There and back again: a dendrimer's tale.

The development of molecular nanostructures with well-defined particle size and shape is of eminent interest in biomedicine. Among many studied nanostructures, dendrimers represent the group of those most thoroughly characterized ones. Due to their unique structure and properties, dendrimers are very attractive for medical and pharmaceutical applications. Owing to the controllable cavities inside the dendrimer, guest molecules may be encapsulated, and highly reactive terminal groups are susceptible to further modifications, e.g., to facilitate target delivery. To understand the potential of these nanoparticles and to predict and avoid any adverse cellular reactions, it is necessary to know the mechanisms responsible for an efficient dendrimer uptake and the destination of their intracellular journey. In this article, we summarize the results of studies describing the dendrimer uptake, traffic, and efflux mechanisms depending on features of specific nanoparticles and cell types. We also present mechanisms of dendrimers responsible for toxicity and alteration in signal transduction pathways at the cellular level.

Neurotoxicity of poly(propylene imine) glycodendrimers.

Published results of studies on poly(propylene imine) (PPI) dendrimers indicate their potential use in the treatment of brain cancer or neurodegenerative diseases due to their ability to cross the blood-brain barrier. However, depending on dose, neurotoxicity may occur. Here, we discuss the impact of maltotriose modified PPI dendrimers on rat's nervous system. Wistar rats were treated intravenously for 14 consecutive days with densely (dense-shell; DS) and partly (open-shell; OS) modified PPI dendrimers at doses established as safe in the previous experiment following a single DS or OS administration. The examination included an estimation of the motility and the clinical symptoms of the respiratory, nervous, and cardiovascular systems. Both DS and OS glycodendrimers (GDs) induced adverse effects at the doses tested. Multiple administrations of PPI-OS had a detrimental influence on rats' survival. These findings suggest that the dendrimers adversely influence the nervous system and their toxic effects accumulate over time. In PPI-DS treated animals, the harmful effects were less severe but still present. However, with each treatment day, the clinical symptoms in both groups were less severe as if the animals developed tolerance to GDs. We hypothesize that the neurotoxicity of tested dendrimers is related to nanoparticles-induced autophagy.

All That Glitters Is Not Silver-A New Look at Microbiological and Medical Applications of Silver Nanoparticles.

Silver and its nanoparticles (AgNPs) have different faces, providing different applications. In recent years, the number of positive nanosilver applications has increased substantially. It has been proven that AgNPs inhibit the growth and survival of bacteria, including human and animal pathogens, as well as fungi, protozoa and arthropods. Silver nanoparticles are known from their antiviral and anti-cancer properties; however, they are also very popular in medical and pharmaceutical nanoengineering as carriers for precise delivery of therapeutic compounds, in the diagnostics of different diseases and in optics and chemistry, where they act as sensors, conductors and substrates for various syntheses. The activity of AgNPs has not been fully discovered; therefore, we need interdisciplinary research to fulfil this knowledge. New forms of products with silver will certainly find application in the future treatment of many complicated and difficult to treat diseases. There is still a lack of appropriate and precise legal condition regarding the circulation of nanomaterials and the rules governing their safety use. The relatively low toxicity, relative biocompatibility and selectivity of nanoparticle interaction combined with the unusual biological properties allow their use in animal production as well as in bioengineering and medicine. Despite a quite big knowledge on this topic, there is still a need to organize the data on AgNPs in relation to specific microorganisms such as bacteria, viruses or fungi. We decided to put this knowledge together and try to show positive and negative effects on prokaryotic and eukaryotic cells.

Maltotriose-modified poly(propylene imine) Glycodendrimers as a potential novel platform in the treatment of chronic lymphocytic Leukemia. A proof-of-concept pilot study in the animal model of CLL.

Cancer nanotherapeutics have shown promise in resolving some of the limitations of conventional drug delivery systems such as nonspecific biodistribution and targeting, lack of water solubility, and low therapeutic indices, Among the various nanoparticles that are available, dendrimers, highly branched macromolecules with a specific size and shape, are one of the most promising ones. In this preliminary study, we tested the anti-tumor activity of maltotriose-modified fourth-generation poly(propylene imine) glycodendrimers (PPI-G4-M3) in vivo in the subcutaneous MEC-1 xenograft model of human chronic lymphocytic leukemia (CLL) in NOD scid gamma mice. Fludarabine was used for model validation and as a positive treatment control. The anti-tumor response was calculated as tumor volume, tumor control ratio, and tumor growth inhibition. The study showed that PPI-G4-M3 inhibited subcutaneous tumor growth more efficiently than fludarabine. The anti-tumor response was dose-dependent. Cationic PPI-G4-M3 showed the highest anti-tumor activity but also higher toxicity than the neutral dendrimers and fludarabine. These first promising results warrant further studies in the optimization of dendrimers charge, dose, route and schedule of administration to combat CLL.

Affecting NF-κB cell signaling pathway in chronic lymphocytic leukemia by dendrimers-based nanoparticles.

The complex genetic diversity of chronic lymphocytic leukemia (CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven, maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) modulate BCR, TRAIL and WNT signaling pathway gene expression in CLL cells and strongly influence their survival by inducing apoptosis and inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60 K microarray, as it is one of the major factors in the pathogenesis of B-cell neoplasms. The findings were compared with those obtained with Fludarabine (FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sized macromolecules for closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties.

The influence of maltotriose-modified poly(propylene imine) dendrimers on the chronic lymphocytic leukemia cells in vitro: dense shell G4 PPI.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Europe and North America. For many years scientists and doctors have been working on introducing the most effective therapy into CLL as prognosis of survival time and the course of the disease differ among patients, which might pose a problem in treating. Nanotechnology is providing new insights into diagnosis and, compared with conventional treatments, more efficient treatments, which might improve patients' comfort by decreasing side effects. Among the various nanoparticles that are available, dendrimers are one of the most promising. The aim of this study was a preliminary assessment of the clinical value of treating CLL patients with fourth generation poly(propylene imine) (PPI) dendrimers-either unmodified (PPI-G4) or approximately 90% maltotriose-modified (PPI-G4-DS-Mal-III). PPI-G4-DS-Mal-III dendrimers have, in contrast to the cationic PPI-G4, a neutral surface charge and are characterized by low cyto-, geno-, and hematotoxicity in vitro and in vivo. For the in vitro study we used blood mononuclear cells collected from both untreated CLL patients and from healthy donors. Apoptosis was measured by an annexin-V (Ann-V)/propidium iodide (IP) assay, and mitochondrial membrane potential was estimated with use of Mito Tracker Red CMXRos. Presented results confirm the influence of dendrimers PPI-G4 and PPI-G4-DS-Mal-III on apoptosis and CLL lymphocytes viability in in vitro cultures. Both tested dendrimers demonstrated higher cytotoxicity to CLL cells than to healthy donors cells, whereas unmodified dendrimers were more hematotoxic. The surface modification clearly makes glycodendrimers much more suitable for biomedical applications than unmodified PPI-G4; therefore further biological evaluations of these nanoparticles are conducted in our laboratories.

Type of serum influences the rituximab dependent cytotoxicity and apoptosis of chronic lymphocytic leukemia cells in vitro.

PURPOSE: The aim of the study was to compare the influence of types of serum on the in vitro viability and on either spontaneous or rituximab (RIT)-induced apoptosis of chronic lymphocytic leukemia (CLL) cells. METHODS: The influence of fetal calf serum (FCS), patients' autologous serum (AS) and human AB-serum (ABS), used alone and in combinations consisting of two of them (v/v-1:1), on RIT-dependent cytotoxicity, apoptosis, detection of active forms of caspases-3,-9,-8 and disruption of mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. RIT was used at the concentration of 10 µg/ml. The spontaneous apoptosis was assessed in culture without RIT. RESULTS: AS revealed the protective action on CLL cells, however this serum added in vitro to the culture either alone or in combination with FCS was the only one to allow RIT to exert its cytotoxic action against CLL cells. RIT-induced apoptosis involved changes in ΔΨm and activation of caspases-3,-8,-9 when AS+FCS was applicated. Drug induced apoptosis (DIA) was 6.02 and 0.34, when FCS+AS and FCS alone were used, respectively (p<0.01). The RIT-dependent cytotoxic effect decreased when FCS+AS or FCS+ABS were used, as compared to effect of AS used separately. The cytotoxic effect of RIT did not depend on drug concentration, but on the type of serum added to the culture. CONCLUSIONS: The strongest cytotoxic effect of RIT in the presence of AS suggests that this drug activity towards CLL cells is enhanced by known cytotoxic mechanisms, caspase-dependent apoptotic pathway and possible influence of other extracellular factors present in the patients' sera.

Activated B Cells and Plasma Cells Are Resistant to Radiation Therapy.

PURPOSE: B cells play a key role in outcomes of cancer patients and responses to checkpoint blockade immunotherapies. However, the effect of radiation therapy on B cell populations is poorly understood. Here we characterize the effects of radiation on the development, survival, and phenotype of physiological B-cell subsets. METHODS AND MATERIALS: Naïve and immunized tumor bearing and nontumor bearing mice were treated with large-field or focal stereotactic radiation and distinct B-cell subsets of varying developmental stages were analyzed by flow cytometry and real-time reverse transcription polymerase chain reaction. RESULTS: We first report that focal stereotactic radiation is highly superior to large-field radiation at inducing tumor infiltration of B cells, CD8+ T cells, and macrophages. We observed that radiation affects B cell development in the bone marrow, increasing frequencies of early pro-B cells and late pro-B cells while inducing upregulation of programmed cell death protein 1. We then demonstrate that class switched B cells and plasma cells are highly resistant to radiation therapy compared with naïve B cells and upregulate activation markers programmed cell death 1 ligand 2 and major histocompatibility complex class II) after radiation. Mechanistically, radiation upregulates Xbp1 and Bcl6 in plasma cells, conferring radioresistance. Furthermore, using an immunization approach, we demonstrate that radiation enhances activation-induced cytidine deaminase mediated class switching and somatic hypermutation in primed B cells. CONCLUSIONS: These data demonstrate that stereotactic radiation is superior to large-field radiation at inducing infiltration of immune cells into tumors and that plasma cells and class switched B cells are highly resistant to radiation therapy. These results represent the most comprehensive analysis of the effects of radiation on B cells to date and identify novel mechanisms by which radiation modulates immune cells within the tumor microenvironment.

Monomethyl auristatin antibody and peptide drug conjugates for trimodal cancer chemo-radio-immunotherapy.

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control.

Anti-Tumour Activity of Glycodendrimer Nanoparticles in a Subcutaneous MEC-1 Xenograft Model of Human Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic Lymphocytic Leukaemia (CLL) is an indolent disorder, which mainly affects older adults. Since the advent of chemoimmunotherapy, great progress has been made in its treatment. However, some patients develop a more aggressive form of the disease and are included in the group of high-risk CLL patients with a dismal prognosis and a need for new therapies. OBJECTIVE: Maltotriose-modified poly(propylene imine) dendrimers were presented as potential agents in targeted therapy for CLL in the murine xenograft model. METHODS: Tumour, brain and internal organs resected from NOD scid gamma mice were subjected to gross and histopathological evaluation. RESULTS: The results of ex vivo tissue examination indicated that open-shell glycodendrimers prevented/inhibited the spread of CLL into the brain and internal organs and its transformation into a more aggressive form. CONCLUSION: The results of the study have a potentially important impact on the design of future personalized therapies as well as clinical trials.

B Cells Improve Overall Survival in HPV-Associated Squamous Cell Carcinomas and Are Activated by Radiation and PD-1 Blockade.

PURPOSE: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations. EXPERIMENTAL DESIGN: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV+ HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade. RESULTS: RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade. CONCLUSIONS: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells.

Blockage of Wnt/ß-Catenin Signaling by Nanoparticles Reduces Survival and Proliferation of CLL Cells In Vitro-Preliminary Study.

The Wnt/ß-catenin signaling pathway is shown to play a significant role in the control of the survival, proliferation, and differentiation of hematopoietic cells. Studies have confirmed that aberrant activation of canonical Wnt signaling occurs in various forms of leukemia, and is crucial for chronic lymphocytic leukemia (CLL) pathogenesis. The aim of the study is to evaluate the influence of maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) on Wnt/ß-catenin pathway gene expression in CLL (MEC-1) cells and to compare these findings with those obtained with fludarabine (FA). Microarray data analysis reveals seven of 19 Wnt/ß-catenin pathway genes whose expression changes significantly during dendrimer and FA treatment: WNT10A, WNT6, and CDH1 among others. PPI-G4-M3 is already known to influence MEC-1 cell apoptosis and proliferation. The obtained results suggest that the reduction in cell survival under the influence of glycodendrimers and FA may be due to loss of Wnt signaling.

PPI-G4 Glycodendrimers Upregulate TRAIL-Induced Apoptosis in Chronic Lymphocytic Leukemia Cells.

Although chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western world, it remains incurable with conventional chemotherapeutic agents. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an antitumor candidate in cancer therapy. This study examines the proapoptotic effects of poly(propylene imine) (PPI) glycodendrimers modified with the maltotriose residues (PPI-G4-OS-Mal-III and PPI-G4-DS-Mal-III) on the TNF family in CLL cells. The combination of an understanding of the signaling pathways associated with CLL and the development of a molecular profiling is a key issue for the design of personalized approaches to therapy. Gene expression is determined with two-color microarray 8 × 60K. The findings indicate that PPI-G4-OS/DS-Mal-III affect gene expression from the TRAIL apoptotic pathway and exert a strong effect on CLL cells comparable with fludarabine. Dendrimer-targeted technology may well prove to bridge the gap between the ineffective treatment of today and the effective personalized therapy of the future.

Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment. OBJECTIVE: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier. METHOD: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes. RESULTS: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells. CONCLUSION: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.

Dendrimer-based nanoparticles for potential personalized therapy in chronic lymphocytic leukemia: Targeting the BCR-signaling pathway.

Chronic lymphocytic leukemia (CLL) is one of the most prevalent forms of leukemia in western society. Although classic chemoimmune therapy is still the gold standard of care for leukemic patients, effective therapy of CLL is yet to be achieved. The present study examines the influence of poly(propylene)imine (PPI) dendrimers with primary amino surface groups modified with maltotriose residues in approximately 90% (PPI-G4-DS-Mal-III) or 30% (PPI-G4-OS-Mal-III) of cases on CLL cells (MEC-1 cell line with del(17p)), and confirms that the main trigger in this interaction is the induction of the apoptotic mechanism. The efficacy of each dendrimer was compared using fludarabine (FA). Gene expression profiling (GEP) by microarray identified a group of genes in the BCR signaling pathway characterized by different levels of expression directly associated with the tested agent and type of interaction. Network analysis revealed the potential patterns involved in potential personalized therapy of CLL. The expression of most BCR genes decreased under the influence of dendrimers, which might translate into decreased maturation and proliferation of CLL lymphocytes. Moreover, PPI-G4-OS/DS-Mal-III dendrimers affected gene expression and CLL cells in a different way to FA. Thanks to unique properties, dendrimers may be specifically targeted, thus improving the effectiveness of CLL therapy.

Toxicity and proapoptotic activity of poly(propylene imine) glycodendrimers in vitro: considering their contrary potential as biocompatible entity and drug molecule in cancer.

Since the first dendrimers were synthesized, scientists around the world have studied their properties and potential applications. Cationic dendrimers are characterized by significant toxicity due to their interactions with cells components. The replacement of all cationic surface groups by neutral ones and therefore diminishing positive charge reduces the toxicity but may also lead to loss of dendrimers' desirable properties and restrict their biomedical applications. We have compared the cytotoxicity, as well as proapoptotic and antiproliferative activity of unmodified fourth generation PPI dendrimer (PPI-G4) and dendrimers modified with maltose (Mal) or maltotriose (Mal-III) - for full (dense shell - DS) or partial (open shell - OS) surface modifications. We have proved that among glycodendrimers, the OS-Mal PPI-G4 dendrimer is the most toxic, whereas DS-Mal-III molecule shows relatively weak or even no effect. We have also confirmed that OS dendrimers, both maltotriose and maltose modified, not only reduce cancer cells viability by inducing apoptosis but also inhibit their proliferation. The use of dendrimers as an active substance, which may be a drug per se is one of the most exciting and clinically important applications of cancer nanotechnology, therefore a partial modification of the surface appears to be a perfect solution for this purpose.

Toward personalized therapy for chronic lymphocytic leukemia: DSC and cDNA microarray assessment of two cases.

The differences in clinical course of chronic lymphocytic leukemia could have an impact on variations in a patient's response to therapy. Our published results revealed that thermal transition (95 ± 5°C) in differential scanning calorimetry profiles appear to be characteristic for the advanced stage of CLL. Moreover, a decrease/loss of this transition in nuclei from leukemic cells exposed to drugs ex vivo could indicate their diverse efficacy. It seems that the lack of changes in thermal profile could predict patient's drug resistance. In this study, we demonstrate the results obtained after drug treatment of leukemic cells by calorimetry, apoptosis-related parameters involved in expression of genes using cDNA microarray and western blot. These data were compared with the patients' clinical parameters before and after RCC therapy (rituximab + cladribine + cyclophosphamide). The complementary analysis of studied cases with opposite clinical response (CR or NR) revealed a strong relationship between clinical data, differences in thermal scans and apoptosis-related gene expression. We quantified expression of eight of apoptosis-related 89 genes, i.e., NOXA, PUMA, APAF1, ESRRBL1, CASP3, BCL2, BCL2A1 and MCL1. Particular differences in NOXA and BCL2 expression were revealed. NOXA expression in cells of patients who achieved a complete response to RCC therapy was 0.44 times higher in comparison to control ones. Interestingly, in the case of patients who did not respond to immunotherapy, NOXA expression was highly downregulated (RQ = 4.39) as compared with untreated cells. These results were confirmed by distinct cell viability, protein expression as well as by differences in calorimetry profiles.

Apoptotic gene expression under influence of fludarabine and cladribine in chronic lymphocytic leukemia-microarray study.

BACKGROUND: A deep insight into gene expression profiling (GEP) is a key to understanding the background of disease. It can lead to identification of diagnostic and prognostic factors and then to a selection of the most appropriate therapy. The aim of this study was to evaluate differences in apoptotic gene expression in chronic lymphocytic leukemia (CLL) cells influenced by fludarabine (FA) or cladribine (2-CdA). METHODS: GEP was performed in cells obtained from 10 untreated CLL patients and cultured in vitro with FA or 2-CdA. Ninety-three selected apoptotic genes were analyzed using 384 TaqMan® Low Density Arrays in pooled RNA. RESULTS: Relevant results were found in a set of 27 genes, however, the most striking differences between FA and 2-CdA were observed in the following 5 genes: BAD, TNFRSF21, DAPK1, CARD 6 and CARD 9. CONCLUSION: We have found some differences in apoptotic gene expression between FAand 2-CdA. These findings give prominence to genes qualifying for further studies currently conducted in our Department.

Changes in the apoptotic gene expression profile in CLL patients treated with rituximab combined with cladribine and cyclophosphamide-preliminary results.

The study was aimed to investigate modifications of apoptotic gene expression profile by microarray technique in 10 patients with chronic lymphocytic leukemia by treatment with rituximab, cladribine and cyclophosphamide (RCC) according to IGHV mutational status. The TaqMan Low Density Array for 96 gene transcripts was used. Those modifications followed two distinctive patterns largely overlapping the IGHV mutational status. In the IGHV-mutated group, the expression of many proapoptotic genes increased after treatment as compared to initial value. Our results suggest that RCC drugs may act through influence on the expression of some apoptosis-involved genes dependently on the IGVH mutational status.