Abstract
Abstract 4601
Treatment of chronic lymphocytic leukemia (CLL), including conventional therapy based on alkylating agents, purine nucleoside analogues (cladribine, fludarabine (FA)) or monoclonal antibodies (rituximab (Rit), alemtuzumab), significantly improved overall and complete responses. Although there are many treatment possibilities, this disease still remains incurable. Therefore, searching for new therapeutical strategies in CLL is vital.
Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles (polymers) of dendritic architecture, used as carriers of drugs, nucleic acid and photosensitizers for targeted delivery, contrast agents in magnetic resonance imaging (MRI) have already been reported. It seems likely that dendrimers themselves might be damaging for neoplastic cells.
The aim of our study was to preliminarly assess the clinical value of treating CLL patients with poly(propylene imine) (PPI) dendrimers. The assessment was based on the in vitro induction of cytotoxicity and apoptosis by fourth generation PPI dendrimers, with the surface amino groups substituted with maltotriose [Mal-III] residues. PPI-G4-OS-Mal-III dendrimers have, in contrast to the parental cationic PPI-G4, a virtually neutral surface charge. Therefore, they are preferentially involved in H-bond driven interactions.
The study was conducted in 15 untreated CLL patients (7 men, 8 women) who were diagnosed in accordance with IWCLL criteria and followed at the Hematology Department, Medical University, Lodz, Poland. The Ethics Committee of the Medical University of Lodz, Poland approved the study (RNN/75/10/KE). Informed consent was obtained from all patients involved in the study. The mean age of CLL patients was 63.80 yrs.
Peripheral blood mononuclear cells (MNCs) were separated from EDTA fresh blood. MNCs were incubated with dendrimers at concentrations of 4, 6 and 8 mg/ml.
The cultures without dendrimers served as controls.
Dendrimers in which approximately 35% of peripheral amino groups were coated with Mal-III (Leibniz Institute of Polymer Research, Dresden, Germany) have been defined as PPI-G4-OS-Mal-III. The abbreviation OS describes the open shell structure of carbohydrate-modified dendrimers (Fig. 1). The molar mass of this PPI dendrimer was 31000 g/mol.
Apoptosis was measured by the Annexin-V/Propidium Iodide test. A decrease in mitochondrial membrane potential (one of the earliest events in the apoptotic pathway) was evaluated by the CMXRos technique, using flow cytometry. The percentage of cells with lower mitochondrial membrane potential (MMP) (DYmlow/Gly-A−) was determined.
The percentage of apoptotic MNCs induced by PPI-G4-OS-Mal-III after 24 h and 48 h incubations was significantly higher than the percentage of spontaneous apoptotic leukemic cells. PPI glycodendrimers induced MNC apoptosis to a greater degree after 48 h than 24 h. The largest differences were observed for Ann-V+JP+ cells at a concentration of 8 mg/ml (p = 0.007). The IC50 value after 48-h-incubation was calculated as 8.24 mg/ml.
Moreover, the CMXRos technique revealed apoptosis induction by PPI-G4-OS-Mal-III at each examined concentration in comparison with control cultures (Table 1).
Table 1. 24h Control PPI-G4-OS-Mal-III 4 mg/ml PPI-G4-OS-Mal-III 6 mg/ml PPI-G4-OS-Mal-III 8 mg/ml FA 1.6 μM Rit 10 μg/ml X 23.7 51.0 60.1 64.4 62.9 28.0 48h X 28.3 67.8 71.9 79.3 89.0 29.4
X – mean percentage of cells with lower mitochondrial potential (DYmlow/Gly-A− [%])
Finally, PPI-G4-OS-Mal-III was not observed to have any harmful effects on erythrocytes (RBCs) or platelets (PLTs). The studied dendrimer induced PLT aggregation at a concentration of 50 mg/ml. Hemolysis induced by the PPI-G4-OS-Mal-III dendrimer is not important from the biological point of view.
To summarise, the PPI-G4-OS-Mal-III dendrimer demonstrated higher cytotoxicity towards CLL cells than healthy donor cells. Its potency to trigger apoptosis is similar to many PNAs and monoclonal antibodies widely used in CLL therapy. Thus, dendrimers are a potential tool for CLL treatment.
The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371 from the National Science Centre, Poland.
Fig. 1. Fig. 1.
Disclosures:
No relevant conflicts of interest to declare.
Characterization of the phytohemagglutinin-induced proliferating lymphocyte subpopulations in chronic lymphocytic leukemia patients using a clonogenic agar technique and monoclonal antibodies