Angus P. R. Johnston

Next generation, sequentially assembled ultrathin films: beyond electrostatics

Over the last 15 years, the layer-by-layer (LbL) assembly technology has proven to be a versatile method for surface modification. This approach is likely to find widespread application because of its simplicity and versatility; however, the conventional use of highly charged materials with limited responsive behaviour presents some key limitations. In this tutorial review, the formation of multilayer thin films prepared through non-electrostatic interactions is reviewed. We discuss the assembly of films via a number of different methodologies, with particular emphasis on those that provide enhanced orientational control, stimuli-responsive behaviour, and improved film stability.

Layer-by-layer-assembled capsules and films for therapeutic delivery.

Polymeric materials formed via layer-by-layer (LbL) assembly have promise for use as drug delivery vehicles. These multilayered materials, both as capsules and thin fi lms, can encapsulate a high payload of toxic or sensitive drugs, and can be readily engineered and functionalized with specific properties. This review highlights important and recent studies that advance the use of LbL-assembled materials as therapeutic devices. It also seeks to identify areas that require additional investigation for future development of the field. A variety of drug-loading methods and delivery routes are discussed. The biological barriers to successful delivery are identified, and possible solutions to these problems are discussed. Finally, state-of-the-art degradation and cargo release mechanisms are also presented.

A general approach for DNA encapsulation in degradable polymer microcapsules.

We report a general and facile method for the encapsulation of DNA in nanoengineered, degradable polymer microcapsules. Single-stranded (ss), linear double-stranded (ds), and plasmid DNA were encapsulated into disulfide-cross-linked poly(methacrylic acid) (PMA) capsules. The encapsulation procedure involves four steps: adsorption of DNA onto amine-functionalized silica (SiO(2)(+)) particles; sequential deposition of thiolated PMA (PMA (SH)) and poly(vinylpyrrolidone) to form multilayers; cross-linking of the thiol groups of the PMA (SH) in the multilayers into disulfide linkages; and removal of the sacrificial SiO(2)(+) particles. Multilayer growth was dependent on the surface coverage of DNA on the SiO(2)(+) particles, with stable capsules formed from particles with up to 50% DNA surface coverage. The encapsulation strategy applies to nucleic acids with varied size and conformation and allows DNA to be concentrated over 100-fold from dilute solutions into monodisperse, uniformly loaded polymer capsules. The capsule loading can be controlled by the DNA:SiO(2)(+)particle ratio, and for 1 microm diameter capsules, loadings of approximately 1000 chains of 800 bp dsDNA and more than 10,000 chains of 20-mer ssDNA can be achieved. The encapsulated DNA was released and successfully used in polymerase chain reactions as both templates (linear dsDNA and plasmid DNA) and primer sequences (ssDNA), confirming the functionality and structural integrity of the encapsulated DNA. These DNA-loaded polymer microcapsules hold promise as delivery vehicles for gene therapy and diagnostic applications.

A Protective Vaccine Delivery System for In Vivo T Cell Stimulation Using Nanoengineered Polymer Hydrogel Capsules

Successful delivery of labile vaccine antigens, such as peptides and proteins, to stimulate CD4 and CD8 T cell immunity could improve vaccine strategies against chronic infections such as HIV and Hepatitis C. Layer-by-layer (LbL)-assembled nanoengineered hydrogel capsules represent a novel and promising technology for the protection and delivery of labile vaccine candidates to antigen-presenting cells (APCs). Here we report on the in vitro and in vivo immunostimulatory capabilities of LbL-assembled disulfide cross-linked poly(methacrylic acid) (PMA(SH)) hydrogel capsules as a delivery strategy for protein and peptide vaccines using robust transgenic mice models and ovalbumin (OVA) as a model vaccine. We demonstrate that OVA protein as well as multiple OVA peptides can be successfully encapsulated within nanoengineered PMA(SH) hydrogel capsules. OVA-containing PMA(SH) capsules are internalized by mouse APCs, resulting in presentation of OVA epitopes and subsequent activation of OVA-specific CD4 and CD8 T cells in vitro. OVA-specific CD4 and CD8 T cells are also activated to proliferate in vivo following intravenous vaccination of mice with OVA protein- and OVA peptide-loaded PMA(SH) hydrogel capsules. Furthermore, we show that OVA encapsulated within the PMA(SH) capsules resulted in at least 6-fold greater proliferation of OVA-specific CD8 T cells and 70-fold greater proliferation of OVA-specific CD4 T cells in vivo compared to the equivalent amount of OVA protein administered alone. These results highlight the potential of nanoengineered hydrogel capsules for vaccine delivery.

Targeting of Cancer Cells Using Click-Functionalized Polymer Capsules

Targeted delivery of drugs to specific cells allows a high therapeutic dose to be delivered to the target site with minimal harmful side effects. Combining targeting molecules with nanoengineered drug carriers, such as polymer capsules, micelles and polymersomes, has significant potential to improve the therapeutic delivery and index of a range of drugs. We present a general approach for functionalization of low-fouling, nanoengineered polymer capsules with antibodies using click chemistry. We demonstrate that antibody (Ab)-functionalized capsules specifically bind to colorectal cancer cells even when the target cells constitute less than 0.1% of the total cell population. This precise targeting offers promise for drug delivery applications.

Uptake and Intracellular Fate of Disulfide-Bonded Polymer Hydrogel Capsules for Doxorubicin Delivery to Colorectal Cancer Cells

Understanding the interactions between drug carriers and cells is of importance to enhance the delivery of therapeutics. The release of therapeutics into different intracellular environments, such as the lysosomes or the cell cytoplasm, will impact their pharmacological activity. Herein, we investigate the intracellular fate of layer-by-layer (LbL)-assembled, submicrometer-sized polymer hydrogel capsules in a human colon cancer derived cell line, LIM1899. The cellular uptake of the disulfide-stabilized poly(methacrylic acid) (PMA(SH)) capsules by colon cancer cells is a time-dependent process. Confocal laser scanning microscopy and transmission electron microscopy reveal that the internalized capsules are deformed in membrane-enclosed compartments, which further mature to late endosomes or lysosomes. We further demonstrate the utility of these redox-responsive PMA(SH) capsules for the delivery of doxorubicin (DOX) to colon cancer cells. The DOX-loaded PMA(SH) capsules demonstrate a 5000-fold enhanced cytotoxicity in cell viability studies compared to free DOX.

Influence of Size, Surface, Cell Line, and Kinetic Properties on the Specific Binding of A33 Antigen-Targeted Multilayered Particles and Capsules to Colorectal Cancer Cells

There has been increased interest in the use of polymer capsules formed by the layer-by-layer (LbL) technique as therapeutic carriers to cancer cells due to their versatility and ease of surface modification. We have investigated the influence of size, surface properties, cell line, and kinetic parameters such as dosage (particle concentration) and incubation time on the specific binding of humanized A33 monoclonal antibody (huA33 mAb)-coated LbL particles and capsules to colorectal cancer cells. HuA33 mAb binds to the A33 antigen present on almost all colorectal cancer cells and has demonstrated great promise in clinical trials as an immunotherapeutic agent for cancer therapy. Flow cytometry experiments showed the cell binding specificity of huA33 mAb-coated particles to be size-dependent, with the optimal size for enhanced selectivity at approximately 500 nm. The specific binding was improved by increasing the dosage of particles incubated with the cells. The level of specific versus nonspecific binding was compared for particles terminated with various polyelectrolytes to examine the surface dependency of antibody attachment and subsequent cell binding ability. The specific binding of huA33 mAb-coated particles is also reported for two colorectal cancer cell lines, with an enhanced binding ratio between 4 and 10 obtained for the huA33 mAb-functionalized particles. This investigation aims to improve the level of specific targeting of LbL particles, which is important in targeted drug and gene delivery applications.

Engineering particles for therapeutic delivery: prospects and challenges.

Nanoengineered particles that can facilitate drug formulation and passively target tumors have reached the clinic in recent years. These early successes have driven a new wave of significant innovation in the generation of advanced particles. Recent developments in enabling technologies and chemistries have led to control over key particle properties, including surface functionality, size, shape, and rigidity. Combining these advances with the rapid developments in the discovery of many disease-related characteristics now offers new opportunities for improving particle specificity for targeted therapy. In this Perspective, we summarize recent progress in particle-based therapeutic delivery and discuss important concepts in particle design and biological barriers for developing the next generation of particles.

Nanoescapology: progress toward understanding the endosomal escape of polymeric nanoparticles

Using nanoparticles to deliver drugs to cells has the potential to revolutionize the treatment of many diseases, including HIV, cancer, and diabetes. One of the major challenges facing this field is controlling where the drug is trafficked once the nanoparticle is taken up into the cell. In particular, if drugs remain localized in an endosomal or lysosomal compartment, the therapeutic can be rendered completely ineffective. To ensure the design of more effective delivery systems we must first develop a better understanding of how nanoparticles and their cargo are trafficked inside cells. This needs to be combined with an understanding of what characteristics are required for nanoparticles to achieve endosomal escape, along with methods to detect endosomal escape effectively. This review is focused into three sections: first, an introduction to the mechanisms governing internalization and trafficking in cells, second, a discussion of methods to detect endosomal escape, and finally, recent advances in controlling endosomal escape from polymer- and lipid-based nanoparticles, with a focus on engineering materials to promote endosomal escape. WIREs Nanomed Nanobiotechnol 2017, 9:e1452. doi: 10.1002/wnan.1452 For further resources related to this article, please visit the WIREs website.

Tuning the formation and degradation of layer-by-layer assembled polymer hydrogel microcapsules.

Engineered polymer capsules are finding widespread importance in the delivery of encapsulated toxic or fragile drugs. The effectiveness of polymer capsules as therapeutic delivery vehicles is often dependent on the degradation behavior of the capsules because it is often necessary to release the encapsulated drugs at specific times and in certain locations. Herein we investigate the parameters that govern the formation and degradation of a recently introduced new class of polymer hydrogel capsules based on disulfide cross-linked poly(methacrylic acid). We report a new and efficient method for the synthesis of thiol-functionalized poly(methacrylic acid) (PMA(SH)), the main component of the capsules. Polymeric capsules were synthesized by the layer-by-layer deposition of PMA(SH) and poly(vinylpyrrolidone) (PVPON) on silica particle templates, followed by cross-linking the PMA(SH) layers and removing PVPON and the template particles. The disulfide cross-links provided a redox-active trigger for degradation that was initiated by a cellular concentration of glutathione. We demonstrate that increasing the degree of PMA(SH) thiol modification affords direct control over the thickness of the polymer film and the degradation rate of the polymer capsules. Furthermore, the degradation rate of the PMA(SH) capsules was independent of film thickness, suggesting a bulk erosion process.