Dalin Wu

Polymer nanocompartments in broad-spectrum medical applications

The field of nanoscience is expected to make significant contributions to contemporary medicine by providing unique solutions to critical problems. These solutions require the design of hybrid materials/systems with new properties and functionalities. This review focuses on spherical polymer nanocompartments (capsules and vesicles) and describes their potential in a wide variety of medical applications that range from passive drug carriers to active nanoreactors to artificial organelles. Here, we place emphasis on the complex requirements that a polymer assembly must fulfill for consideration in the medical domain. In terms of stability and chemical diversity, synthetic polymer compartments are superior to currently marketed liposomes, thereby supporting their modification for targeting approaches, stimuli-responsiveness, and multifunctionality. The authors present the latest concepts and examples based on the encapsulation/entrapment of biomolecules (e.g., enzymes and proteins) for the development of active nanosystems for application in the medical domain.

Nanomimics of Host Cell Membranes Block Invasion and Expose Invasive Malaria Parasites

The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.

Polymersomes conjugated to 83-14 monoclonal antibodies: in vitro targeting of brain capillary endothelial cells.

The blood-brain barrier (BBB) remains an obstacle for many drugs to reach the brain. A strategy to cross the BBB is to modify nanocarrier systems with ligands that bind to endogenous receptors expressed at the BBB to induce receptor-mediated transport. The aim of the present study was to investigate the potential of polymersomes composed of the amphiphilic diblock copolymer poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline), PDMS-b-PMOXA, for active targeting of brain capillary endothelial cells. We conjugated PDMS-b-PMOXA polymersomes to the anti-human insulin receptor antibody 83-14 and studied their uptake by brain capillary endothelial cells. Transmission electron micrography and light scattering measurements revealed the self-assembly of the block copolymers into 200 nm vesicles after extrusion. Fluorescence correlation spectroscopy was employed to calculate the number of antibodies coupled to one polymersome. Binding and uptake of the polymersomes conjugated to 83-14 mAb were studied in the human BBB in vitro model hCMEC/D3 expressing the human insulin receptor. Competitive inhibition with an excess of free 83-14 mAb demonstrated the specificity of cellular binding and uptake. Our results suggest that PDMS-b-PMOXA polymersomes conjugated to 83-14 mAb may be suitable nanocarriers for drug delivery to the brain.

Hybrid Polymer–Lipid Films as Platforms for Directed Membrane Protein Insertion

Hybrids composed of amphiphilic block copolymers and lipids constitute a new generation of biological membrane-inspired materials. Hybrid membranes resulting from self-assembly of lipids and polymers represent adjustable models for interactions between artificial and natural membranes, which are of key importance, e.g., when developing systems for drug delivery. By combining poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) amphiphilic copolymers (PDMS-b-PMOXA) with various phospholipids, we obtained hybrid films with modulated properties and topology, based on phase separation, and the formation of distinct domains. By understanding the factors driving the phase separation in these hybrid lipid-polymer films, we were able to use them as platforms for directed insertion of membrane proteins. Tuning the composition of the polymer-lipids mixtures favored successful insertion of membrane proteins with desired topological distributions (in polymer or/and lipid regions). Controlled insertion and location of membrane proteins in hybrid films make these hybrids ideal candidates for numerous applications where specific spatial functionality is required.

Functional surface engineering by nucleotide-modulated potassium channel insertion into polymer membranes attached to solid supports.

Planar solid-supported membranes based on amphiphilic block copolymers represent promising systems for the artificial creation of structural surfaces. Here we introduce a method for engineering functional planar solid-supported membranes through insertion of active biomolecules. We show that membranes based on poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) amphiphilic diblock copolymers, which mimic natural membranes, are suitable for hosting biomolecules. Our strategy allows preparation of large-area, well-ordered polymer bilayers via Langmuir-Blodgett and Langmuir-Schaefer transfers, and insertion of biomolecules by using Bio-Beads. We demonstrate that a model membrane protein, the potassium channel from the bacterium Mesorhizobium loti, remains functional after insertion into the planar solid-supported polymer membrane. This approach can be easily extended to generate a platform of functional solid-supported membranes by insertion of different hydrophobic biomolecules, and employing different types of solid substrates for desired applications.

Polymersomes containing quantum dots for cellular imaging

Quantum dots (QDs) are highly fluorescent and stable probes for cellular and molecular imaging. However, poor intracellular delivery, stability, and toxicity of QDs in biological compartments hamper their use in cellular imaging. To overcome these limitations, we developed a simple and effective method to load QDs into polymersomes (Ps) made of poly(dimethylsiloxane)-poly(2-methyloxazoline) (PDMS-PMOXA) diblock copolymers without compromising the characteristics of the QDs. These Ps showed no cellular toxicity and QDs were successfully incorporated into the aqueous compartment of the Ps as confirmed by transmission electron microscopy, fluorescence spectroscopy, and fluorescence correlation spectroscopy. Ps containing QDs showed colloidal stability over a period of 6 weeks if stored in phosphate-buffered saline (PBS) at physiological pH (7.4). Efficient intracellular delivery of Ps containing QDs was achieved in human liver carcinoma cells (HepG2) and was visualized by confocal laser scanning microscopy (CLSM). Ps containing QDs showed a time- and concentration-dependent uptake in HepG2 cells and exhibited better intracellular stability than liposomes. Our results suggest that Ps containing QDs can be used as nanoprobes for cellular imaging.

Angiopep2-functionalized polymersomes for targeted doxorubicin delivery to glioblastoma cells

A targeted drug delivery nanosystem for glioblastoma multiforme (GBM) based on polymersomes (Ps) made of poly(dimethylsiloxane)-poly(2-methyloxazoline) (PDMS-PMOXA) diblock copolymers was developed to evaluate their potential to actively target brain cancer cells and deliver anticancer drugs. Angiopep2 was conjugated to the surface of preformed Ps to target the low density lipoprotein receptor-related protein 1 that are overexpressed in blood brain barrier (BBB) and glioma cells. The conjugation efficiency yield for angiopep2 was estimated to be 24%. The angiopep2-functionalized Ps showed no cellular toxicity after 24h and enhanced the cellular uptake around 5 times more in U87MG glioblastoma cells compared to the non-targeted Ps. The encapsulation efficiency of doxorubicin (DOX) in Ps was 13% by co-solvent method, compared to a film rehydration method (4%). The release profiles of the DOX from Ps showed a release of 42% at pH 5.5 and 40% at pH 7.4 after 24h, indicating that Ps can efficiently retain the DOX with a slow release rate. Furthermore, the in vitro antiproliferative activity of DOX-loaded Ps-Angiopep2 showed enhanced toxicity to U87MG glioblastoma cells, compared to non-targeted Ps. Overall, our in vitro results suggested that angiopep2-conjugated Ps can be used as nanocarriers for efficient targeted DOX delivery to glioblastoma cells.

Biocompatible polymer-Peptide hybrid-based DNA nanoparticles for gene delivery.

Currently, research on polymers to be used as gene delivery systems is one of the most important directions in both polymer science and biomedicine. In this report, we describe a five-step procedure to synthesize a novel polymer-peptide hybrid system for gene transfection. The block copolymer based on the biocompatible polymer poly(2-methyl-2-oxazoline) (PMOXA) was combined with the biocleavable peptide block poly(aspartic acid) (PASP) and finally modified with diethylenetriamine (DET). PMOXA-b-PASP(DET) was produced in high yield and characterized by (1)H NMR and FT-IR. Our biopolymer complexed plasmid DNA (pDNA) efficiently, and highly uniform nanoparticles with a slightly negative zeta potential were produced. The polymer-peptide hybrid system was able to efficiently transfect HEK293 and HeLa cells with GFP pDNA in vitro. Unlike the commonly used polymer, 25 kDa branched poly(ethylenimine), our biopolymer had no adverse effects on cell growth and viability. In summary, the present work provides valuable information for the design of new polymer-peptide hybrid-based gene delivery systems with biocompatible and biodegradable properties.

Poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) for a combined study of molecular recognition and spatial constraints in protein binding and interactions.

Many biological processes require precise regulation and synergy of proteins, and consequently involve molecular recognition and spatial constraints between biomolecules. Here, a library of poly(N-isopropylacrylamide-co-tris-nitrilotriacetic acid acrylamide) (PNTs) has been synthesized and complexed with Cu(2+) in order to serve as models for investigation of the combined effects of molecular recognition and spatial constraints in biomolecular interactions. The average distance between Cu(2+)-trisNTA binding sites in PNTs polymers was varied from 4.3 to 31.5 nm by adjusting their trisNTA contents. His tag (His6), His-tagged enhanced yellow fluorescent protein (His6-eYFP), and His6-tagged collagenase G (His6-ColG), with sizes ranging from 1 to 11 nm, were used as models to assess whether the binding ability is influenced by a cooperative topology based on molecular recognition interactions with Cu(2+)-trisNTA binding sites, and spatial constraints created by decreasing average distance between trisNTAs. His-tagged molecules bound to all PNTs polymers due to their molecular recognition interaction involving histidines and Cu(2+)-trisNTA pockets, but with a binding ability that was highly modulated by the average distance between the trisNTA binding sites. Small molecular mass molecules (His6) exhibit a high binding ability to all PNTs polymers, whereas his-tagged proteins bind to PNTs efficiently only when the average distance between trisNTA binding sites is larger than the protein dimensions.