Cytotoxicity Of Dendrimers Research Articles

Data-Driven Approaches to Predict Dendrimer Cytotoxicity.

Dendrimers are employed as functional elements in contrast agents and are proposed as nontoxic vehicles for drug delivery. Toxicity is a property that is to be evaluated for this novel class of bionanomaterials for in vivo applications. The current research is hampered due to the lack of structured data sets for toxicity studies for dendrimers. In this work, we have built a data set by curating literature for toxicity data and augmented it with structural and physicochemical features. We present a comprehensive, feature-rich database of dendrimer toxicity measured across various cell lines for prediction, design, and optimization studies. We have also explored novel computational approaches for predicting dendrimer cytotoxicity. We demonstrate superior outcomes for toxicity prediction using essential regression in the space of small data sets.

The Potential of Dendrimers as Alzheimer’s Disease Nanotherapeutics – Evidence of Antiamyloidogenic Properties Against Aβ42

AbstractBackgroundToday, available therapies for Alzheimer’s disease (AD) fail to modify the disease progression for the major AD clinical stages [1]. Therefore, new therapeutic approaches are an urgent need. Within nanomedicine, the use of nanomaterials, and dendrimers in particular, pose as promising AD therapies [2]. Dendrimers are globular macromolecules, with a well‐defined and hyperbranched structure, controllable nanosize (<15 nm) and multivalent surface. Due to their intrinsic characteristics, they are first‐in‐class for drug delivery as they can act as efficient and multivalent carriers of different therapeutic molecules. In addition, dendrimers can be used as a drug per se. Different types of dendrimers display antioxidant, anti‐inflammatory, anti‐prion and anti‐amyloidogenic properties [2]. With that in mind, a proprietary family of dendrimers was explored to hamper Aβ (1‐42) peptide aggregation.MethodThe dendrimer was synthesized following a divergent strategy up to generation 3 and characterized by nuclear magnetic resonance and Fourier‐transform infrared spectroscopy. To analyse the dendrimer’s effect on fibrillation, the aggregation kinetics of the Aβ (1‐42) peptide was studied by thioflavin T (ThT) in the presence of different dendrimer concentrations. The aggregates’ morphology was assessed by transmission electron microscopy. The cytotoxicity of dendrimers was evaluated in SH‐SY5Y cells by metabolic assays (resazurin).ResultThe dendrimers showed to interfere with Aβ fibrillation in a concentration‐dependent manner. Their behaviour was similar to fibrillation inhibitors that can break fibrils as well [3]. When at a lower molar ratio than Aβ, dendrimers hampered fibrillation and small/nonfibrillar aggregates were present. For a higher molar ratio (dendrimer/peptide ratio >1), they promoted Aβ fibrillation as more fibrils were present. More importantly, they were shown non‐toxic for neuroblastoma cells.ConclusionIn conclusion, these results show that these dendrimers can interfere with Aβ fibrillation. Future studies will be performed to understand the mechanism of action of this new family of dendrimers.

Biodistribution and toxicity assessment of methoxyphenyl phosphonium carbosilane dendrimers in 2D and 3D cell cultures of human cancer cells and zebrafish embryos

The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.

The application advances of dendrimers in biomedical field

AbstractDendrimers are a family of nano‐sized three‐dimensional polymers with unique dendritic branching structures and compact spherical geometries. In recent years, dendrimers have made a series of breakthroughs in the biomedical field. In this review, we introduce the synthesis principles, modification methods, and new materials designed based on dendrimers; discuss the importance of cytotoxicity of dendrimers for applications; and elaborate on their applications in the field of molecular assembly and cancer diagnosis and treatment. We speculate that in the near future, more new materials based on dendrimers will be applied in the biomedical field.

Interaction of Cationic Carbosilane Dendrimers and Their siRNA Complexes with MCF-7 Cells Cultured in 3D Spheroids.

Cationic dendrimers are effective carriers for the delivery of siRNA into cells; they can penetrate cell membranes and protect nucleic acids against RNase degradation. Two types of dendrimers (CBD-1 and CBD-2) and their complexes with pro-apoptotic siRNA (Mcl-1 and Bcl-2) were tested on MCF-7 cells cultured as spheroids. Cytotoxicity of dendrimers and dendriplexes was measured using the live–dead test and Annexin V-FITC Apoptosis Detection Kit (flow cytometry). Uptake of dendriplexes was examined using flow cytometry and confocal microscopy. The live–dead test showed that for cells in 3D, CBD-2 is more toxic than CBD-1, contrasting with the data for 2D cultures. Attaching siRNA to a dendrimer molecule did not lead to increased cytotoxic effect in cells, either after 24 or 48 h. Measurements of apoptosis did not show a high increase in the level of the apoptosis marker after 24 h exposure of spheroids to CBD-2 and its dendriplexes. Measurements of the internalization of dendriplexes and microscopy images confirmed that the dendriplexes were transported into cells of the spheroids. Flow cytometry analysis of internalization indicated that CBD-2 transported siRNAs more effectively than CBD-1. Cytotoxic effects were visible after incubation with 3 doses of complexes for CBD-1 and both siRNAs.

Nuclear-Targeting Delivery of CRISPRa System for Upregulation ofβ-Defensin against Virus Infection by Dexamethasone and Phenylalanine Dual-Modified Dendrimer

The dual-modified dendrimer containing dexamethasone (DET) and phenylalanine (Phe) was prepared to deliver plasmid DNA encoding dCas9 and single-guide RNA (sgRNA) for specific upregulation ofβ-defensin. DET and Phe moieties synergistically enhanced the transfection efficiency and reduced cytotoxicity of dendrimers. Combination of three sgRNAs targetingβ-defensin gene demonstrated higher activation efficacy ofβ-defensin than any single sgRNA and combinations of any two sgRNAs, showing an efficient inhibition of virus infection and replication. The titer of vesicular stomatitis virus (VSV) in the cells treated with dCas9-sgRNA targetingβ-defensin was reduced by about 100-fold compared to that of cells treated with dCas9-scramble sgRNA (dCas9-scr sgRNA).In vivoexperiments demonstrated that the DET- and Phe-modified dendrimer effectively delivered plasmid DNA encoding dCas9 protein into the airway epithelium, inducingβ-defensin expression. Delivery of the CRISPR activation system by a dendrimer modified with DET and Phe was a promising approach against viral disease.

Poly(lysine) Dendrimers Form Complexes with siRNA and Provide Its Efficient Uptake by Myeloid Cells: Model Studies for Therapeutic Nucleic Acid Delivery.

The disruption of the cellular pathways of protein biosynthesis through the mechanism of RNA interference has been recognized as a tool of great diagnostic and therapeutic significance. However, in order to fully exploit the potential of this phenomenon, efficient and safe carriers capable of overcoming extra- and intracellular barriers and delivering siRNA to the target cells are needed. Recently, attention has focused on the possibility of the application of multifunctional nanoparticles, dendrimers, as potential delivery devices for siRNA. The aim of the present work was to evaluate the formation of dendriplexes using novel poly(lysine) dendrimers (containing lysine and arginine or histidine residues in their structure), and to verify the hypothesis that the use of these polymers may allow an efficient method of siRNA transfer into the cells in vitro to be obtained. The fluorescence polarization studies, as well as zeta potential and hydrodynamic diameter measurements were used to characterize the dendrimer:siRNA complexes. The cytotoxicity of dendrimers and dendriplexes was evaluated with the resazurin-based assay. Using the flow cytometry technique, the efficiency of siRNA transport to the myeloid cells was determined. This approach allowed us to determine the properties and optimal molar ratios of dendrimer:siRNA complexes, as well as to demonstrate that poly(lysine) dendrimers may serve as efficient carriers of genetic material, being much more effective than the commercially available transfection agent Lipofectamine 2000. This outcome provides the basis for further research on the application of poly(lysine) dendrimers as carriers for nucleic acids in the field of gene therapy.

New Poly(Propylene Imine) Dendrimer Modified with Acridine and Its Cu(II) Complex: Synthesis, Characterization and Antimicrobial Activity

A second-generation poly(propylene imine) dendrimer modified with acridine and its Cu(II) complex have been synthesized for the first time. It has been found that two copper ions form complexes with the nitrogen atoms of the dendrimeric core by coordinate bonds. The new compounds have been characterized by nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), fourier-transform infrared spectroscopy (FTIR) and fluorescence spectroscopy. The spectral characteristics of the modified dendrimer have been measured in different organic solvents, and a negative fluorescence solvatochromism has been observed. The antimicrobial activity of the dendrimers has been tested against model pathogenic microorganisms in agar and by broth dilution method. The cotton fabric treated with both dendrimers has been evaluated towards pathogenic microorganisms. The obtained modified cotton fabrics have been shown to hamper bacterial growth and to prevent biofilm formation. Dendrimer cytotoxicity has been investigated in vitro in the model HEp-2 cell line.

Cytotoxicity of Dendrimers.

Drug delivery systems are molecular platforms in which an active compound is packed into or loaded on a biocompatible nanoparticle. Such a solution improves the activity of the applied drug or decreases its side effects. Dendrimers are promising molecular platforms for drug delivery due to their unique properties. These macromolecules are known for their defined size, shape, and molecular weight, as well as their monodispersity, the presence of the void space, tailorable structure, internalization by cells, selectivity toward cells and intracellular components, protection of guest molecules, and controllable release of the cargo. Dendrimers were tested as carriers of various molecules and, simultaneously, their toxicity was examined using different cell lines. It was discovered that, in general, dendrimer cytotoxicity depended on the generation, the number of surface groups, and the nature of terminal moieties (anionic, neutral, or cationic). Higher cytotoxicity occurred for higher-generation dendrimers and for dendrimers with positive charges on the surface. In order to decrease the cytotoxicity of dendrimers, scientists started to introduce different chemical modifications on the periphery of the nanomolecule. Dendrimers grafted with polyethylene glycol (PEG), acetyl groups, carbohydrates, and other moieties did not affect cell viability, or did so only slightly, while still maintaining other advantageous properties. Dendrimers clearly have great potential for wide utilization as drug and gene carriers. Moreover, some dendrimers have biological properties per se, being anti-fungal, anti-bacterial, or toxic to cancer cells without affecting normal cells. Therefore, intrinsic cytotoxicity is a comprehensive problem and should be considered individually depending on the potential destination of the nanoparticle.

Pyrrolidone Modification Prevents PAMAM Dendrimers from Activation of Pro-Inflammatory Signaling Pathways in Human Monocytes.

The biological features of dendrimers are affected by the character of highly reactive terminal moieties. In some polyamine dendrimer types the surface charge makes them bioincompatible and prevent their direct medical application. Moreover, foreign particles can induce the immune response which is undesirable due to the adverse side effects in vivo. The reduction of cytotoxicity of positively charged macromolecules is possible through chemical modifications of terminal groups. In our study, we have developed new derivatives of PAMAM dendrimers modified with 4-carbomethoxypyrrolidone and evaluated their immunomodulatory properties. The experiments were conducted on two human cancer myeloid cell lines: THP-1 and U937. To evaluate the cytotoxicity of dendrimers, the reasazurin assay was applied. The expression level of NF-κB targets (NFKBIA, BTG2) and cytokine genes (IL1B, TNF) was determined by quantitative real-time RT-PCR. The measurement of binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The ELISA cytokine assay was performed to measure protein concentration of IL-1β and TNFα. We have found that PAMAM-pyrrolidone dendrimers did not impact THP-1 and U937 viability even at high concentrations (up to 200 μM). The surface modification prevented PAMAM dendrimers from stimulating NF-κB-related signal transduction, which have been determined on the level of nuclear translocation, gene expression and protein secretion. Pyrrolidone modification efficiently prevents PAMAM dendrimers from stimulating pro-inflammatory response in human cancer myeloid cell lines, thus it can be used to improve the biocompatibility of positively charged dendrimers and to broaden the scope of their biological applications.

Fabrication of Low-Generation Dendrimers into Nanostructures for Efficient and Nontoxic Gene Delivery.

Dendrimers with well-defined molecular structure and high monodispersity have gained tremendous interest in gene delivery. However, current gene carriers based on dendrimers are either not effective or are too toxic on the transfected cells. The efficacy and cytotoxicity of dendrimers are strongly correlated with their molecular weight or generation. High-generation dendrimers are reported with relatively high transfection efficacy but serious cytotoxicity due to the excess positive charges on the polymers, while low-generation dendrimers with minimal toxicity have poor polyplex stability and thus weak transfection efficacy. To break up the correlation between efficacy and toxicity, low-generation dendrimers were fabricated into various nanostructures by several strategies to improve their gene-binding capacity, polyplex stability, and transfection efficacy without inducing additional toxicity. In this review article, we will highlight recent advances in the development of assembled dendrimer nanostructures for efficient and non-toxic gene delivery. Specifically, the principles and strategies in the fabrication of dendrimer nanostructures are intensively reviewed.

Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-\u03baB Pathway in a Myeloid Cell Line

PurposeFourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors.MethodsTo estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4.ResultsWe found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 μM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25–100 μM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected.ConclusionWe conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).

Gadobutrol-dendrimer effects on metastatic and apoptotic gene expression

Dendrimers are one of the most appropriate nanocaries for imaging moieties in imaging applications.The purpose of this study was the evalution of cytotoxicity and inducing apoptosis of dendrimers. This study was conducted in order to investigate the metastasis suppression effect of dendrimer in human breast MCF-7 cell line and finding the nanoparticle protein corona in biological enviromental. Dendrimer cytotoxicity effect was assessed by MTT assay. The mRNA experession level of KAI1 as a metastasis suppressor gene, Bax as Pro- apoptotic gene, Bcl-2 as an anti-apoptotic gene and GAPDH as a housekepping gene were determined by real-time PCR assays.concentration-dependent nanoparticle cytotoxicity effect was proofed at range of 1-2 mg/mL in 24 hours, significant upregulation of mRNA expression of Bax, was observed whereas expression of anti-apoptotic Bcl-2 was down-regulated, also expression of metastasis suppressor gene KAI1 was up-regulated. So far a few studies confirmed apoptosis enhancement effect of dendrimers in MCF-7 cell line via bax/bcl-2 pathways. dendrimer nanoparticles was able to act as metastase inhibitor via upregulation of KAI1 gene.

Predicting cytotoxicity of PAMAM dendrimers using molecular descriptors.

The use of data mining techniques in the field of nanomedicine has been very limited. In this paper we demonstrate that data mining techniques can be used for the development of predictive models of the cytotoxicity of poly(amido amine) (PAMAM) dendrimers using their chemical and structural properties. We present predictive models developed using 103 PAMAM dendrimer cytotoxicity values that were extracted from twelve cancer nanomedicine journal articles. The results indicate that data mining and machine learning can be effectively used to predict the cytotoxicity of PAMAM dendrimers on Caco-2 cells.

Specific gene delivery mediated by poly(ethylene glycol)-grafted polyamidoamine dendrimer modified with a novel HER2-targeting nanobody

As compared to other nanobiopolymers used in gene delivery applications, polyamidoamine dendrimers possess significantly improved physical and chemical properties such as monodispersity, well-defined size and shape, and biocompatibility, which make them ideal vectors for biomedical purposes. The aim of this study was fine-tuning of an anti–human epidermal growth factor receptor 2 (HER2) nanobody–polyethylene glycol (PEG)–G5 polyamidoamine gene delivery vector for potential in vivo applications. Polyethylene glycol was conjugated to polyamidoamine dendrimers at three different molar ratios of 10, 20, and 30 (polyethylene glycol:polyamidoamine). Anti-HER2 nanobody was chosen as our targeting agents and attached to polyethylene glycol–polyamidoamine conjugates. Cytotoxicity assays and transfection studies were carried out to find the most efficient conjugate in terms of both low cytotoxicity and high transfection rate. Our data indicated that PEGylation decreased the cytotoxicity of dendrimers alone and when complexed with DNA (dendriplexes). Among all of the polyethylene glycol–polyamidoamine dendrimers, polyethylene glycol(10)–polyamidoamine resulted in the most efficient gene transfection vector in both BT-474 and MCF-10A cell lines. Incorporation of anti-HER2 nanobodies could further increase their cellular uptake up to 1.7-fold compared to native dendrimers in HER2-overexpressing BT-474 cells but not in HER2-negative MCF-10A cells. It can be concluded from our results that nanobody–polyethylene glycol–polyamidoamine conjugates might be a promising new bioconjugate for the targeted gene delivery to HER2-positive tumor cells in vivo.

Synthesis, biodistribution, and imaging of PEGylated-acetylated polyamidoamine dendrimers.

Polyamidoamine (PAMAM) dendrimers have been widely used as drug carriers, non-viral gene vectors and imaging agents. However, the use of dendrimers in biological system is constrained because of inherent toxicity and organ accumulation. In this study, the strategy of acetylation and PEGylation-acetylation was used to minimize PAMAM dendrimers toxicities and to improve their biodistribution and pharmacokinetics for medical application. PEGylated-acetylated PAMAM (G4-Ac-PEG) dendrimers were synthesized by PEGylation of acetylated PAMAM dendrimer of generation 4 (G4) with acetic anhydride and polyethylene glycol (PEG) 3.4 k. To investigate the cytotoxicity and in vivo biodistribution of the conjugates, in vitro cell viability analysis, Iodine-125 (125I) imaging, tissue distribution and hematoxylin-eosin (HE) staining were performed. We find that acetylation and PEGylation-acetylation essentially eliminates the inherent dendrimer cytotoxicity in vitro. Planar gamma (gamma) camera imaging revealed that all the conjugates were slowly eliminated from the body, and higher abdominal accumulation of acetylation PAMAM dendrimer was observed. Tissue distribution analysis showed that PEGylated-acetylated dendrimers have longer blood retention and lower accumulation in organs such as the kidney and liver than the non-PEGylated-acetylated dendrimers, but acetylation only can significantly increase the accumulation of G4 in the kidney and decrease the concentration in blood. Histology results reveal that no obvious damage was observed in all groups after high dose administration. This study indicates that PEGylation-acetylation could improve the blood retention, decrease organ accumulation, and improve pharmacokinetic profile, which suggests that PEGylation-acetylation provides an alternative method for PAMAM dendrimers modification.

Novel aldehyde-terminated dendrimers; synthesis and cytotoxicity assay.

Polyamidoamine (PAMAM) dendrimers are a unique family of dendritic polymers with numerous pharmaceutical and biomedical applications. One major problem with these polymers is their cytotoxicity. The purpose of this study was to synthesize novel dendrimers with aldehyde terminal groups and compare their cytotoxicity with that of dendri¬mers containing amine-terminated groups. G1(first generation) and G2 (second generation) dendrimers with amine-terminated groups were synthesized by divergent method and then the amine-terminated groups were converted to the aldehyde groups using surface modification of the functional group inversion (FGI) method. The cytotoxicity of the novel G1 and G2 polyamidoaldehyde (PAMAL) dendrimers together with that of G1 and G2 PAMAM-NH2 dendrimers was investigated by MTT assay using MCF-7 cell line. The results showed that cytotoxicity of dendrimers with aldehyde-terminated groups is much lower than that of G1 and G2 PAMAM-NH2 dendri¬mers. Dendrimers with aldehyde-terminated groups could be used as novel and convenient carriers for drug delivery with low cytotoxic effect compared with the amine-terminated dendrimers. The results revealed that the same generations of the dendri¬mers with aldehyde-terminated groups are far less toxic than the corresponding amine-terminated dendrimers.

Polyamidoamine dendrimer nanoparticle cytotoxicity, oxidative stress, caspase activation and inflammatory response: experimental observation and numerical simulation

Mechanisms underlying the in vitro cytotoxicity of Polyamidoamine nano-dendrimers in human keratinocytes are explored. Previous studies demonstrated a systematic, dendrimer-generation-dependent cytotoxicity, oxidative stress, and genotoxicity. The emerging picture is of dendrimer endocytosis, endosomal rupture and subsequent mitochondrial attack and cell death. To understand the underlying mechanisms, the evolution of reactive oxygen species, intracellular glutathione, caspase activation, mitochondrial membrane potential decay, and inflammatory responses have been examined. Early-stage responses are associated with endosomal encapsulation, later-stage with mitochondrial attack. In all cases, the magnitude and evolution of responses depend on dendrimer generation and dose. The early-stage response is modelled using a rate equation approach, qualitatively reproducing the time, dose and generation dependences, using only two variable parameters. The dependence of the response on the nanoparticle physicochemical properties can thus be separated from internal cellular parameters, and responses can be quantified in terms of rate constants rather than commonly employed effective concentrations. From the Clinical EditorThis contribution reports on the intracellular mechanism of PAMAM dendrimer cytotoxicity in human keratinocytes. In all cases, the magnitude and evolution of responses depend on dendrimer generation and dose. Experimental data were supported by numerical simulation using only two variables. It is suggested that responses can be quantified in terms of rate constants rather than effective concentrations.

Taxol-Conjugated Pamam Dendrimers Utilize Three Modes of Action on Microtubule Structure

Paclitaxel (Taxol) is a cancer drug that causes cell death by stabilizing microtubules and consequently arresting cell division. Previously, the cytotoxicity of taxol-conjugated PAMAM dendrimers was demonstrated in cancer cells. The exact mode(s) of action responsible for the potent cytotoxicity of these dendrimers were not examined but the literature provides uncertainty that the taxol could be released from the dendrimer carrier after cellular entry. Accordingly, we asked whether the taxol-dendrimer conjugate itself is able to bind microtubules. To address this question, we investigated the effect of these conjugates on microtubules in vitro using total internal reflection fluorescence microscopy (TIRFM) and transmission electron microscopy (TEM). We find that the taxol-dendrimer conjugate affects microtubule structure in two ways: (1) the conjugate can bind tubulin during tubulin polymerization and stabilize it into a tubular structure and (2) the conjugate can bundle microtubules in a manner that is not dependent on taxol, but dendrimer electrostatics. Both of these modes of action would arrest cell division and consequently kill the cell. This is the first time that the binding of taxol-conjugated dendrimers to microtubules has been demonstrated in vitro. Furthermore, our results provide both mechanistic insights into the cytotoxicity of the previously characterized taxol-conjugated PAMAM dendrimers and additional evidence for the potential of these and similar conjugates as cancer therapeutics.

Effects of PEGylation and Acetylation of PAMAM Dendrimers on DNA Binding, Cytotoxicity and in Vitro Transfection Efficiency

Poly(amidoamine) (PAMAM) dendrimers are promising multipotent gene delivery vectors, providing favorable DNA condensation properties also in combination with the possibility of conjugation of different targeting ligands to their surface. They have been used for transfection both in vitro and in vivo, but their application is currently somewhat limited due to inherent cytotoxicity. In this work we investigate how two types of surface modification, acetylation and PEGylation, affect the DNA binding characteristics, the cytotoxicity and the in vitro transfection efficiency of generation 4 and 5 PAMAM dendrimers. Particularly, we address how the morphology of DNA-dendrimer complexes, formed under low salt conditions, changes upon dilution in cell growth medium, an event that inevitably occurs before the complexes reach the cell surface in any transfection experiment. We find that acetylation and PEGylation essentially eliminates the inherent dendrimer cytotoxicity. However, the transfection efficiency of the modified dendrimers is lower than that of the corresponding unmodified dendrimers, which can be rationally understood by our observations that DNA is less condensed when complexed with these modified dendrimers. Although small DNA-dendrimer particles are formed, the availability for ethidium intercalation and nuclease degradation is significantly higher in the modified DNA-dendrimer complexes than in unmodified ones. Dilution in cell growth medium has a drastic effect on these electrostatically assembled complexes, resulting in increase in size and DNA availability. Our results strongly add to the notion that it is of importance to perform a biophysical characterization under conditions as close to the transfection situation as possible, to enable conclusions regarding structure-activity relations of gene delivery vectors.