": Particle and Heavy Ion Transport Code System-Based Application for Radionuclide Dosimetry in Meshes), a freeware application for implementing tetrahedral mesh-type phantoms in absorbed dose calculations. It considers all medically relevant radionuclides, including α, ß, γ, positron, and Auger/conversion electron emitters, and handles calculation of mean dose to individual regions, as well as 3-dimensional dose distributions for visualization and analysis in a variety of medical imaging software. This work describes the development of PARaDIM, documents the measures taken to test and validate its performance, and presents examples of its uses. Methods: Human, small-animal, and cell-level dose calculations were performed with PARaDIM and the results compared with those of widely accepted dosimetry programs and literature data. Several tetrahedral phantoms were developed or adapted using computer-aided modeling techniques for these comparisons. Results: For human dose calculations, agreement of PARaDIM with OLINDA 2.0 was good-within 10%-20% for most organs-despite geometric differences among the phantoms tested. Agreement with MIRDcell for cell-level S value calculations was within 5% in most cases. Conclusion: PARaDIM extends the use of Monte Carlo dose calculations to the broader community in nuclear medicine by providing a user-friendly graphical user interface for calculation setup and execution. PARaDIM leverages the enhanced anatomic realism provided by advanced computational reference phantoms or bespoke image-derived phantoms to enable improved assessments of radiation doses in a variety of radiopharmaceutical use cases, research, and preclinical development. PARaDIM can be downloaded freely at www.paradim-dose.org.

Synthesis and characterization of pHLIP® coated gold nanoparticles.

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP®) were introduced. The presence of a tumor-targeting pHLIP® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ∼43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.