Acid- and salt-triggered multifunctional poly(propylene imine) dendrimer as a prospective drug delivery system

A novel dendrimeric compound is designed with the objective of simultaneously addressing issues commonly encountered in drug delivery, i.e., stability in biological milieu as well as targeting. For this purpose, a multifunctional dendrimeric system derived from diaminobutane poly(propylene imine) dendrimers (DAB) is prepared bearing at its external surface poly(ethylene glycol) chains and guanidinium moieties. For these moieties, it has been established that they exhibit protective and targeting properties, respectively. The release of encapsulated compounds is triggered by titration with acids followed by the addition of sodium chloride solution. Specifically for pyrene, the solubilization site of which can be clearly traced, protonation leads to a distribution between the core and the poly(ethylene glycol) chains in the periphery of the dendrimer while it is released to the aqueous bulk solution by the addition of sodium chloride. The release of betamethasone valerate is also triggered by the addition of sodium chloride solution.     

pH response of conformation of poly(propylene imine) dendrimer in water: a molecular simulation study

This work presents the first molecular dynamics simulation of poly(propylene imine) (PPI) dendrimer in explicit water under various pH conditions. The sizes, shapes, surfaces/volumes and density profiles of the PPI dendrimer are analysed. The PPI dendrimer essentially approaches the perfect sphere under all pH conditions, and higher pH leads to more globular structure. The radius of gyration, solvent-accessible surface area (SASA) and solvent-excluded volume (SEV) are all found to increase significantly from high pH to neutral pH and to level thereafter until low pH, which illustrate the dramatic changes in the whole conformation of the dendrimer. These behaviours of the PPI dendrimer quite differ from those of polyamidoamine [Liu, et al. J. Am. Chem. Soc. 2009, 131, 2798–2799], which can be explained by the favourable interactions arising from the additional amide groups. The density profiles have also been calculated to confirm the shifts of density and back-folding of terminals and penetrations of water.     

Genotoxicity of poly(propylene imine) dendrimers

Dendrimers are highly branched macromolecules with the potential in biomedical applications. Due to positively charged surfaces, several dendrimers reveal toxicity. Coating peripheral cationic groups with carbohydrate residues can reduce it. In this study, the cytotoxicity and genotoxicity of three types of 4th generation poly(propylene imine) dendrimers were investigated. Peripheral blood mononuclear cells (PBMCs) were treated with uncoated (PPI-g4) dendrimers, and dendrimers in which approximately 40% or 90% of peripheral amino groups were coated with maltotriose (PPI-g4-OS or PPI-g4-DS) at concentration of 0.05, 0.5, 5 mg/ml. Abbreviations OS and DS stand for open shell and dense shell respectively, that describes the structure of carbohydrate modified dendrimers. After 1 h of cell incubation at 37°C, the MTT and comet assays were performed. PPI dendrimers demonstrated surface-modification-degree dependent toxicity, although genotoxicity of PPI-g4 and PPI-g4-OS measured by the comet assay was concentration dependent up to 0.5 mg/ml and at 5 mg/ml the amount of DNA that left comet's head decreased. Results may suggest a strong interaction between dendrimers and DNA, and furthermore, that coating PPI dendrimers by maltoriose is an efficient method to reduce their genotoxicity what opens the possibilities to use them as therapeutic agents or drug carriers.     

Zirconia-poly(propylene imine) dendrimer nanocomposite based electrochemical urea biosensor

In this article we report a selective urea electrochemical biosensor based on electro-co-deposited zirconia-polypropylene imine dendrimer (ZrO₂-PPI) nanocomposite modified screen printed carbon electrode (SPCE). ZrO₂ nanoparticles, prepared by modified sol–gel method were dispersed in PPI solution, and electro-co-deposited by cyclic voltammetry onto a SPCE surface. The material and the modified electrodes were characterised using FTIR, electron microscopy and electrochemistry. The synergistic effect of the high active surface area of both materials, i.e. PPI and ZrO₂ nanoparticles, gave rise to a remarkable improvement in the electrocatalytic properties of the biosensor and aided the immobilisation of the urease enzyme. The biosensor has an ampereometric response time of ∼4 s in urea concentration ranging from 0.01 mM to 2.99 mM with a correlation coefficient of 0.9985 and sensitivity of 3.89 μA mM⁻¹ cm⁻². The biosensor was selective in the presence of interferences. Photochemical study of the immobilised enzyme revealed high stability and reactivity.     

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.     

Biophysical characterization of a riboflavin-conjugated dendrimer platform for targeted drug delivery

The present study describes the biophysical characterization of generation-five poly(amidoamine) (PAMAM) dendrimers conjugated with riboflavin (RF) as a cancer-targeting platform. Two new series of dendrimers were designed, each presenting the riboflavin ligand attached at a different site (isoalloxazine at N-3 and d-ribose at N-10) and at varying ligand valency. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the binding activity for riboflavin binding protein (RfBP) in a cell-free solution. The ITC data shows dendrimer conjugates have K(D) values of ≥ 465 nM on a riboflavin basis, an affinity ~93-fold lower than that of free riboflavin. The N-3 series showed greater binding affinity in comparison with the N-10 series. Notably, the affinity is inversely correlated with ligand valency. These findings are also corroborated by DSC, where greater protein-conjugate stability is achieved with the N-3 series and at lower ligand valency.     

Coarse-grained modelling of urea-adamantyl functionalised poly(propylene imine) dendrimers

To investigate the behaviour of poly(propylene imine) dendrimers – and urea–adamantyl functionalised ones – in solution using molecular dynamics simulations, we developed a coarse-grained model to tackle the relatively large system sizes and time scales needed. Harmonic bond and angle potentials were derived from atomistic simulations using an iterative Boltzmann inversion scheme, modified to incorporate Gaussian fits of the bond and angle distributions. With the coarse-grained model and accompanying force field simulations of generations 1–7 of both dendrimer types in water were performed. They compare favourably with atomistic simulations and experimental results on the basis of size, shape, monomer density, spacer back-folding and atomic form factor measurements. These results show that the structural dynamics of these dendrimers originate from flexible chains constrained by configurational and spatial requirements. Large dendrimers are more rigid and spherical, while small ones are flexible, alternatively rod-like and globular.     

Stepwise self-assembled poly(amidoamine) dendrimer and poly(styrenesulfonate) microcapsules as sustained delivery vehicles

Hollow microcapsules comprised of poly(styrenesulfonate) (PSS) and a fourth generation poly(amidoamine) dendrimer (4G PAMAM) were prepared by depositing PSS/4G PAMAM multilayers on melamine formaldehyde (MF) colloid particles by the layer-by-layer self-assembly technique and subsequently dissolving the templated cores. The PSS/4G PAMAM layers were unstable toward the core removal procedure (pH approximately 1), resulting in a low yield of intact hollow capsules (<10% for 3.5 microm diameter MF templates). Stretching of the multilayer film due to core swelling during MF core dissolution leads to partial or complete destruction of capsules, giving discrete PSS-4G PAMAM complexes. Yields were increased by increasing inter- and intramolecular attractive forces between the PSS chains in the capsules through electrostatic, hydrophobic, and a combination of these interactions. The yields, however, were practically unaffected by enhancing such effects between dendrimer molecules. Transmission electron microscopy and scanning force microscopy measurements show no deformation for 3.5 microm capsules stabilized through the various interactions stated above. Further, capsules were filled with low molecular weight dextran sulfate and subsequently loaded with a model, therapeutically active molecule, doxorubicin hydrochloride (DOX). Release of DOX from the capsules was also studied to highlight the drug delivery potential of the dendrimer-based microcapsules.     

Direct plant gene delivery with a poly(amidoamine) dendrimer

Plant gene delivery is challenging due to the presence of plant cell walls. Conventional means such as Agrobacterium infection, biolistic particle bombardment, electroporation, or polyethylene glycol attachment are often characterized by high cost, labor extensiveness, and a significant perturbation to the growth of cells. We have succeeded in delivering GFP-encoding plasmid DNA to turfgrass cells using poly(amidoamine) dendrimers. Our new scheme utilizes the physiochemical properties as well as the nanosize of the poly(amidoamine) dendrimer for direct and noninvasive gene delivery. The GFP gene was expressed in the plant cells as observed by confocal fluorescence microscopy. The transfection efficiency may be further improved by optimizing the pH of the cell culture medium and the molar ratio of the dendrimer to DNA. The use of the current delivery system can be extended to virtually all plant species having successful regeneration systems in place.     

Dendritic poly(ether imine) based gene delivery vector

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether imine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.     

Synthesis of 6-O-methotrexylhyaluronan as a drug delivery system

Selective halogenation of hyaluronan and partial halogen substitution by methotrexate led to 6-chloro-6-deoxy-6-O-methotrexylhyaluronan, a potential antitumor drug. The remaining halogen could be further substituted by a second organic carboxylate, leading to mixed esters. 6-O-Acetyl-6-O-methotrexylhyaluronan and 6-O-butyryl-6-O-methotrexylhyaluronan were thus synthesized and characterized by NMR spectroscopy.     

Epidermal growth factor (EGF) receptor targeted delivery of PEGylated adenovirus

A biotin-polyethylene glycol (PEG)-epidermal growth factor (EGF) conjugate was immobilized onto the surface of avidin-modified adenovirus (ADV-Avi) via biotin-avidin interaction to deliver ADV specifically to EGF receptor over-expressing cancer cells. ADV-Avi/biotin-PEG-EGF complexes showed greatly enhanced intracellular uptake of ADV particles for an EGF receptor positive cell line (A431 cells), compared to naked or PEG alone immobilized ADV. ADV coding an exogenous GFP gene was used to quantitatively evaluate the level of GFP expression. ADV-Avi/biotin-PEG-EGF complexes also exhibited significantly increased extent of GFP expression for A431 cells, but not for MCF-7 cells (an EGF receptor deficient cell line), suggesting that retargeting of ADV to specific cells occurred by tethering of a cell-specific targeting ligand to the distal end of a PEG chain anchored onto the surface of ADV. This study demonstrates that ADV-Avi/biotin-PEG-EGF construct systems can be applied for cell-specific delivery of ADV with simultaneously reducing innate immune responses.     

Synthesis and in vitro characterization of oxytocin receptor targeted PEGylated immunoliposomes for drug delivery to the uterus

Abstract

Targeted delivery of therapeutics to the uterus is an important goal in the treatment of obstetric complications, such as preterm labour, postpartum hemorrhage, and dysfunctional labour. Current treatment for these obstetric complications is challenging, as there are limited effective and safe therapeutic options available. We have developed a targeted drug delivery system for the uterus by conjugating anti-oxytocin receptor (OTR) antibodies to the surface of PEGylated liposomes (OTR-PEG-ILs). The functionality of the OTR-PEG-ILs has previously been evaluated on human and murine myometrial tissues as well as in vivo in a murine model of preterm labour. The aim of this study was to report the pharmaceutical synthesis and characterization of the OTR-PEG-ILs and investigate their specific cellular interaction with OTR-expressing myometrial cells in vitro. Immunoliposomes composed of 1,2-distearoyl-sn-glycero-2-phosphocholine (DSPC) and cholesterol were prepared using an optimized method for the coupling of low concentrations of antibody to liposomes. The liposomes were characterized for particle size, antibody conjugation, drug encapsulation, liposome stability, specificity of binding, cellular internalization, mechanistic pathway of cellular uptake, and cellular toxicity. Cellular association studies demonstrated specific binding of OTR-PEG-ILs to OTRs and significant cellular uptake following binding. Evaluation of the mechanistic pathway of cellular uptake indicated that they undergo internalization through both clathrin- and caveolin-mediated mechanisms. Furthermore, cellular toxicity studies have shown no significant effect of OTR-PEG-ILs or the endocytotic inhibitors on cell viability. This study further supports oxytocin receptors as a novel pharmaceutical target for drug delivery to the uterus.     

Synthesis of RGD analogs as potential vectors for targeted drug delivery

RGD analogs bind to integrin receptors with high affinity and therefore have the potential to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites. Critical to this application is the ability to synthesize RGD analogs with different side chain functional groups that allow for the ready tethering of pharmaceutical agents without sacrificing their affinity for the target receptor significantly. A series of RGD analogs intended to be used as delivery vectors of pharmaceutical agents were prepared and evaluated for their ability to inhibit platelet aggregation by binding to glycoprotein IIb/IIIa. Among them, compound 11 showed the lowest IC50 against platelets activated by ADP. It was found that such RGD analogs could tolerate side chain modification fairly well with various functional groups attached such as amide, amine, ester, protected amine and poly(ethylene glycol). The fact that the compound with a side chain modification of poly(ethylene glycol) retained high affinity for glycoprotein IIb/IIIa (IC50 150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents to such RGD analogs without significant sacrifice of their affinity to the intended receptor.     

Modulation of biogenic amines content by poly(propylene imine) dendrimers in rats

Biogenic amines and polyamines participate in all vital organism functions, their levels being important function determinants. Studies were performed to check whether repeated administration of poly(propylene imine) (PPI) dendrimers, synthetic macromolecules with diaminobutane core, and peripheral primary amine groups, may influence the endogenous level of amines, as represented by the two of them: spermidine, a natural derivative of diaminobutane, and histamine. The experiment was carried out on Wistar rats. Fourth generation PPI dendrimer, as well as maltotriose-modified fourth generation PPI dendrimers with (a) cationic open sugar shell and (b) neutral dense sugar shell that possess a higher biocompatibility, was used. Applying the combination of column chromatography on Cellex P and spectrofluorimetric assays of o-phthaldialdehyde, the final amine condensation products were employed to analyze tissue spermidine and histamine outside the central nervous system. Furthermore, radioenzymatic assay was used to measure histamine levels in the brain. The obtained results indicate that in some tissues, the endogenous concentrations of histamine and spermidine may be affected by dendrimers depending on their dose and type of dendrimers.     

Synthesis and evaluation of a star amphiphilic block copolymer from poly(epsilon-caprolactone) and poly(ethylene glycol) as a potential drug delivery carrier

A star polymer composed of amphiphilic block copolymer arms has been synthesized and characterized. The core of the star polymer is polyamidoamine (PAMAM) dendrimer, the inner block in the arm is lipophilic poly(epsilon-caprolactone) (PCL), and the outer block in the arm is hydrophilic poly(ethylene glycol) (PEG). The star-PCL polymer was synthesized first by ring-opening polymerization of epsilon-caprolactone with a PAMAM-OH dendrimer as initiator. The PEG polymer was then attached to the PCL terminus by an ester-forming reaction. Characterization with SEC, (1)H NMR, FTIR, TGA, and DSC confirmed the star structure of the polymers. The micelle formation of the star copolymer (star-PCL-PEG) was studied by fluorescence spectroscopy. Hydrophobic dyes and drugs can be encapsulated in the micelles. A loading capacity of up to 22% (w/w) was achieved with etoposide, a hydrophobic anticancer drug. A cytotoxicity assay demonstrated that the star-PCL-PEG copolymer is nontoxic in cell culture. This type of block copolymer can be used as a drug delivery carrier.     

Synthesis,spectral properties and antimicrobial activity of a new cationic water‐soluble pH‐dependent poly(propylene imine) dendrimer modified with 1,8‐naphthalimides

A three‐step synthesis was implemented to prepare a quaternary ammonium functionalized blue fluorescent poly(propylene imine) dendrimer modified with pyridinium salt of 4‐acylamino‐1,8‐naphthalimide. The new cationic dendrimer absorbs in the ultraviolet light region and emits blue fluorescence. Its spectral characteristics in organic solvents and in an aqueous solution were studied. The influence of pH on the fluorescence intensity of the dendrimer was established with regard to its use as a pH sensor. The effect of hydroxyl ions on the absorption and fluorescence spectra in dry N,N‐dimethylformamide was also investigated. The antimicrobial activity of the dendrimer was assessed against model pathogenic microorganisms in agar, liquid medium, and after its deposition on cotton fabric.     

Synthesis and self-assembly of a DNA-conjugated poly(propylene glycol) derivative

Due to several characteristic features of DNA (e.g., genetic coding transfer and nanoscaled accuracy or so), DNA based compounds have been recently highlighted in a variety of research fields, including physics, chemistry, engineering or so. To date, they have been expanded into a hybrid form conjugated with conventional polymer groups. Such synthetic hybrid conjugates can be organized into multi-dimensional nanoor micro-structures; rod, sheet, and spherical shaped nanomaterials [1]. In this study, we showed a novel block copolymer composed of DNA and poly(propylene glycol) (PPG) (designated as DNA-b-PPG) for the self-assembled construction into three-dimensional vesicular structures.     

Synthesis of cholesterol-carborane conjugate for targeted drug delivery

The cholesterol-carborane conjugate has been designed and synthesized to selectively deliver boron to tumor cells by means of reconstituted low-density lipoprotein. The chemical stability and cytotoxicity of the new compound have been examined. Several methods have been evaluated for incorporation of the compound into LDL.     

Synthesis and biological evaluation of paclitaxel-C225 conjugate as a model for targeted drug delivery

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2'-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% +/- 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosine-kinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% +/- 6.0% and 7.1% +/- 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 microg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.