Lajos Balogh

Nanoparticle Targeting of Anticancer Drug Improves Therapeutic Response in Animal Model of Human Epithelial Cancer

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

In vivo, in situ tissue analysis using rapid evaporative ionization mass spectrometry

The analysis of intact biological tissues by mass spectrometry (MS) has been pursued for more than three decades. However, mass spectrometric methods have always put strong constraints on the geometry and the preparation of these samples. Even with the recent advent of ambient ionization methods, not all of these restrictions have been lifted. MS analysis of biomolecules in tissue has traditionally been achieved by desorption ionization methods including secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption (MALDI), 19, 20] and desorption electrospray ionization (DESI) 5,18] methods. While desorption ionization methods are not appropriate for the analysis of vital (living) tissues, rapid thermal evaporation has the potential to establish the in situ, in vivo ionization of tissue constituents. The possible formation of organic ions from condensed-phase samples in a purely thermal process was initially proposed by Holland et al., and it was successfully demonstrated later. The rationale of rapid heating was to achieve molecular evaporation rates comparable to the rate of decomposition, which results in the formation of a considerable quantity of gaseous molecules or molecular ions. The quest for efficient thermal evaporation methods has led to the development of various thermally assisted ionization methods, including thermospray ionization. Since collisional cooling of nascent ions at higher pressure is more effective, thermal evaporation at atmospheric pressure is expected to suppress thermal decomposition. Atmospheric pressure thermal desorption ionization was demonstrated recently by the desorption of organic cations with minimal thermal degradation. 27] The present study is based on the discovery that rapid thermal evaporation of biological tissues yields gaseous molecular ions of the major tissue components, for example, phospholipids. As thermal evaporation of tissues is widely used in surgery (i.e., electrosurgery and laser surgery), it was sensible to use dedicated surgical instruments for the experiments. Combination of surgical and MS techniques also offers a possibility for in situ chemical analysis of tissue during surgery. Since the key feature of the technique is the fast evaporation of a sample, it was termed “Rapid Evaporative Ionization Mass Spectrometry” (REIMS). The tentative mechanism of ion formation is described in the Supporting Information. Electrosurgical dissection is based on the Joule heating and evaporation of tissues by an electric current. The presence of ionized water molecules during electrosurgical dissection raises the possibility of an alternative ionization mechanism involving neutral desorption and chemical ionization in the gas phase. For more details, see the Supporting Information. An electrosurgical electrode was used as an ion source coupled to a distant mass spectrometer employing a Venturi gas jet pump and 1–2 m long polytetrafluoroethylene (PTFE) tubing (Figure 1).

3H Dendrimer Nanoparticle Organ/Tumor Distribution

AbstractPurpose. To determine the in vivo biodistribution for differently charged poly(amidoamine) (PAMAM) dendrimers in B16 melanoma and DU145 human prostate cancer mouse tumor model systems. Methods. Neutral (NSD) and positive surface charged (PSD) generation 5 (d =5 nm) PAMAM dendrimers were synthesized by using 3H-labeled acetic anhydride and tested in vivo. Dendrimer derivatives were injected intravenously, and their biodistribution was determined via liquid scintillation counting of tritium in tissue and excretory samples. Mice were also monitored for acute toxicity. Results. Both PSD and NSD localized to major organs and tumor. Dendrimers cleared rapidly from blood, with deposition peaking at 1 h for most organs and stabilizing from 24 h to 7 days postinjection. Maximal excretion occurred via urine within 24 h postinjection. Neither dendrimer showed acute toxicity. Conclusions. Changes in the net surface charge of polycationic PAMAMs modify their biodistribution. PSD deposition into tissues is higher than NSD, although the biodistribution trend is similar. Highest levels were found in lungs, liver, and kidney, followed by those in tumor, heart, pancreas, and spleen, while lowest levels were found in brain. These nanoparticles could have future utility as systemic biomedical delivery devices.

Identification of Biological Tissues by Rapid Evaporative Ionization Mass Spectrometry

The newly developed rapid evaporative ionization mass spectrometry (REIMS) provides the possibility of in vivo, in situ mass spectrometric tissue analysis. The experimental setup for REIMS is characterized in detail for the first time, and the description and testing of an equipment capable of in vivo analysis is presented. The spectra obtained by various standard surgical equipments were compared and found highly specific to the histological type of the tissues. The tissue analysis is based on their different phospholipid distribution; the identification algorithm uses a combination of principal component analysis (PCA) and linear discriminant analysis (LDA). The characterized method was proven to be sensitive for any perturbation such as age or diet in rats, but it was still perfectly suitable for tissue identification. Tissue identification accuracy higher than 97% was achieved with the PCA/LDA algorithm using a spectral database collected from various tissue species. In vivo, ex vivo, and post mortem REIMS studies were performed, and the method was found to be applicable for histological tissue analysis during surgical interventions, endoscopy, or after surgery in pathology.

Formation of silver and gold dendrimer nanocomposites

Structural types of dendrimer nanocomposites have been studied and the respective formation mechanisms have been described, with illustration of nanocomposites formed from poly(amidoamine) PAMAM dendrimers and zerovalent metals, such as gold and silver. Structure of {(Au(0))n−PAMAM} and {(Ag(0))n−PAMAM} gold and silver dendrimer nanocomposites was found to be the function of the dendrimer structure and surface groups as well as the formation mechanism and the chemistry involved. Three different types of single nanocomposite architectures have been identified, such as internal (‘I’), external (‘E’) and mixed (‘M’) type nanocomposites. Both the organic and inorganic phase could form nanosized pseudo-continuous phases while the other components are dispersed at the molecular or atomic level either in the interior or on the surface of the template/container. Single units of these nanocomposites may be used as building blocks in the synthesis of nanostructured materials.

Characterization of crystalline dendrimer-stabilized gold nanoparticles

Monodispersed, highly crystalline dendrimer-stabilized gold nanoparticles (Au DSNPs) were synthesized via hydrazine reduction chemistry and stabilized using primary amine-terminated poly(amidoamine) (PAMAM) dendrimers of different generations (generations 2–6) with the same molar ratios of dendrimer terminal nitrogen ligands/gold atoms. The sizes of the synthesized Au DSNPs decrease with the increase of the number of dendrimer generations. These Au DSNPs are fluorescent and display strong blue emission intensity at 458 nm. Polyacrylamide gel electrophoresis (PAGE) analysis indicates that all Au DSNPs are stable and both metal NPs and dendrimer stabilizers do not separate from each other during the electrophoresis process. The synthesized inorganic/organic hybrid Au DSNPs provide new nanoplatforms that will be further modified with various biological ligands for the application of biosensing and targeted cancer therapeutics.

Absorption, uptake and tissue affinity of high-molecular-weight hyaluronan after oral administration in rats and dogs

The purpose of this study was to determine the absorption, distribution and excretion of (99m)technetium-labeled, high-molecular-weight hyaluronan (((99m)Tc-HA) and (99m)technetium pertechnetate ((99m)Tc-P) after single dose, oral administration to Wistar rats and Beagle dogs. A pilot study utilized (99m)Tc-HA alone, and a second confirmatory study compared uptake of labeled (99m)Tc-HA with (99m)Tc-P. Urinary and fecal excretion after (99m)Tc-HA ingestion by rats showed 86.7-95.6% of radioactivity was recovered, almost all in feces. All tissues examined showed incorporation of radioactivity from (99m)Tc-HA starting at 15 min and persisting for 48 h, in a pattern significantly different from (99m)Tc-P. Whole-body scintigraphs and close-ups of the ventral chest region showed nonalimentary radioactivity from (99m)Tc-HA concentrated in joints, vertebrae and salivary glands four hours after administration. Autoradiography of skin, bone and joint tissue pieces after 24 h showed incorporation of radioactivity from (99m)Tc-HA, but not from (99m)Tc-P. Conversely, absorption, distribution and excretion of (99m)Tc was completely different from (99m)Tc-HA, showing an expected pattern of rapid absorption and excretion in urine, with accumulation in thyroid glands, stomach, kidney and bladder. This report presents the first evidence for uptake and distribution to connective tissues of orally administered, high-molecular-weight HA.

Imaging {Au0-PAMAM} Gold-dendrimer Nanocomposites in Cells

Dendrimer nanocomposites (DNC) are hybrid nanoparticles formed by the dispersion and immobilization of guest atoms or small clusters in dendritic polymer matrices. They have a great potential in biomedical applications due to their controlled composition, predetermined size, shape and variable surface functionalities. In this work, d=5–25 nm spherical nanoparticles composed of gold and poly(amidoamine) (PAMAM) dendrimers have been selected to demonstrate this nanoparticle based concept. {Au(0)n-PAMAM} gold dendrimer nanocomposites with a well-defined size were synthesized and imaged by transmission electron microscopy both in vitro and in vivo. DNC have also the potential to be used for imaging and drug delivery vehicles either by utilizing bioactive guests or through the incorporation of radioactive isotopes, such as Au-198.

In Vivo Biodistribution of Dendrimers and Dendrimer Nanocomposites — Implications for Cancer Imaging and Therapy

Our results indicate that the surface chemistry, composition, and 3-D structure of nanoparticles are critical in determining their in vivo biodistribution, and therefore the efficacy of nanodevice imaging and therapies. We demonstrate that gold/dendrimer nanocomposites in vivo, present biodistribution characteristics different from PAMAM dendrimers in a B16 mouse tumor model system. We review important chemical and biologic uses of these nanodevices and discuss the potential of nanocomposite devices to greatly improve cancer imaging and therapy, in particular radiation therapy. We also discuss major issues confronting the use of nanoparticles in the near future, with consideration of toxicity analysis and whether biodegradable devices are needed or even desirable.

Strong synergy of heat and modulated electromagnetic field in tumor cell killing

Background and Purpose:Hyperthermia is an emerging complementary method in radiooncology. Despite many positive studies and comprehensive reviews, the method is not widely accepted as a combination to radiotherapy. Modulated electrohyperthermia (mEHT; capacitive, electric field modulated, 13.56 MHz) has been used in clinical practice for almost 2 decades in Germany, Austria and Hungary. This in vivo study in nude mice xenograft tumors compares mEHT with “classic” radiative hyperthermia (radHT).Material and Methods:Nude mice were xenografted with HT29 human colorectal carcinoma cells. 28 mice in four groups with seven animals each and two tumors per animal (totally 56 tumors) were included in the present study: group 1 as untreated control; group 2 treated with radHT at 42 °C; group 3 treated with mEHT at identical 42 °C; group 4 treated with mEHT at 38 °C (by intensively cooling down the tumor). 24 h after treatment, animals were sacrificed and the tumor cross sections studied by precise morphological methods for the respective relative amount of “dead” tumor cells.Results:The effect of mEHT established a double effect as a synergy between the purely thermal (temperature-dependent) and nonthermal (not directly temperature-dependent) effects. The solely thermal enhancement ratio (TER) of cell killing was shown to be 2.9. The field enhancement ratio (FER) at a constant temperature of 42 °C was measured as 3.2. Their complex application significantly increased the therapeutic enhancement to 9.4.Conclusion:mEHT had a remarkable cancer cell-killing effect in a nude mice xenograft model.Hintergrund und Ziel:Die Hyperthermie ist eine aufstrebende ergänzende Therapie in der Radioonkologie. Trotz zahlreicher positiver Studien und umfassender Reviews ist diese Methode immer noch nicht als Kombination zur Radiotherapie anerkannt. Die modulierte Elektrohyperthermie (mEHT; kapazitiv mit moduliertem elektrischem Feld, 13,56 MHz) wird seit fast 2 Jahrzehnten in Deutschland, Österreich und Ungarn klinisch angewandt. Die vorliegende In-vivo-Studie vergleicht in einem Xenograft-Nacktmaus-Tumormodell die mEHT mit der „klassischen“ radiativen Hyperthermie (radHT).Material und Methodik:Nacktmäuse wurden mit humanen kolorektalen HT29-Tumorzellen xenotransplantiert. 28 Mäuse in vier Gruppen zu je sieben Tieren mit zwei Tumoren pro Tier (gesamt 56 Tumoren) wurden in diese Studie einbezogen: Gruppe 1 als unbehandelte Kontrollgruppe; Gruppe 2 behandelt mit radHT bei 42 °C; Gruppe 3 behandelt mit mEHT ebenfalls bei 42 °C; Gruppe 4 behandelt mit mEHT bei 38 °C (durch intensive Kühlung des Tumors). 24 h nach der Behandlung wurden die Tiere getötet und die Tumorquerschnitte morphologisch auf den jeweiligen Anteil „toter“ Tumorzellen untersucht.Ergebnisse:Die Behandlung mit mEHT zeigte eine doppelte Wirkung als Synergie zwischen dem ausschließlich thermalen (temperaturabhängigen) und dem nichtthermalen (nicht direkt temperaturabhängigen) Effekt. Folgende Faktoren wurden gemessen: die durch alleinige Hyperthermie bedingte Verstärkung der Zellzerstörung („thermal enhancement ratio“ [TER]) mit dem Faktor 2,9; der alleinige Feldverstärkungseffekt („field enhancement ratio“ [FER]) bei konstanter Temperatur von 42 °C mit dem Faktor 3,2; die Kombination beider Effekte mit einem signifikant erhöhten Faktor von 9,4.Schlussfolgerung:Die durch ein moduliertes elektrisches Feld (13,56 MHz) erzeugte mEHT hatte in einem Nacktmaus-Xenograft-Tumormodell einen ausgeprägten tumorzellabtötenden Effekt.