modular single-chain polymer nanoparticles in drug delivery
Pia Kröger is a PhD student in the research group Biomolecular Nanotechnology. Her supervisors are prof.dr. D.W. Grijpma and prof.dr. J.J.L.M. Cornelissen from the faculty of Science and Technology.
In this thesis, thiol-Michael addition was employed in the intramolecular crosslinking of thiol polymers to yield a variety of single-chain polymer nanoparticles (SCNPs). These polymer nanoparticles are ~10 nm in diameter, which is markedly smaller than conventional polymer nanoparticles. Furthermore, altered biodistribution behavior is anticipated, with improved targeting characteristics towards certain areas in the human body.
In Chapter 2, different literature approaches towards water-soluble SCNPs and their prospected medical applications are presented. Chapter 3 introduces thiol-Michael addition as an intramolecular crosslinking method in the preparation of SCNPs. Based on xanthate and thioacetate monomers, a range of thiol-functional (meth)acrylate and styrene polymers were prepared. Both xanthate and thioacetate moieties proved suitable for RAFT polymerization and the thiol moiety was readily accessible through aminolysis with primary amines. Chain collapse and size of the SCNPs were directly related to the polymer precursor chain length and the degree of intramolecular crosslinking. All of the obtained nanoparticles in Chapter 3 are soluble in organic solvents.
In Chapter 4, pentafluorophenyl methacrylate was used as the primary monomer in the preparation of precursor polymers and subsequent SCNP formation, enabling facile post-formation modification of SCNPs with a range of different amines. This approach was utilized to render the obtained SCNPs water-soluble and, beyond that, for the addition of fluorescent labels and alkyne moieties for click conjugation. Likewise, conjugation of amino acid moieties and peptides onto the particles is an important first step in the development of protein mimicking SCNPs.
In Chapter 5, solketal methacrylate was employed as the basis of the polymer backbone in SCNPs. The acid-labile acetal protection group allowed either the polymer precursor or the formed SCNPs to be rendered water-soluble. Using this feature, a dual pathway synthesis for water-soluble SCNPs was developed, allowing SCNP formation in polar and apolar solvents. The potential of drug encapsulation was demonstrated by incorporation of the solvatochromic dye Nile red into the glycol-SCNPs. Shifts in the fluorescence spectra of Nile red confirmed the dye encapsulation, which was more pronounced in SCNPs via the organic pathway – presumably because of the far better solubility of Nile red in organic solvents. Encapsulation and release study of the antibiotic Rifampicin revealed controlled release over 24 h. Evaluation on human brain endothelial cells (hCMEC/D3) showed cellular uptake, while no cytotoxicity of the SCNPs was observed. Fluorescent-labeled SCNPs loaded with Nile red co-localized with the dye in the cells. Accordingly, the SCNPs may be utilized as drug carriers. Alternatively, drugs may be conjugated to the SCNPs as demonstrated for curcumin in Chapter 8.
Chapter 6 compares the cellular effects of SCNPs which are based on three different glucose monomers. C1- and C6-conjugated glucose methacrylates, in the pyranose or partly open-chain form, were obtained by enzymatic-catalyzed reactions as a building block for gluco-polymers and gluco-SCNPs. These glucose/glucoside SCNPs were observed inside the endosomes and lysosomes of HeLa cells. Whereas the C1-conjugated glucose macromolecules showed an increased binding affinity to the lectin Concanavalin A, increased cellular uptake in HeLa cells was observed for the C6-conjugated SCNPs. The difference between glucose in its partly open-chain form and in its fixated cyclic form played a minor role in the uptake efficiency. Consequently, conjugation of glucose at different positions influences the uptake behavior of the SCNPs, which might be used as a targeting strategy.
In Chapters 4, 5 and 6, a variety of water-soluble SCNPs with different targeting moieties has been developed. However, in order to evaluate the biological effect of nanoparticles prior to animal studies, there is still a lack of readily available, valid assays and models. Therefore, a blood-brain barrier (BBB) model, based on hCMEC/D3 cells cultured in a transwell system, was produced as described in Chapter 7. The in vivo function of the BBB is to protect the brain against endo- and exogenous substances and thus the BBB is not easily passed by drugs and nanocarriers. Accordingly, nanoparticles that pass the BBB are of great interest. The here applied BBB model proved to have appropriate tightness to constrain paracellular passage of 2-12 nm-sized dextrans and was therefore considered suitable for analysis of the transcellular passage activity of SCNPs. However, interaction between the collagen coating of the wells and the SCNPs impeded further evaluation. Current work is focused on altering the coating to minimize the SCNP interaction while maintaining cell growth. Once a valid model of the BBB which is suitable for SCNPs has been developed, the full potential of SCNPs may be explored and their efficacy determined. The here obtained SCNPs will be investigated for brain uptake and drug transport in our future work as highlighted in Chapter 8.
