Trastuzumab targeted micellar delivery of docetaxel using dendron-polymer conjugates.

Incorporation of a therapeutic antibody into nanosized drug delivery systems can improve their target specificity. This work reports an antibody-conjugated targeted delivery system composed of polymer-dendron conjugates. Trastuzumab is chosen as the targeting moiety, since it is clinically used against tumor cells expressing HER2 receptors. A micellar delivery system was generated using amphiphilic polymer-dendron conjugates containing a fourth-generation polyester dendron as the hydrophobic block and a linear poly(ethylene glycol) (PEG) chain as the hydrophilic block. After preparation of docetaxel loaded (ca. 10% wt) micelles, trastuzumab was conjugated onto the micellar shell using an amidation reaction. Micelles remained stable after conjugation of the antibody, with a slight increase in size from 179 nm to 185 nm upon functionalization. Docetaxel release was determined to be responsive to acidic pH, and over the course of 30 h, 54% drug release was measured in acidic media, whereas it was around 30% under neutral conditions. Cytotoxicity experiments on MCF-7 and SK-OV-3 cell lines displayed improved toxicity levels for targeted micelles in comparison with the non-targeted counterparts, whereas pulse-chase experiments indicated effectiveness of micellar formulations and the presence of targeting groups. Cellular internalization experiments using fluorescence microscopy and flow cytometry further demonstrated the enhanced cellular uptake of antibody conjugated targeted micelles.

Diels-Alder "Clickable" Biodegradable Nanofibers: Benign Tailoring of Scaffolds for Biomolecular Immobilization and Cell Growth.

Biodegradable polymeric nanofibers have emerged as promising candidates for several biomedical applications such as tissue engineering and regenerative medicine. Many of these applications require modification of these nanofibers with small ligands or biomolecules such as peptides and other growth factors, which necessitates functionalization of these materials in mild and benign fashion. This study reports the design, synthesis, and functionalization of such nanofibers and evaluates their application as a cell culture scaffold. Polylactide based copolymers containing furan groups and triethylene glycol (TEG) units as side chains were synthesized using organocatalyzed ring opening polymerization. The furan moiety, an electron rich diene, provides "clickable" handles required for modification of nanofibers since they undergo facile cycloaddition reactions with maleimide-containing small molecules and ligands. The TEG units provide these fibers with hydrophilicity, enhanced biodegradability, and antibiofouling characteristics to minimize nonspecific adsorption. A series of copolymers with varying amounts of TEG units in their side chains were evaluated for fiber formation and antibiofouling characteristics to reveal that an incorporation of 7.5 mol % TEG-based monomer was optimal for nanofibers containing 20 mol % furan units. Facile functionalization of these nanofibers in a selective manner was demonstrated through attachment of a dienophile containing fluorophore, namely, fluorescein maleimide. To show efficient ligand-mediated bioconjugation, nanofibers were functionalized with a maleimide appended biotin, which enabled efficient attachment of the protein, Streptavidin. Importantly, the crucial role played by the TEG-based side chains was evident due to lack of any nonspecific attachment of protein to these nanofibers in the absence of biotin ligand. Furthermore, these nanofibers were conjugated with a cell adhesive cyclic peptide, cRGDfK-maleimide, at room temperature without the need of any additional catalyst. Importantly, comparison of the cell attachment onto nanofibers with and without the peptide demonstrated that fibers appended with the peptides promoted cells to spread nicely and protrude actin filaments for enhanced attachment to the support, whereas the cells on nonfunctionalized nanofibers showed a rounded up morphology with limited cellular spreading.

Dendrons and Multiarm Polymers with Thiol-Exchangeable Cores: A Reversible Conjugation Platform for Delivery.

Disulfide exchange reaction has emerged as a powerful tool for reversible conjugation of proteins, peptides and thiol containing molecules to polymeric supports. In particular, the pyridyl disulfide group provides an efficient handle for the site-specific conjugation of therapeutic peptides and proteins bearing cysteine moieties. In this study, novel biodegradable dendritic platforms containing a pyridyl disulfide unit at their focal point were designed. Presence of hydroxyl groups at the periphery of these dendrons allows their elaboration to multivalent initiators that yield poly(ethylene glycol) based multiarm star polymers via controlled radical polymerization. The pyridyl disulfide unit at the core of these star polymers undergoes efficient reaction with thiol functional group containing molecules such as a hydrophobic dye, namely, Bodipy-SH, glutathione, and KLAK sequence containing peptide. While conjugation of the hydrophobic fluorescent dye to the PEG-based multiarm polymer renders it water-soluble, it can be cleaved off the construct through thiol-disulfide exchange in the presence of an external thiol such as dithiothreitol. The multiarm polymer was conjugated with a thiol group containing apoptotic peptide to increase its solubility and cellular transport. In vitro cytotoxicity and apoptosis assays demonstrated that the resultant peptide-polymer conjugate had almost five times more apoptotic potential primarily through triggering apoptosis by disrupting mitochondrial membranes of human breast cancer cell line (MDA-MB-231) compared to naked peptide. The novel dendritic platform disclosed here offers an attractive template that can be modified to multiarm polymeric constructs bearing a "tag and release" characteristic.

Targeting to the Bone: Alendronate-Directed Combretastatin A-4 Bearing Antiangiogenic Polymer-Drug Conjugates.

Selective targeting of tumor site with chemotherapeutic agents appears to be one of the most effective methods to address many of the problems encountered with conventional chemotherapy. In this work, poly(oligoethylene glycol)methacrylate (POEGMA) based bone-targeting polymers bearing an antiangiogenic drug combretastatin A4 (CA4) were synthesized using free radical polymerization. Targeted and nontargeting copolymers were evaluated for their bone targeting efficiency, cytotoxicities against endothelial cells, namely, HUVECs and U2-OS and Saos-2 cancerous cell lines, as well as their antiangiogenic activity against endothelial cell tube formation by HUVECs. It is observed that the drug conjugated polymers conjugated with the bisphosphonate groups containing drug alendronate (ALN) have remarkably high affinity for bone mineral when compared to the polymer-drug conjugates devoid of the bisphosphonate groups. Both targeted and nontargeted polymer-drug conjugates show a sustained drug release in rat plasma with an overall release of 80-93% over 5 days. In vitro studies revealed high levels of cytotoxicity of the polymer-drug conjugates against HUVECs and U2-OS, and moderate cytotoxicity toward Saos-2. Importantly, the CA4 conjugated copolymers displayed excellent level of antiangiogenic activity as deduced from in vitro endothelial cell tube formation assay using HUVECs. Overall, a novel bone-targeting antiangiogenic polymer-drug conjugate that can be further elaborated to carry additional anticancer drugs is disclosed.

Dexamethasone - PAMAM dendrimer conjugates for retinal delivery: preparation, characterization and in vivo evaluation.

OBJECTIVE: Ocular diseases affecting retina, such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and glaucoma are the major causes of blindness, and their treatment is still a challenge due to the special structure of the eye. The purpose of this study was to prepare a sustained release DEX conjugate formulation with enhanced ocular permeation using poly(amidoamine) (PAMAM) dendrimers and to evaluate the effects of conjugation on DEX release and ocular residence time. METHODS: PAMAM G3.5 and PAMAM G4.5 dendrimers were used to prepare DEX conjugates, and conjugation was confirmed using (1) H-NMR. Formulations were evaluated in terms of drug release in the presence of ocular enzymes and cytotoxicity on ARPE19 cell lines. Fluorotron analysis was performed and ocular pharmacokinetic properties of DEX-PAMAM conjugates were studied in Sprague Dawley rats following intravitreal and subconjunctival applications. KEY FINDINGS: The results indicated that DEX-PAMAM conjugates were able to enhance ocular permeability and ocular tissue levels of DEX following subconjunctival injection, and results were encouraging when compared to the literature that has reported DEX getting cleared from vitreous in 3 h. CONCLUSION: Current studies are focused on formulation improvement to enhance hydrolysis and clearance time.

Combretastatin A-4 Conjugated Antiangiogenic Micellar Drug Delivery Systems Using Dendron-Polymer Conjugates.

Employment of polymeric nanomaterials in cancer therapeutics is actively pursued since they often enable drug administration with increased efficacy along with reduced toxic side effects. In this study, drug conjugated micellar constructs are fabricated using triblock dendron-linear polymer conjugates where a hydrophilic linear polyethylene glycol (PEG) chain is flanked by well-defined hydrophobic biodegradable polyester dendrons bearing an antiangiogenic drug, combretastatin-A4 (CA4). Variation in dendron generation is utilized to obtain a library of micellar constructs with varying sizes and drug loadings. In particular, a family of drug appended dendron-polymer conjugates based on polyester dendrons of generations ranging from G1 to G3 and 10 kDa linear PEG were obtained using [3 + 2] Huisgen type "click" chemistry. The final constructs benefit from PEG's hydrophilicity and antibiofouling character, as well as biodegradable nature of the hydrophobic polyester dendrons. The hydrophobic-hydrophilic-hydrophobic character of these constructs leads to the formation of flower-like micelles in aqueous media. In addition to generation-dependent subnanomolar range critical micelle concentrations, the resulting micelles possess hydrodynamic diameters suitable for passive tumor targeting through enhanced permeability and retention (EPR) effect; thereby they are suitable candidates as controlled drug delivery agents. For all constructs, in vitro cytotoxicities were investigated and inhibitory effect of Comb-G3-PEG on tube formation was shown on human umbilical vein endothelial cells (HUVECs).