Matthew T. Basel

Cell-delivered magnetic nanoparticles caused hyperthermia-mediated increased survival in a murine pancreatic cancer model

Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.

Protease-sensitive, polymer-caged liposomes: a method for making highly targeted liposomes using triggered release.

Liposomes have become useful and well-known drug delivery vehicles because of their ability to entrap drugs without chemically modifying them and to deliver them somewhat selectively to tumorous tissue via the enhanced permeation and retention (EPR) effect. Although useful, liposome preparations are still less than ideal because of imperfect specificity, slow release kinetics in the tumor, and leakiness prior to reaching the tumor site. Cancer-associated proteases (CAPs), which are differentially expressed in tumors, have also gained traction recently as a method for tumor targeting and drug delivery. By combining the EPR effect with CAPs sensitivity, a much more specific liposome can be produced. The method described here creates an improved liposome system that can target more specifically, with faster release kinetics and lower general leaking, by deliberately producing a very unstable liposome (loaded with hyperosmotic vehicle) that is subsequently stabilized by a cross-linked polymer shell containing consensus sequences for cancer-associated proteases (protease-triggered, caged liposomes). A cholesterol-anchored, graft copolymer, composed of a short peptide sequence for urokinase plasminogen activator (uPA) and poly(acrylic acid), was synthesized and incorporated into liposomes prepared at high osmolarities. Upon cross-linking of the polymers, the protease-triggered, caged liposomes showed significant resistance to osmotic swelling and leaking of contents. Protease-triggered, caged liposomes also showed significant and substantial differential release of contents in the presence of uPA, while bare liposomes showed no differential effect in the presence of uPA. Thus a protease-sensitive liposome system with fast release kinetics was developed that could be used for more specific targeting to tumors.

Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs.

Abstract Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments; however, therapies tested in such models often fail to translate into clinical settings. Therefore, a better preclinical model for cancer treatment testing is needed. Here we demonstrate that an immunodeficient line of pigs can host and support the growth of xenografted human tumors and has the potential to be an effective animal model for cancer therapy. Wild-type and immunodeficient pigs were injected subcutaneously in the left ear with human melanoma cells (A375SM cells) and in the right ear with human pancreatic carcinoma cells (PANC-1). All immunodeficient pigs developed tumors that were verified by histology and immunohistochemistry. Nonaffected littermates did not develop tumors. Immunodeficient pigs, which do not reject xenografted human tumors, have the potential to become an extremely useful animal model for cancer therapy because of their similarity in size, anatomy, and physiology to humans.

A cell-delivered and cell-activated SN38-dextran prodrug increases survival in a murine disseminated pancreatic cancer model.

Enzyme-activated prodrugs have been investigated and sought after as highly specific, low-side-effect treatments, especially for cancer therapy. Unfortunately, excellent targets for enzyme-activated therapy are rare. Here a system based on cell delivery that can carry both a prodrug and an activating enzyme to the cancer site is demonstrated. Raw264.7 cells (mouse monocyte/macrophage-like cells, Mo/Ma) are engineered to express intracellular rabbit carboxylesterase (InCE), which is a potent activator of the prodrug irinotecan to SN38. InCE expression is regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline, is present. Concurrently, an irinotecan-like prodrug, which is conjugated to dextran and can be loaded into the cytoplasm of Mo/Ma, is synthesized. To test the system, a murine pancreatic cancer model is generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma are loaded with the prodrug and are injected i.p. Two days later, doxycycline was given i.p. to activate InCE, which activated the prodrug. A survival study demonstrates that this system significantly increased survival in a murine pancreatic cancer model. Thus, for the first time, a prodrug/activating enzyme system, which is self-contained within tumor-homing cells and can prolong the life of i.p. pancreatic tumor bearing mice, is demonstrated.

Luminol-based bioluminescence imaging of mouse mammary tumors

Polymorphonuclear neutrophils (PMNs) are the most abundant circulating blood leukocytes. They are part of the innate immune system and provide a first line of defense by migrating toward areas of inflammation in response to chemical signals released from the site. Some solid tumors, such as breast cancer, also cause recruitment and activation of PMNs and release of myeloperoxidase. In this study, we demonstrate that administration of luminol to mice that have been transplanted with 4T1 mammary tumor cells permits the detection of myeloperoxidase activity, and consequently, the location of the tumor. Luminol allowed detection of activated PMNs only two days after cancer cell transplantation, even though tumors were not yet palpable. In conclusion, luminol-bioluminescence imaging (BLI) can provide a pathway towards detection of solid tumors at an early stage in preclinical tumor models.

Cells as delivery vehicles for cancer therapeutics

Cell-based therapeutics have advanced significantly over the past decade and are poised to become a major pillar of modern medicine. Three cell types in particular have been studied in detail for their ability to home to tumors and to deliver a variety of different payloads. Neural stem cells, mesenchymal stem cells and monocytes have each been shown to have great potential as future delivery systems for cancer therapy. A variety of other cell types have also been studied. These results demonstrate that the field of cell-based therapeutics will only continue to grow.

Hexagonal magnetite nanoprisms: preparation, characterization and cellular uptake

The capacity of iron oxide nanocrystals to heat tissue when subjected to an alternating magnetic field (AMF hyperthermia) is shape-selective. Although iron oxide nanostructures with numerous shapes have been synthesized to date, hexagonal Fe3O4 prisms of low toxicity remained elusive. Here, we report the use of a dual ligand system permitting feasible reaction conditions to synthesize nearly perfect hexagonal Fe3O4 nanoplatelet structures, with edge length of 45 ± 5 nm and thickness of 5 to 6 nm. Their Specific Absorption Rate (SAR) is >750 W g(Fe)-1. The Fe3O4 hexagons were coated with a dopamine-based ligand to increase dispersibility in aqueous buffers. The Fe3O4 hexagons were only minimally toxic to RAW264.7 cells, which can be utilized in cell-based cancer targeting approaches.

Channel blocking of MspA revisited.

Porin A from Mycobacterium smegmatis (MspA) is a highly stable, octameric channel protein, which acts as the main transporter of electrolytes across the cell membrane. MspA features a narrow, negatively charged constriction zone, allowing stable binding of various analytes thereby blocking the channel. Investigation of channel blocking of mycobacterial porins is of significance in developing alternate treatment methods for tuberculosis. The concept that ruthenium(II)quaterpyridinium complexes have the capability to act as efficient channel blockers for MspA and related porins, emerged after very high binding constants were measured by high-performance liquid chromatography and steady-state luminescence studies. Consequently, the interactions between the ruthenium(II) complex RuC2 molecules and MspA, leading to RuC2@MspA assemblies, have been studied utilizing time-resolved absorption/emission, atomic force microscopy, dynamic light scattering, ζ potential measurements, and isothermal titration calorimetry. The results obtained provide evidence for the formation of clusters/large aggregates of RuC2 and MspA. The results are of interest with respect to utilizing prospective channel blockers in porins. The combination of results from conceptually different techniques shed some light onto the chemical nature of MspA-channel blocker interactions thus contributing to the development of a paradigm for channel blocking.

Direct Observation of Gold Nanoparticle Assemblies with the Porin MspA on Mica

The octameric porin MspA from Mycobacterium smegmatis is sufficiently stable to form a nonmembrane-supported stand-alone porin on mica surfaces. About 98% of all MspA octamers were found to stand upright on mica, with their periplasmic loop regions bound to the hydrophilic mica surface. Both, small (d = 3.7 nm) and large (d = 17 nm) gold nanoparticles bind to MspA, however, in different positions: small gold nanoparticles bind within the MspA pore, whereas the large gold nanoparticles bind to the upper region of MspA. These experiments demonstrate that gold nanoparticles can be positioned at different, well-defined distances from the underlying surface using the MspA pore as a template. These findings represent a significant step toward the use of electrically insulating stable proteins in combination with metal nanoparticles in nanodevices.

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

Summary Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.