Micelles composed of block copolymers of poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) have shown great promise as drug delivery carriers due to their excellent stability and high loading capacity. In the present study, parameters influencing micelle size were investigated to tailor sizes in the range of 25 to 100 nm. Micelles were prepared by a nanoprecipitation method and their size was modulated by the block copolymer properties such as molecular weight, their hydrophilic to hydrophobic ratio, homopolymer content, as well as formulation and processing parameters. It was shown that the micelles have a core-shell structure using a combination of dynamic light scattering and transmission electron microscopy analysis. By varying the degree of polymerization of the hydrophobic block (NB) between 68 and 10, at a fixed hydrophilic block mPEG5K (NA=114), it was shown that the hydrophobic core of the micelle was collapsed following the power law of (NB×Nagg)1/3. Further, the calculated brush height was similar for all the micelles examined (10 nm), indicating that crew-cut micelles were made. Both addition of homopolymer and preparation of micelles at lower concentrations or lower rates of addition of the organic solvent to the aqueous phase increased the size of micelles due to partitioning of the hydrophobic homopolymer chains to the core of the micelles and lower nucleation rates, respectively. Furthermore, it was shown that by using different solvents, the size of the micelles substantially changed. The use of acetone, acetonitrile, ethanol, THF and dioxane, resulted in micelles in the size range from 45 to 60 nm after removal of the organic solvents. The use of DMF and DMSO led to markedly larger sizes of 75 and 180 nm respectively. In conclusion, the results show that by modulating polymer properties and processing conditions, micelles with tailorable sizes can be obtained.

The careful design of nanoparticles, in terms of size and morphology, is of great importance to developing effective drug delivery systems. The ability to precisely tailor nanoparticles in size and morphology during polymer self-assembly was therefore investigated. Four poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) mPEG-b-p(HPMA-Bz) block copolymers with a fixed hydrophilic block of mPEG 5 kDa and a varying molecular weight of the hydrophobic p(HPMA-Bz) block (A: 17.1, B: 10.0, C: 5.2 and D: 2.7 kDa) were self-assembled into nanoparticles by nanoprecipitation under well-defined flow conditions, using microfluidics, at different concentrations. The nanoparticles from polymer A, increased in size from 55 to 90 nm using lower polymer concentrations and slower flow rates and even polymer vesicles were formed along with micelles. Similarly, nanoparticles from polymer D increased in size from 35 to 70 nm at slower flow rates and also formed vesicles along with micelles, regardless of the used concentration. Differently, polymers B and C mainly self-assembled into micelles at the different applied flow rates with negligible size difference. In conclusion, this study demonstrates that the self-assembly of mPEG-b-p(HPMA-Bz) block copolymers can be easily tailored in size and morphology using microfluidics and is therefore an attractive option for further scaled-up production activities.

An efficient, scalable and good manufacturing practice (GMP) compatible process was developed for the production of docetaxel-loaded poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) micelles. First, the synthesis of the mPEG-b-p(HPMA-Bz) block copolymer was optimized through step-by-step investigation of the batch synthesis procedures. This resulted in the production of 1 kg of mPEG-b-p(HPMA-Bz) block copolymer with a 5 kDa PEG block and an overall molecular weight of 22.5 kDa. Second, the reproducibility and scalability of micelle formation was investigated for both batch and continuous flow setups by assessing critical process parameters. This resulted in the development of a new and highly efficient continuous flow process, which led to the production of 100 mL of unloaded micelles with a size of 55 nm. Finally, the loading of the micelles with the anti-cancer drug docetaxel was successfully fine-tuned to obtain precise control on the loaded micelle characteristics. As a result, 100 mL of docetaxel loaded micelles (20 mg/mL polymer and 5 mg/mL docetaxel in the feed) with a size of 55 nm, an encapsulation efficiency of 65 %, a loading capacity of 14 % and stable for at least two months in water at room temperature, were produced with the newly developed continuous flow process. In conclusion, this study paves the way for efficient and robust large-scale production of docetaxel loaded micelles with high encapsulation efficiencies and stability, which is crucial for their applicability as a clinically relevant drug delivery platform.

Virus capsids, i.e. viruses devoid of their genetic material, are suitable nanocarriers for biomedical applica-tions such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the com-pounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for co-valent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the pro-teins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The applica-tion of capsid assembly conditions on the functionalized proteins led to successful particle formation, show-ing the potential of this covalent encapsulation strategy.