G.M.J.P.C. Coué

Functionalized linear poly(amidoamine)s are efficient vectors for intracellular protein delivery

An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense β-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. β-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing β-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery.

Bioresponsive poly(amidoamine)s designed for intracellular protein delivery

Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reversal of the polymeric carrier from negative to positive. The concomitant endosomal buffering effect and increased polymer-endosomal membrane interactions are considered to lead to increased protein delivery into the cytosol. Besides destabilization of the polymer-protein nanoparticles by the charge reversal effect, intracellular cleavage of disulfide linkages in the polymer ensure further unpacking of the protein in the cytosol. Cellinternalization and cytotoxicity experiments with primary human umbilical vein endothelial cells (HUVEC) showed that the SS-PAA-based nanocomplexes were essentially non-toxic, and that lysozyme is successfully internalized into HUVEC. The results indicate that these charge reversal SS-PAAs have excellent properties as non-toxic intracellular delivery systems for cationic proteins.

Bioreducible insulin-loaded nanoparticles and their interaction with model lipid membranes

To improve design processes in the field of nanomedicine, in vitro characterization of nanoparticles with systematically varied properties is of great importance. In this study, surface sensitive analytical techniques were used to evaluate the responsiveness of nano-sized drug-loaded polyelectrolyte complexes when adsorbed to model lipid membranes. Two bioreducible poly(amidoamine)s (PAAs) containing multiple disulfide linkages in the polymer backbone (SS-PAAs) were synthesized and used to form three types of nanocomplexes by self-assembly with human insulin, used as a negatively charged model protein at neutral pH. The resulting nanoparticles collapsed on top of negatively charged model membranes upon adsorption, without disrupting the membrane integrity. These structural rearrangements may occur at a cell surface which would prevent uptake of intact nanoparticles. By the addition of glutathione, the disulfide linkages in the polymer backbone of the SS-PAAs were reduced, resulting in fragmentation of the polymer and dissociation of the adsorbed nanoparticles from the membrane. A decrease in ambient pH also resulted in destabilization of the nanoparticles and desorption from the membrane. These mimics of intracellular environments suggest dissociation of the drug formulation, a process that releases the protein drug load, when the nanocomplexes reaches the interior of a cell.

Bioreducible poly(amidoamine)s with charge-reversal properties for intracellular protein delivery

An effective intracellular protein delivery system was developed using bioreducible disulfide-containing poly(amidoamine)s with negatively charged citraconic side groups that can give charge-reversal upon pH decrease. These water-soluble and linear polymers efficiently self-assemble with proteins into nanocomplexes by charge attraction. Intracellular-mimicking protein release from the particles is triggered by the reduction of polymer disulfide linkages, causing polymer degradation, and inversion of protein-polymer interaction at endosomal pH due to the charge-reversal of the citraconic side group.

Cationic polymers for intracellular delivery of proteins

Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering cell transdifferentiation processes, within other curative applications. However, several hurdles have to be overcome. For this purpose the use of polymers, with their ease of modification in physical and chemical properties, is attractive in protein drug carriers. They can protect their therapeutic protein cargo from degradation and enhance their bioavailability at targeted sites. In this chapter, potential and currently used polymers for fabrication of protein delivery systems and their applications for intracellular administration are discussed. Special attention is given to the use of cationic polymers for their ability to promote the cellular uptake of therapeutic proteins.

Design and physicochemical characterization of poly(amidoamine) nanoparticles and the toxicological evaluation in human endothelial cells: applications to peptide delivery to the brain

In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles were characterized in terms of size, surface charge, and loading efficiency, and were applied on human cerebral microvascular endothelial cells (hCMEC/D3) and human umbilical vein endothelial cells (Huvec) cells. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially nontoxic, and the peptide was successfully internalized into cells. The results indicate that these PAAs have an excellent property as nontoxic carriers for intracellular protein and peptide delivery, and provide opportunities for novel applications in the delivery of peptides to endothelial cells of the brain.

Development and in vitro Evaluation of Antigen-Loaded Poly(amidoamine) Nanoparticles for Respiratory Epithelium Applications

A poly(amidoamine) with disulfide linkages in the main chain and 4‐hydroxybutyl and ω‐carboxy‐PEG groups (9:1 ratio) as side chains was prepared by Michael addition polymerization of cystamine bisacrylamide with 4‐hydroxybutylamine and ω‐carboxy‐PEG‐amine. To develop therapeutic protein formulations for improved delivery of antigen via the intranasal route, nanoparticles were prepared from this polymer by self‐assembly with p24 or ovalbumin as the model proteins and CpG as the adjuvant. The nanoparticles incorporated the antigens and adjuvant from the feed solution with high efficiency (∼90u2009%) and have sizes of 112 and 169u2005nm, respectively, with low positive surface charge (∼+2u2005mV). Formulations of the nanoparticles were shown to be nontoxic and stable for at least 10 days at room temperature. Their capacity to pass through epithelial and endothelial cell layers was evaluated in vitro by using a respiratory mucosa‐like barrier model in which monolayers of NCIu2009H441 respiratory epithelial cells and ISO‐HAS‐1 endothelial cells were co‐cultured on both sides of a transwell filter membrane. It was shown that p24 incorporated in the nanoparticles was transported with >140u2009% greater efficiency through the two contact‐inhibited layers than p24 in its free form, whereas incorporation of ovalbumin in the nanoparticles leads to a 40u2009% decrease in transport efficiency relative to the free antigen.

Perspectives on nanoparticulate delivery of therapeutic proteins by oral administration

The oral route is the most common and preferred route of drug delivery in view of its convenience and patient acceptance. However for oral administration of therapeutic proteins, several hurdles have to be overcome. For this purpose the use of polymers, with their ease of modification in physical and chemical properties, are attractive in protein drug carriers. They can protect their therapeutic protein loading from degradation in the gastrointestinal tract, and enhance their bioavailability at targeted sites of the body. In this review, potential and currently used polymers for fabrication of protein delivery systems and their applications for oral administration will be discussed.