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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.

Antibody-drug conjugates have demonstrated enhanced efficacy and reduced toxicity by targeted delivery of toxic payloads, yet they can also be used to deliver non-toxic payloads (peptides and oligonucleotides) tailored for disease-specific needs. We have previously developed an adaptable drug conjugate strategy using a highaffinity single-chain variable fragment specific for a short unstructured synthetic peptide tag (pTag). When this fragment is fused to an antibody structure, payload loading can be performed by a simple mixing step provided that the pTag is part of the payload. To assess the impact on conjugate stability and biological responses, we evaluated variants of the pTag by introducing amino acid changes at a central position for affinity binding. In a competition ELISA, a 2.4-fold reduction in IC50 was noted for a non-conservative amino acid alteration (pTagK8L), whereas a conservative amino acid substitution (pTagK8H) resulted in a 1.3-fold decrease compared to the pTag. The non-conservative amino acid change (pTagK8L) negatively influenced the stability of the conjugate, illustrated in a hydrogel model. Additionally, the pTagK8L alteration led to increased CD8+ T cell proliferation and a slight decrease in CD4+ T cell proliferation in vitro. This was irrespective of whether formulated with the bispecific antibody or not. In vivo, the data displayed that the pTag led to significantly higher T cell expansion than the pTagK8L, suggesting that lower affinity may impair immune activation and that conjugation stability is key to achieving the desired targeted delivery capacity.

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.

Extracellular vesicles (EVs) derived from corneal epithelial cells have shown great promise in promoting corneal wound healing and stromal regeneration, but they face challenges with rapid clearance from the eye. This study addresses these challenges by developing a biocompatible collagen-hydrogel sustained delivery system. We successfully isolated, purified, and characterized corneal epithelial EVs (CE-EVs), assessed their efficacy in corneal epithelial healing in vitro, and demonstrated their sustained delivery over 10 days followed by an on-demand release through enzymatic degradation of the hydrogel, which mimics the in vivo scenario. To develop a microscale understanding of the EV diffusion inside the hydrogel matrix, we probed the hydrogel network with several model compounds and nanoparticles by using advanced confocal microscopy analyses, followed by fitting our results to established diffusion models. Our findings suggest this innovative approach offers a safe and effective strategy to promote corneal wound healing. This technology has the potential to revolutionize corneal injury treatment and improve patient outcomes. Moreover, the possibility to tailor EV-release kinetics broadens the scope of EV research in clinical practices, as varying short- and long-term release profiles will be required for diverse medical applications.

Polyelectrolyte-surfactant systems of opposite charge have been widely studied because of their relevance in applications ranging from pharmaceuticals to advanced materials. However, the role of hydrophobic interactions in such systems remains debated, particularly for sodium poly(styrenesulfonate) (NaPSS) and cationic surfactants. This study investigates the binding behavior of NaPSS with the conventional hydrocarbon surfactant dodecylpyridinium chloride (DPC) or the fluorinated surfactant 1H,1H,2H,2H-perfluorodecylpyridinium chloride (HFDePC), with the objective of elucidating the importance of hydrophobic interactions in such systems. The binding isotherms of both surfactants were measured in both linear NaPSS solutions and covalently cross-linked NaPSS hydrogels. HFDePC exhibited a binding isotherm with a negative slope, indicative of unique binding behavior. Thermodynamic modeling revealed that the negative slope arises from the formation of metastable colloidal states. For DPC, modeling indicated the formation of mixed micelles with NaPSS with a consistent surfactant/polyion charge ratio, explaining its lower cooperativity and atypical phase behavior. In NaPSS hydrogels, the swelling isotherms revealed a uniform hydrogel collapse in the case of DPC and the formation of biphasic core-shell structures in the case of HFDePC. Small-angle X-ray scattering showed rod-like micelle formation for both systems, with increased micelle length at higher binding ratios and the emergence of hexagonal packing of micelles near coil saturation. Overall, the findings of this study underscore the importance of surfactant and polyelectrolyte structure and hydrophobicity and offer new insights into the nature of interactions in polyelectrolyte-surfactant systems of opposite charge.

The objective was to derive indications of the microstructure of lactose powder compacts was studied by smallangle and ultra small-angle X-ray scattering experiments (SAXS/USAXS). Measurements were performed on a series of lactose powder compacts formed at four different pressures in the interval 300-1000 MPa. Porod analyses of the 1-D scattering profiles, i.e. intensity vs. magnitude of scattering vector (q), enabled accurate determinations of the specific surface area. From these, the interparticle contact area was estimated and compared to the compact tensile strength. Measures of the volume fraction and sizes of the voids were obtained from a fit of a model of polydisperse cylindrical pores. These were found to be approximately disc-shaped, with one dimension significantly smaller than the other two. The scattering data were consistent with an isotropic distribution of pores. The void volume fractions, void sizes and specific surface areas showed anti-correlating trends with the pressure applied.

For many biopharmaceuticals, subcutaneous (sc) administration is the only viable route. However, there is no in vitro method available accurately predicting the absorption profiles of subcutaneously injected pharmaceuticals. In this work, we show that a recently developed microfluidics method for interaction studies (MIS) has the potential to be useful in this respect. The method utilises the responsiveness of polyelectrolyte microgel networks to oppositely charged molecules as a means to monitor the interaction between peptides and hyaluronic acid (HA), a major constituent of the subcutaneous extracellular matrix. We use the method to determine parameters describing the strength of interaction between peptide and HA as well as the peptide's aggregation tendency and transport properties in HA networks. The results from MIS studies of the peptide drugs exenatide, pramlintide, vancomycin, polymyxin B, lanreotide, MEDI7219 and AZD2820 are compared with results from measurements with the commercially available SCISSOR system and in vivo absorption and bioavailability data from the literature. We show that both MIS and SCISSOR reveal differences in the peptides' diffusivity and tendency to aggregate in the presence of HA. We show that MIS is particularly good at discriminating between peptides forming aggregates stabilised by non-electrostatic forces in the presence of HA, and peptides forming complexes stabilised by electrostatic interactions with HA. The method provides two parameters that can be used to quantify the peptides' aggregation tendency, the one describing the peptide packing density in complexes with HA and the other the apparent diffusivity upon release in a medium of physiological ionic strength and pH. The order of the peptides when ranked by increasing binding strength at pH 7.4 determined with MIS is shown to be in agreement with the order when ranked by the apparent 1st order absorption rate constant (k_a) after sc administration in humans: lanreotide (Autogel) < exenatide (IRF) < AZD2820 < pramlintide < lanreotide (IRF) (IRF: Immediate release formulation). A correlation is found between the 1st order release rate constant determined with SCISSOR and k_a for lanreotide (Autogel), exenatide and AZD2820. A mechanism relating the magnitude of k_a to the peptides’ charge is proposed.

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.

We have synthesized and characterized a collagen-hyaluronic acid hybrid network. The aim was to create a hydrogel mimicking the extracellular matrix of adipose tissue, primarily for use in in vitro studies of protein drug transport in the subcutaneous interstitial space. The network was created by covalently crosslinking methacryloyl-functionalized collagen type I and thiol-functionalized hyaluronic acid by means of thiol-Michael and thiol-ene photo-click reaction. The degree of modification corresponded to 74 % of the lysine and arginine groups on collagen, and 16 to 29 % of the carboxylate groups on hyaluronic acid, as determined with H-1 NMR. Circular dichroism measurements showed that the triple helix of modified collagen remained intact. Oscillatory shear rheology tests showed that the hydrated networks displayed viscoelastic properties characteristic of hydrogels. The storage modulus, measured at 1 Hz frequency in the linear viscoelastic range (<5%), varied in a controllable way between 1.5 and 4 kPa depending on the collagen concentration and collagen-to-hyaluronic acid ratio. The hydrogels had a lower collagen content (0.6--1.2 wt%) but similar hyaluronic acid content and shear modulus at low strain rates as the extracellular matrix in adipose tissue and were penetrable by albumin and lysozyme. The results show that the hydrogels are promising as model systems for investigations of drug transport.

Subcutaneous (SC) injection is a common route of administration for drug compounds with poor oral bioavailability. However, bioavailability is often variable and incomplete, and there is as yet no standard accepted medium for simulation of the human SC environment. In this work we evaluate a FRAP based method for quantitative determination of local self-diffusion coefficients within extracellular matrix (ECM) mimetic hydrogels, potentially useful as in vitro models for drug transport in the ECM after SC injection. Gels were made consisting of either agarose, cross-linked collagen (COL) and hyaluronic acid (HA) or cross-linked HA. The diffusivities of uncharged FITC-dextran (FD4), the highly charged poly-lysine (PLK20) and poly-glutamic acid (PLE20) as well as the GLP-1 analogue exenatide were determined within the gels using FRAP. The diffusion coefficients in uncharged agarose gels were in the range of free diffusion in PBS. The diffusivity of cationic PLK20 in gels containing anionic HA was substantially decreased due to strong electrostatic interactions. Peptide aggregation could be observed as immobile fractions in experiments with exenatide. We conclude that the FRAP method provides useful information of peptides’ interactions and transport properties in hydrogel networks, giving insight into the mechanisms affecting absorption of drug compounds after subcutaneous injection.