W.E. Hennink

Thermosensitive polymers as carriers for DNA delivery.

Copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) and N-isopropylacryl amide (NIPAAm) of various monomer ratios and molecular weights were evaluated as carrier systems for DNA delivery. All copolymers, even with a low DMAEMA content of 15 mol%, were able to bind to DNA at 25 degrees C. Light-scattering measurements indicate that complexation is accompanied by precipitation of the (co)polymer in the complex caused by a drop of the lower critical solution temperature of the (co)polymer. The (co)polymer/plasmid ratio at which complexes with a size of around 200 nm were formed increased with increasing NIPAAm content of the copolymer and was independent of molecular weight of the (co)polymer. However, complexes containing (co)polymers of low molecular weight or high NIPAAm content prepared at 25 degrees C aggregated rapidly when the temperature was raised to 37 degrees C, whereas complexes containing (co)polymers of high molecular weight or lower NIPAAm content were relatively stable at 37 degrees C. The zeta potential of the complexes was also independent of molecular weight of the (co)polymer and increased with increasing (co)polymer/plasmid ratio until a plateau value was reached. The (co)polymer/plasmid ratio at which this plateau was reached increased with increasing NIPAAm content. The plateau values decreased from around 26 mV to around 13 mV when the NIPAAm content of the copolymer was increased from 0 to 85 mol%. The cytotoxicity of the complexes strongly decreased with increasing NIPAAm content and was independent of molecular weight of the (co)polymer. The transfection efficiency of complexes with poor stability was in general much lower than that of complexes with good stability. The transfection efficiency as a function of the (co)polymer/plasmid ratio showed a bell-shaped curve. The (co)polymer/plasmid ratio at which the transfection efficiency was maximal increased with increasing NIPAAm content, while the maximum transfection efficiency strongly decreased with increasing NIPAAm content of the copolymer. The results of this study show that the formation of stable (co)polymer/plasmid complexes with a size of around 200 nm is a prerequisite for efficient transfection. Furthermore, the transfection efficiency and cytotoxicity strongly decreased with decreasing zeta potential. Therefore, besides the size, the zeta potential can also be used as a characteristic to predict the behavior of this type of (co)polymer/plasmid complexes in transfection. Copolymers of DMAEMA and NIPAAm provided with a homing device may be interesting carrier systems for gene targeting because these copolymers can condense DNA to small particles, and the resulting complexes show a low cytotoxicity and aspecific transfection.

Targeted core-crosslinked polymeric micelles with controlled release of covalently entrapped doxorubicin

Doxorubicin (DOX) is clinically applied in cancer therapy, but its use is associated with dose limiting severe side effects. Core-crosslinked biodegradable polymeric micelles composed of poly(ethylene glycol)-b-poly(N-(2-hydroxypropyl) methacrylamide-lactate) (mPEG-b-p(HPMAm-Lacn)) diblock copolymers have shown prolonged circulation in the blood stream upon intravenous administration and enhanced tumor accumulation through the enhanced permeation and retention (EPR) effect. However a (physically) entrapped anticancer drug (paclitaxel) was previously shown to be rapidly eliminated from the circulation, likely because the drug was insufficiently retained in the micelles. To fully exploit the EPR effect for drug targeting, a DOX methacrylamide derivative (DOX-MA) was covalently incorporated into the micellar core by free radical polymerization. The structure of the doxorubicin derivative is susceptible to pH-sensitive hydrolysis, enabling controlled release of the drug in acidic conditions (in either the intratumoral environment and/or the endosomal vesicles). 30e40% w/w of the added drug was cova- lently entrapped, and the micelles with covalently entrapped DOX had an average diameter of 80 nm. The entire drug payload was released within 24 h incubation at pH 5 and 37 � C, whereas only around 5% release was observed at pH 7.4. DOX micelles showed higher cytotoxicity in B16F10 and OVCAR-3 cells compared to DOX-MA, likely due to cellular uptake of the micelles via endocytosis and intracellular drug release in the acidic organelles. The micelles showed better anti-tumor activity than free DOX in mice bearing B16F10 melanoma carcinoma. The results presented in this paper show that mPEG-b-p(HPMAm- Lacn) polymeric micelles with covalently entrapped doxorubicin is a system highly promising for the targeted delivery of cytostatic agents.

Microwave-assisted click polymerization for the synthesis of Aβ(16–22) cyclic oligomers and their self-assembly into polymorphous aggregates

We report on the design, synthesis, and structural analysis of cyclic oligomers with an amyloidogenic peptide sequence as the repeating unit to obtain novel self-assembling bionanomaterials. The peptide was derived from the Alzheimer Abeta(16-22) sequence since its strong tendency to form antiparallel beta-sheets ensured the formation of intermolecular hydrogen bridges on which the supramolecular assembly of the individual cyclic oligomers was based. The synthesis of the cyclic oligomers was performed via a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide as the monomer. The formation of cyclic oligomers, up to pentamers (35 amino acid residues), was verified by MALDI-TOF analysis and the individual cyclic monomer and dimer could be isolated by HPLC. Gelation behavior and the self-assembly of the linear monomer and the cyclic monomer and dimer were studied by TEM, FTIR and CD. Significant differences were observed in the morphology of the supramolecular aggregates of these three peptides that could be explained by alterations of the hydrogen bond network.

In-situ forming hydrogels by simultaneous thermal gelling and Michael addition reaction between methacrylate bearing thermosensitive triblock copolymers and thiolated hyaluronan

summary This study reports on the synthesis, characterization and peptide release behavior of a simultaneously physically and chemically crosslinked hydrogel. Thermosensitive methacrylate bearing polymers and thiolated hyaluronan were combined to prepare a biodegradable but structurally stable and biocompatible hydrogel system. This tandem system, displaying in situ physical gelation, triggered by temperature, and chemical gelation, through Michael type addition reaction between thiol and methacrylate groups, showed favorable properties for the diffusion-controlled delivery of peptides. Introduction Injectable, self-assembling hydrogels are receiving increasing attention because of their desirable properties for drug delivery and tissue engineering applications. Their advantages over preformed chemically crosslinked polymer hydrogels are the use of minimally invasive methods for administration, shape adaptation in vivo and ease of drug loading. In this study an ABA-triblock copolymer consisting of a poly (ethylene glycol) (PEG)middle block, flanked by thermosensitive blocks of random N-isopropylacrylamide (pNIPAm)/N-(2-hydroxypropyl) methacrylamide dilactate (pHPMAmlac2) and exhibiting lower critical solution temperature behavior in aqueous solutionwas synthesized and used for the preparation of body-temperature-induced self-assembling hydrogels. A general drawback of self-assembling hydrogels, however, is their limited mechanical strength and low stability due to the rapid swelling and subsequent dissolutionof the polymers. Therefore, in order to stabilize the structure of the above described hydrogel, the free hydroxyl groups in the side chains of HPMAmlac2 units were partially derivatized with methacrylate groups to obtain a polymer, abbreviated as pNHPtma (Fig. 1), able to form chemical cross-links through Michael addition reaction with thiolated compounds. Michael addition is a catalyst-free reaction occurring betweenmethacrylate and thiol groups in physiological conditions [1]. Fig. 1. Chemical structure of pNHPtma triblock copolymer. In our approach, hyaluronic acid, a natural linear polysaccharide known for its favorable physical (e.g. viscosity, hydration) and biological (protein and cell interactions) properties was derivatized with thiol moieties (Fig. 2) and used as a curing agent for thermosensitive pNHPtma based hydrogels. This novel combination of thermal gelling and Michael addition cross-linking represents an interesting approach to the design of a new generation of hydrogels, displaying several advantageous aspects, such as injectability, in situ gelling, biodegradability, initial structural stability and biocompatibility. Fig. 2. Chemical structure of thiol derivatized hyaluronic acid. Experimental methods The thermosensitive triblock copolymer was synthesized by radical polymerization using HPMAmlac2 and NIPAm as monomers at a feed ratio of 25/75 and PEG-ABCPA (4,4-azobis-(4-cyanopentanoic acid)) as macroinitiator. Next, methacrylic side groups were introduced using methacrylic anhydride. The synthesized polymer Abstracts / Journal of Controlled Release 148 (2010) e21–e56 e28

NanoDDS 2013: The 11th International Nano Drug Delivery Symposium

Nano-scale drug delivery systems (NDDSs) are both very important and very special. They are important because they are being applied to the most challenging diseases facing the world, including cancer, cardiovascular diseases, and infectious diseases. NDDSs are special because they alter biodistribution, pharmacokinetics, and pharmacodynamics in ways that cannot be achieved using traditional therapeutics. The size scale of these NDDSs ranges from a few nm up to several hundreds of nm. The molecular weight (or size) and biodegradability of the nanocarrier are both very important to its eventual clearance from the body. A nanocarrier may consist of a drug conjugated to a polymer (e.g., polyethylene glycol (PEG), poly(N-2-hydroxypropyl methacrylamide) (PHPMA)) or to a nanoparticle (gold, silica, carbon nanotubes), or complexed within polymer or lipid nanoparticles in a number of ways: ionically to a species of the opposite charge (e.g., polyplexes, lipoplexes), by hydrophobic forces (albumin, micellar, and liposomal membranes), or by aqueous solubility (within liposomes). The nanocarrier may also be biologically (‘actively’) targeted to specific cells using ligands (monoclonal antibodies, peptide ligands,metabolites) or physically (‘passively’) targeted to tumors via leaky blood vessels (enhanced permeability and retention effect, EPR). More recently, nano-scale features are becoming increasingly important in the development of local therapies, which include drug depots, stents, regenerative medicines, and other devices. Most clinically approved NDDS formulations have been designed for treating cancer; however, technologies are also being developed to treat a range of other chronic and life-threatening diseases. The topics covered by this special issue are an excellent representation of the diversity both in disease targets as well as in technology (Table 1). Many of these systems are composed of water-soluble and hydrophilic polymers, although they may be copolymerized with hydrophobic components. Some newer approaches utilize inorganic nanoparticles as well.