Chemistry and Analysis of Nanoparticle-Modified Living Cells

Conventional therapeutics are often limited by their targeting ability, resulting in harmful and potential fatal side-effects for the patients. Recently, new strategies have been developed to improve target specificity of drugs in order to generate more efficient therapeutics. An innovative concept, examined in this thesis, is the use of cells as therapeutic carriers. This concept termed cell-mediated drug delivery, relies on the intrinsic targeting properties of cells to transport the therapeutic drug to the target location. The conjugation of nanoparticles, loaded with drugs or adjuvants, to the surface of cells enables the transport to the desired location and increases the local drug concentration, reducing the required therapeutic leading to further attenuation of side effects. Chapter 1 introduces the concept of cell-mediated drug delivery. It highlights the advantages of using cells decorated with nanoparticles as carriers for therapeutics as opposed to free (polymer) nanoparticles. Examples of various covalent and non-covalent nanoparticle conjugations strategies are discussed and an overview of all approaches, which have been used previously to attach nanoparticles to the cell surface, is presented. The importance of a thorough in vitro characterization of nanoparticle-modified cells is emphasized. The use of common characterization techniques such as flow cytometry, UV-Vis and fluorescence spectroscopy, fluorescence and electron microscopy, and radiolabeling is discussed in detail and examples of qualitative and quantitative in vitro analysis of nanoparticle- modified cells are reviewed. Chapter 2 contains an extensive in vitro analysis of cell-nanoparticle conjugates using fluorescent- and fluorescent label-free methods. The stability of nanoparticles loaded with cargo attached to cells was investigated by encapsulating three different green fluorescent dyes into nanoparticles and tracking the fluorescence signal over 24 h. Two of the dyes revealed a complete loss of fluorescence of a time period of 24 h, whereas a third entrapped dye and a covalently attached dye did not have a decrease in fluorescence over 24 h. Finally, we developed a fluorescent label-free method as an alternative to characterize nanoparticle-decorated cells without the need for any fluorescent label. Chapter 3 systematically explores conjugation chemistries to immobilize two platforms of functional biodegradable polymer nanoparticles (PEG-PLA (poly(ethylene glycol) - poly(lactide)) nanoparticles and carboxylic PLA nanoparticles) on T cells as potential carrier across the blood-brain barrier (BBB). We compared a series of covalent and non-covalent immobilization strategies by flow cytometry (FACS), confocal microscopy, and impact on cell proliferation and cellular viability. We found that T-cell surface coupling of nanoparticles is dependent on the ratio of nanoparticles to cells and that higher ratios led to more particles attached to cells. Furthermore, the most efficient strategy to attach nanoparticles was via ligand-receptor interactions (lectin - sialic acid and biotin - NeutrAvidin). In addition, nanoparticle-decorated T cells were investigated for their ability to cross an in vitro mouse BBB model under static conditions in a two-chamber assay. Decorating the cells with nanoparticles does not affect their ability to bind ICAM-1 (a protein involved in the transmigration process) or to cross the model biological barrier.

Covalent and Noncovalent Conjugation of Degradable Polymer Nanoparticles to T Lymphocytes

Harm-Anton Klok, Tanja Thomsen

Cells are attractive as carriers that can help to enhance control over the biodistribution of polymer nanomedicines. One strategy to use cells as carriers is based on the cell surface immobilization of the nanoparticle cargo. While a range of strategies can be used to immobilize nanoparticles on cell surfaces, only limited effort has been made to investigate the effect of these surface modification chemistries on cell viability and functional properties. This study has explored seven different approaches for the immobilization of poly(lactic acid) (PLA) nanoparticles on the surface of two different T lymphocyte cell lines. The cell lines used were human Jurkat T cells and CD4(+) T-EM cells. The latter cells possess blood-brain barrier (BBB) migratory properties and are attractive for the development of cell-based delivery systems to the central nervous system (CNS). PLA nanoparticles were immobilized either via covalent active ester-amine, azide-alkyne cycloaddition, and thiol-maleimide coupling, or via noncovalent approaches that use lectin-carbohydrate, electrostatic, or biotin-NeutrAvidin interactions. The cell surface immobilization of the nanoparticles was monitored with flow cytometry and confocal microscopy. By tuning the initial nanoparticle/cell ratio, T cells can be decorated with up to similar to 185 nanoparticles/cell as determined by confocal microscopy. The functional properties of the nanoparticle-decorated cells were assessed by evaluating their binding to ICAM-1, a key protein involved in the adhesion of CD4(+) TEM cells to the BBB endothelium, as well as in a two-chamber model in vitro BBB migration assay. It was found that the migratory behavior of CD4(+) T-EM cells carrying carboxylic acid-, biotin-, or Wheat germ agglutinin (WGA)-functionalized nanoparticles was not affected by the presence of the nanoparticle payload. In contrast, however, for cells decorated with maleimide-finictionalized nanoparticles, a reduction in the number of migratory cells compared to the nonmodified control cells was observed. Investigating and understanding the impact of nanoparticle-cell surface conjugation chemistries on the viability and properties of cells is important to further improve the design of cell-based nanoparticle delivery systems. The results of this study present a first step in this direction and provide first guidelines for the surface modification of T cells, in particular in view of their possible use for drug delivery to the CNS.

AMER CHEMICAL SOC2021

Combating HIV Type 1 with Peptide - Synthetic Polymer Conjugates

Maarten Danial

Polymer therapeutics is a class of (bio)hybrid materials that include a plethora of biologically active (synthetic) macromolecules that either consist of polymeric drugs, drug-polymer conjugates, peptide/protein-polymer conjugates or of drug, peptide or proteins covalently linked to self-assembled entities, such as polymer micelles and liposomes. Interest in polymer therapeutics has increased in the last decades particularly as anti-cancer therapeutics, influenza therapeutics, non-viral alternatives to gene delivery and in the advent of biological terrorism as anthrax inoculates. The human immunodeficiency virus (HIV) is one of the most deadly diseases known and despite improvement in education, preventative methods and drug therapies, the virus still affects approximately 33 million people worldwide. Classical therapeutics against HIV consist of low molecular weight drugs that inhibit various viral enzymes in the cell, including reverse transcriptase, integrase and protease. More recently, drugs that target the entry and fusion of the virus have formed a new class of inhibitors, some of which are effective (candidate) drugs. One of the fusion inhibitors that were approved is the drug T-20. T-20 is a 36-amino acid peptide derived from the heptad repeat 2 (HR2) of the HIV-1 glycoprotein gp41and acts as a competitive inhibitor by preventing the fusion of the viral and host cell membrane. Despite the effectiveness of T-20, one major drawback is associated with this drug namely low plasma lifetime. One of the goals of this Thesis is to design and synthesize effective HR2 derived fusion inhibitors through conjugation with synthetic polymers, while extending the lifetime. In addition, a novel concept for macromolecular HIV entry inhibition known as polyvalent inhibition is assessed in this Thesis. This Thesis consists of five chapters in which polymer therapeutics as carriers and polyvalent entities are explored against HIV-1. With the state of the art of HIV-1 therapeutics including polymer therapeutics summarized in Chapter 1, the remainder of the Thesis expands and defines the terrain with regard to novel (bio)hybrid materials as therapeutics against HIV-1. Very few polymer therapeutics that target HIV before cellular entry have been described. Even fewer polymer-based therapeutics inhibit HIV-1 entry via the specific interaction to the HIV-1 envelope proteins. In Chapter 2, novel polyvalent copolymers that specifically target the HIV-1 envelope protein gp120 were generated using a living polymerization technique known as ‘reversible addition-fragmentation chain transfer’ (RAFT), followed by a post-polymerization modification strategy. The strategy involves two synthetic steps, namely an ester/amide exchange followed by a thiol-ene addition of peptide ligands to generate a library of polyvalent copolymers, with varying peptide ligand, 2-hydroxypropyl methacrylamide and allyl methacrylamide content. These novel polyvalent polymers were shown to be effective HIV-1 entry inhibitors. Fusion inhibitors derived from the HR2 domain of gp41 such as T-20, act as competitive inhibitors by binding to the HR1 domain on the envelope glycoprotein gp41, which is involved in HIV-1 – host cell membrane fusion. However, the peptidic nature of these fusion inhibitors restricts their lifetime because they are prone to proteolytic degradation. Chapter 3 explores the effect of attaching synthetic polymer poly(ethylene glycol) (PEG) site-specifically to a HR2 derived peptide fusion inhibitor. The PEGylated peptides were assessed for their biophysical properties, efficacy as membrane fusion inhibitors and stability towards trypsin that was used as a model to show the effectiveness of the PEG to protect the peptide from proteolytic degradation. Although PEGylation causes a reduction in efficacy, this Chapter demonstrates that judicial PEGylation of the peptide can minimize the detrimental effects on inhibitory efficacy, while increasing lifetime against a protease. Conjugation of PEG and the site of PEG conjugation both have a profound effect on the efficacy of HR2 derived membrane fusion inhibitors. However, the effects on the mechanism of HIV-1 infectivity inhibition by polymer conjugated HR2 peptide inhibitors are not known. Chapter 4 investigates the effect of architecturally different synthetic linear PEG, v-shaped PEG or branched poly(glycerol) polymers conjugated to HR2 derived peptides. The peptide-polymer conjugates were then assessed for their inhibition efficacy, affinity and kinetics of binding towards a model of gp41, known as 5-Helix. The results indicate that attachment of a synthetic linear or branched polymer, despite causing a reduction in HIV-1 infectivity inhibition, does not significantly affect affinity towards 5-Helix. However, the rates of association and dissociation towards and from 5-Helix were reduced. Furthermore, synthetic polymer conjugation to HR2 derived peptides prolong their lifetime bound to gp41 and thus potentially reduces HIV-1 inhibition escape. Therefore, this Chapter demonstrates a previously unknown advantage of polymer conjugation to HIV-1 fusion peptide inhibitors. Chapter 5 systematically explores the modification of hydrophilic residues on the hydrophobic face of a HR2 derived peptide. The peptide mutants were assessed for their efficacy against membrane fusion inhibition as well as against fusion of HIV-1 wild type and T-20 resistant variants. Although the peptide mutants did not exhibit any improvement towards the inhibition of wild type membrane fusion, selected peptide mutants with point mutations were shown to be effective inhibitors towards T-20 resistant HIV-1 strains. In conclusion, peptide-polymer hybrid materials can make excellent candidates as long lasting therapeutics against wild type HIV-1 but also against emergent HIV-1 mutant variants. The knowledge from these Chapters will aid the design of innovative, next generation of peptide-polymer hybrid therapeutics.

EPFL2011

Poleable Nanoparticles as Fillers Towards Non-linear Optically Active Actuators

Yee Song Ko

A new type of poleable dielectric elastomer is introduced herein. The elastomer contains polymer nanoparticles with frozen molecular dipoles, which can be oriented at elevated temperatures in an electric field via poling. The aim is to provide a soft material with high, tunable optical properties suitable for actuator and flexible electronics applications. To that end poleable polymeric nanoparticles with high dipole concentrations and glass transition temperatures well above room temperature will be needed to be used as filler in an elastomer matrix. The synthesis and characterization of such particles is presented in this manuscript. Polyhydroxyethyl methacrylate (PHEMA) nanoparticles were synthesized using miniemulsion polymerization. The particles were loaded with 4-[ ethyl (2-hydroxyethyl) amino]-4-nitrobenzene, usually called Disperse Red 1 (DR1), which has a large dipole moment (mu = 7.5 - 9.5 D). (1) The maximum dipole loadings is limited by the solubility of the dipole in the monomer solutions prior to polymerization. All samples show a glass transition temperature around 95 degrees C. Secondary electron microscopy (SEM) revealed spherical particles, the size of which was confirmed by dynamic light scattering (DLS) measurements. A composite was prepared by dispersing the particles in polydimethyl siloxane (PDMS).