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Affibody molecules are protein ligands, that due to their small size (6-19 kDa) and high target affinity exhibit favourable properties for tumour uptake valuable in diagnostic imaging and therapeutic applications. Fusion to a high affinity albumin binding domain (ABD) has been shown to improve circulatory half-life and biodistribution. However, the effect of molecular design is not obvious to predict and in vitro methods to evaluate their transport properties in physiologically relevant environment are needed. In this work we investigated the diffusivities (D) of Affibody molecules, with systematically varied molecular design, in solution and within extracellular matrix mimetic hydrogels composed of either agarose or collagen and hyaluronic acid (COL-HA) using fluorescence recovery after photobleaching. Furthermore, the effect of presence of human serum albumin (HSA) was evaluated. The correlation between D of the tested Affibody molecules in solution and their molecular weight (Mw) was weak, indicating that propensity to form reversible oligomers and the size of the oligomers are more important for their diffusion properties than Mw of the monomer. Positively charged Affibody molecules were enriched in polymer-rich domains of the COL-HA gel accompanied by a decrease in D as a result of electrostatic interactions. Binding to HSA by Affibody molecules containing an ABD was evident as a decrease of D when HSA was present. In COL-HA gels HSA-binding reduced the effect of electrostatic interactions effectively facilitating the transport of those compounds. In conclusion, molecular design especially inclusion of an ABD affected the transport properties of the tested Affibody molecules.

Intestinal motility, including peristalsis and segmentation, drives complex fluid movements critical for the oral delivery of biologics and other macromolecules. Despite advances, oral delivery remains commercially limited by low bioavailability, often attributed to poor epithelial permeability. However, variability in motility patterns may also play a critical role, influencing intraluminal distribution and thus absorption, yet this aspect remains underexplored. Here, we combine computational fluid dynamics and machine learning to evaluate how motility type, intensity, pocket size, contractility, and fluid composition affect the delivery of a model macromolecule (insulin) and a permeation enhancer (sodium caprate, C10). We find that segmentation, especially at light intensity, consistently enhances epithelial colocalisation over peristalsis. Under segmentation, smaller pocket sizes (2 mL versus 10 mL) and stronger contractility (occlusion ratio 0.3) yielded optimal performance. Our extreme gradient boosting regression model identified pocket volume, contractility, and motility type as dominant predictors of colocalisation. In a comparative analysis, segmentation led to 128% and 137% higher maximum normalised concentrations of insulin and C10, respectively, than moderate peristalsis with a nutritional drink. Overall, segmentation achieved 6.7-fold and 8.0-fold higher average maximum normalised concentrations for insulin and C10, respectively. These results emphasise segmentation, characteristic of the fed state, as a superior motility pattern for macromolecular absorption compared to peristalsis during the migrating motor complex (MMC). By elucidating the interplay between motility and transport, our findings may guide the design of more effective oral formulations and support personalised strategies for drug delivery based on individual motility profiles.

Liquid crystal nanoparticles (LCNPs), such as hexosomes based on an internal hexagonal phase (H_II), enhance lipid nanoparticle-mediated drug delivery by improving drug solubility, stability and absorption. LCNPs can also be tailored for specific biological environments by incorporating non-ester-linker lipids into the H_II nanostructure. In this study, we developed an H_II model system with a 90:10 phytantriol:farnesol ratio based on experimental data and conducted all-atom molecular dynamics simulations. The model remained stable across various water-to-lipid ratios, and the structural effects observed were consistent with prior experimental data. We used this model to examine the localization and interactions of antibiotics vancomycin and clarithromycin. Clarithromycin, being highly lipophilic, associated mainly with the lipid phase, while vancomycin localized at the water-lipid interface due to its amphiphilic nature. An extended H_II system with repeating units enclosed in Pluronic F127 polymers was also constructed. Simulations showed that hydrogen bonding between Pluronic F127 and water facilitated water influx into the H_II phase, causing interfacial reorganization. To investigate drug release, we performed umbrella sampling simulations. The resulting energy profiles indicated that polymer-water-lipid interactions lowered the energy barrier for vancomycin release compared to clarithromycin. This was confirmed by in vitro release studies, where vancomycin exhibited a higher release rate. Overall, this model provides molecular-level insights into drug loading, partitioning, and release from H_II systems, supporting the design of more effective drug delivery formulations.

Amphiphilic compounds, such as phospholipids or surface-active substances, are present in biological systems and can be part of pharmaceutical formulations. As a consequence, all pharmaceutically active ingredients will encounter amphiphilic compounds, either in the formulation or after administration. With the growing interest in peptide-based pharmaceuticals, there is a need to enhance the understanding of the interactions between peptides and amphiphilic compounds.

In this particular study, we have chosen to study mixtures of the comparatively small cyclical octapeptide lanreotide and the conventional anionic surfactant sodium dodecylsulfate (SDS). This was done by examining the self-assembly structures formed in lanreotide-SDS mixtures using light scattering and small-angle X-ray scattering (SAXS).

Above the critical micelle concentration (cmc) of SDS, the large excess of SDS could solubilize all lanreotide and form small micelles with lanreotide attached to the interface. Upon dilution to concentrations below the cmc of SDS, a suspension with dispersed solid nanoparticles is formed. The solid nanoparticles grow in size with decreasing concentration and, eventually, precipitate. The precipitated material is arranged in a liquid crystalline micellar phase, consisting of small close-packed SDS micelles with peptide adsorbed at the interface.

We were able to conclude that lanreotide does not form mixed micelles with SDS, indicating that it lacks the amphiphilic properties required to integrate fully with SDS behaving as a cosurfactant. In contrast, lanreotide attaches to the interface of SDS micelles, resembling the interactions of polymers, proteins, and nucleic acids with surfactants.

Subcutaneous (SC) injection is the primary alternative to oral administration for therapeutic proteins and peptides. However, bioavailability and absorption rate are often variable and difficult to predict. Therefore, there is a need for new biorelevant and predictive SC in vitro methods. In this study we systematically investigate the effect of size and charge of a macromolecule on its partitioning and diffusion within extracellular matrix (ECM) mimetic hydrogels in order to gain insight on interactions with the components of the ECM affecting the absorption of a drug after SC injection. Hydrogels consisting of either agarose, cross-linked collagen and hyaluronic acid (HA) or cross-linked HA, were made and equilibrated in solutions of FITC-dextrans of varying sizes (4 to 150 kDa) and model peptides of varying net charge (+2 to +9). Partitioning and diffusion coefficients within gel and solution were determined using confocal laser scanning microscopy and fluorescence recovery after photo bleaching (FRAP), and compared to theoretical models. Generally, the partitioning and diffusivities within the gels decreased with increasing molecular weight, which was in good agreement with models describing the effect of obstruction of the gel network corrected for heterogeneity in the gel structure. The cationic peptides were enriched in the oppositely charged gels and their diffusivities decreased with increasing peptide charge. The experimental results were in semi quantitative agreement with an electrostatic model presented in this work.

Although the term surfactant was coined in the mid-20th century, substances with surfac-tant-like properties have been known to humans for millennia. Comprising two distinct parts, referred to as the tail and the head, surfactants are well known for their ability to lower the surface tension between two phases, such as liquid and air or liquid and liquid. Several natural and synthetic materials exhibit amphiphilic properties similar to conventional surfactants, in-cluding phospholipids, bile salts, certain proteins, and pharmaceuticals. Like ordinary surfac-tants, amphiphilic drugs can self-associate into larger aggregates at higher concentrations. They can be found in several drug categories, for instance analgesics, tranquilizers, antidepressants, and antihistamines. As a result, these compounds can be referred to as drug surfactants. The self-assembly of drug surfactants is influenced by additional molecules such as proteins, lipids, and amphiphilic drugs. Drug surfactants can integrate into biological aggregates, such as mi-celles, bilayers, and liposomes, and can substantially impact their structures, eventually influ-encing their pharmacological activity and toxicity. The overall aim of this thesis was to enhance the understanding of the interactions between amphiphilic drugs and phospholipids, with spe-cial emphasis on the self-assembly of amphiphilic drug-phospholipid binary systems by study-ing their self-associated aggregates. The main focus was on two tricyclic antidepressants (ami-triptyline and doxepin) and a group of selected phosphatidylcholine (PC) phospholipids (DOPC, DEPC, DMPC, and DPPC).Results presented in this thesis demonstrate the enhanced ability of drug surfactants to sol-ubilize PC phospholipids, beyond the capacity of conventional surfactants through the for-mation of mixed micelles. With increasing lipid content, all binary drug-lipid systems reached a micelle-to-bilayer transition point, beyond which bilayer structures, i.e. vesicles and disks were observed in the samples. Within the bilayer region, spontaneous formation of vesicles was observed in different phospholipid-drug binary series. For some samples, vesicles were the pre-dominant self-assembled structure. Vesicle size showed a downward trend as the total concen-tration decreased, in contrast to the behavior of micellar aggregates. Notably, the smallest ves-icles measured less than 10 nm in radius; we have chosen to denote them ultrasmall vesicles. The observed ultrasmall vesicles remained intact for several months, indicating their thermo-dynamic stability and their promise for potential nano-carrier drug delivery and other nanotech-nology applications.In addition, the effects of phospholipid tail (acyl chain) chemical structure, specifically the degree of unsaturation, on the self-assembled aggregates were investigated. Moreover, the in-teraction between a selected drug surfactant (amitriptyline) and supported DOPC bilayer was also studied. In addition, since the properties and behavior of bile salts resemble those of am-phiphilic drugs, they were also investigated in this work.

We have investigated the effect of length and chemical structure of phospholipid tails on the spontaneous formation of unilamellar liposomal vesicles in binary solute mixtures of cationic drug surfactant and zwitterionic phosphatidylcholine phospholipids. Binary drug surfactant-phospholipid mixtures with four different phospholipids with identical headgroups (two saturated phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 14:0) and 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, 16:0), and two unsaturated lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18:1) and 1,2-Dierucoyl-sn-Glycero-3-Phosphatidylcholine (DEPC, 22:1)) combined with two different tricyclic antidepressant drugs (amitriptyline hydrochloride (AMT) and doxepin hydrochloride (DXP)) have been investigated with small-angle neutron scattering (SANS) and cryo-transmission electron microscopy (cryo-TEM). We observe a conspicuous impact of phospholipid tail structure on both micelle-to-vesicle transition point and vesicle size. In particular, ultrasmall unilamellar vesicles, i.e. with a diameter less than 20 nm, were observed in several samples with the two unsaturated phospholipids DOPC and DEPC, but not in any samples with the saturated phospholipids DMPC and DPPC. The smallest vesicles observed in DOPC and DEPC mixtures were smaller than 18 nm in diameter. In contrast, the smallest vesicles observed in DMPC mixtures were about 30 nm in diameter and always larger than 100 nm in DPPC mixtures. The ultrasmall vesicles showed exceptional colloidal stability. Moreover, bilayer vesicles predominated over micelles in a much wider range of concentrations for DOPC and DEPC mixtures as a result of having a smaller phospholipid mole fraction in the aggregates at the micelle-to-vesicle transition. Our results have been theoretically rationalized by combining solution thermodynamics with bending elasticity theory.

The study investigated the sequential relationship between composition, fracture strength, degree of fragmentation, and loss of tabletability (LoT) of dry granulated particles to get a deeper insight into the underlying causes controlling the LoT. Surrogate granules, consistent in dimension and composed of a systematic variation of a brittle (lactose) and plastic powder (microcrystalline cellulose), were prepared by compaction. These granules were then compressed into tablets at low compression pressures, considered to correspond to the compression phase during which the main fragmentation occurs. The mechanically weak tablets were gently deaggregated into compressed granules, and the proportion of formed fragments was determined using dry powder laser diffractometry and by sieving as an indication of the degree of granule fragmentation.

The compressed granules comprised three sub-populations of particles, i.e., non-fragmented granules, fragmented granules, and fine particles, with fragmentation ceasing already within the restricted pressure range applied. The degree of granule fragmentation was dependent on composition and showed an inverted relationship with granule fracture strength. For tablets formed at 100 MPa, the LoT was primarily controlled by the degree of fragmentation, while for tablets formed at 300 MPa, the LoT was affected by a combination of granule fragmentation and deformation. Thus, the relative impact of fragmentation and deformation on the LoT is dependent on the tableting pressure.

Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8⁺ (10-15 fold) and CD4⁺ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

Common ionisable amphiphilic drug molecules form micelles in aqueous solution. Loaded onto oppositely charged polyelectrolyte microgels they associate with the network chains to form dense complex phases. The self-assembling properties control the loading and release properties in drug delivery applications of microgel systems but little is known about the nature of the aggregates and the phase structure. In this paper, we investigated the size and organization of the self-assemblies formed by the hydrochloride salts of amitriptyline (AMT), chlorpromazine (CPZ), and doxepin (DXP) in sodium polyacrylate microgels. Small-angle X-ray scattering (SAXS) was used to determine the microstructure of drug loaded microgels in aqueous environment at ionic strengths relevant for drug loading (0.01 M) and release (0.15 M). The composition of drug loaded microgels was determined by means of a purpose built microscopy cell and UV spectroscopy measurements. Upon drug loading the microgels formed complex phases of low water content. SAXS experiments showed that the drugs formed oblate shaped or spherical micelles displaying local ordering but without long-range ordering even at very high micelle volume fractions. The local ordering resembled the packing of randomly packed hard oblates and spheres. The aggregation number of AMT varied between 10 and 49 depending on the composition. Incorporation of the uncharged base form of the drug caused a transformation of oblate shaped (aspect ratio ∼ 0.4) to spherical micelles, accompanied by an abrupt increase of the aggregation number. Variation of the ionic strength had minor effects on the aggregation number. CPZ formed oblate shape micelles (aspect ratios 0.3–0.4) with aggregation number between 9 and 30. DXP formed oblate shape micelles (aspect ratios 0.3–0.4) with aggregation numbers 10 − 11 at all studied compositions. The results provide a structural basis for, and justification of, previously assumed microstructures underlying mechanistic models of drug-microgel interactions and drug release.