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Previous research shows that thermosensitive small multilamellar lipid nanoparticles (tSMLPs) offer promising features for temperature-triggered cytostatic drug delivery, remaining completely stable at body temperature (37°C) and releasing their payload under mild hyperthermia conditions (42°C). A distinguishing characteristic of tSMLPs is their unique particle morphology - multiple tightly packed bilayers with progressively decreasing intermembrane spacing toward the particle core. In this study, we shift the focus from their thermosensitivity to an in-depth exploration of the particle's morphology. Using in-vial homogenization by dual centrifugation (DC) at very high lipid concentrations (60%), we prepare SMLPs composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG₂). A systematic screening of DPPC/ DPPG₂ 100-x/x (mol/mol) from x = 0 to 100 enabled the formation of SMLPs with sizes below 200 nm, narrow size distribution and well-distinguishable morphologies. These lipid nanoparticles also demonstrated the capacity to entrap hydrophilic compounds, despite their multilamellar structure and thus limited interlamellar aqueous space. We propose that specific headgroup interactions between DPPC and DPPG₂ underlie the observed water influx upon dilution of the initially formed vesicular phospholipid gels (VPGs) during in-vial homogenization by DC. With a combination of biophysical techniques (DLS, Time-resolved fluorescence, SAXS and WAXS) and morphological analysis (cryo-EM), we present a hypothesis to explain the evolving SMLP morphology as a function of increasing DPPG₂ content in the phospholipid blend.

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.

Drug-eluting beads made of responsive polyelectrolyte networks are used in the treatment of liver cancer. Aggregates of loaded drugs in complex with the networks dissolve upon release, causing swelling of the network. According to a recent mechanism the release and swelling rates are controlled by the mass transport of drug through a depletion layer created in the microgel. We hypothesise that the mechanism, in which the stability of the drug aggregates and the swelling properties of the network play crucial roles, offers means to control the release profile also for other drugs. To test this, we investigated the loading and release properties of amitriptyline, chlorpromazine and doxepin in polyacrylate, hyaluronate and DCbeadTM microgels in a microfluidic setup. Loaded drugs could be released to a medium with physiological ionic strength and pH. The binding strength increased with decreasing critical micelle concentration of the drugs and increasing linear charge density of network chains. Microgels displayed drug-rich core/swollen shell coexistence, and swelled during release at a rate in agreement with the depletion layer mechanism, indicating its generality. The results demonstrate the potential of microgels as vehicles for amphiphilic drugs and the usefulness of the microfluidics method for in vitro studies of such systems.

Polyelectrolyte microgels may undergo volume phase transition upon loading and the release of amphiphilic molecules, a process important in drug delivery. The new phase is "born" in the outermost gel layers, whereby it grows inward as a shell with a sharp boundary to the "mother" phase (core). The swelling and collapse transitions have previously been studied with microgels in large solution volumes, where they go to completion. Our hypothesis is that the boundary between core and shell is stabilized by thermodynamic factors, and thus that collapsed and swollen phases should be able to also coexist at equilibrium. We investigated the interaction between sodium polyacrylate (PA) microgel networks (diameter: 400-850 mu m) and the amphiphilic drug amitriptyline hydrochloride (AMT) in the presence of NaCl/phosphate buffer of ionic strength (I) 10 and 155 mM. We used a specially constructed microscopy cell and micromanipulators to study the size and internal morphology of single microgels equilibrated in small liquid volumes of AMT solution. To probe the distribution of AMT micelles we used the fluorescent probe rhodamine B. The amount of AMT in the microgel was determined by a spectrophotometric technique. In separate experiments we studied the binding of AMT and the distribution between different microgels in a suspension. We found that collapsed, AMT-rich, and swollen AMT-lean phases coexisted in equilibrium or as long-lived metastable states at intermediate drug loading levels. In single microgels at I = 10 mM, the collapsed phase formed after loading deviated from the core-shell configuration by forming either discrete domains near the gel boundary or a calotte shaped domain. At I = 155 mM, single microgels, initially fully collapsed, displayed a swollen shell and a collapsed core after partial release of the AMT load. Suspensions displayed a bimodal distribution of swollen and collapsed microgels. The results support the hypothesis that the boundary between collapsed and swollen phases in the same microgel is stabilized by thermodynamic factors.

Polyelectrolyte microgels are used as delivery vehicles for amphiphilic drugs in, e.g., treatments of liver cancer by a method called trans-arterial chemoembolization. The thesis deals with fundamental properties of such delivery systems related to the self-assembling properties of the drug molecules and their interaction with the charged polymer network of the microgel. The main objective was to establish mechanistic models describing the loading and release of drugs under relevant conditions. For that purpose experimental techniques providing thermodynamic, compositional and microstructural information were used to elucidate how the kinetics depend on the stability of the drug self-assemblies and the volume response of the microgels. Micromanipulator-assisted microscopy studies showed that negatively charged microgels phase separated during loading and release of cationic amphiphilic drugs. At intermediate loading levels the drug aggregates and part of the network formed a collapsed phase coexisting with a swollen, drug-lean phase. In particular, during release in a medium of physiological ionic strength, the drug-lean phase formed a depletion layer (shell) surrounding a drug-rich core. Investigations of a series of drugs with different molecular architectures showed that the drug release rate was determined mainly by the stability of the drug aggregates in the core and the diffusive mass transport of drug molecules through the shell. Detailed studies of polyacrylate microgels interacting with amitriptyline hydrochloride showed that swelling of the shell network greatly influenced the release rate. Furthermore, experiments with a specially constructed microscopy cell was used to establish that the collapsed and swollen phases could coexist in equilibrium, and that the swelling of the network in the swollen phase depended on the proportion between them when present in the same microgel. The latter effect was related to the elastic coupling between the phases. Confocal Raman microscopy was employed to demonstrate, for the first time, the related elastic effect, that the concentration of amitriptyline in the swollen phase decreased with increasing proportion of the collapsed phase. Small-angle X-ray scattering showed that the collapsed phase had a disordered microstructure of drug micelles with ellipsoidal shape. The aggregation number increased with increasing concentration of drug in the microgel, most likely by incorporating the uncharged base form. By providing detailed information about thermodynamic properties and microstructures, the results of the thesis provide a basis for rational design of microgel drug delivery systems.

We investigated the loading of an amphiphilic drug, amitriptyline hydrochloride (AMT), onto sodium polyacrylate hydrogels at low ionic strength and its release at high ionic strength. The purpose was to show how the self-assembling properties of the drug and the swelling of the gel network influenced the loading/release mechanisms and kinetics, important for the development of improved controlled-release systems for parenteral administration of amphiphilic drugs. Equilibrium studies showed that single microgels (similar to 100 mu m) in a large solution volume underwent a discrete transition between swollen and dense states at a critical drug concentration in the solution. For single macrogels in a small solution volume, the transition progressed gradually with increasing amount of added drug, with swollen and dense phases coexisting in the same gel; in a suspension of microgels, swollen and collapsed particles coexisted. Time-resolved micropipette-assisted microscopy studies showed that drug self-assemblies accumulated in a dense shell enclosing the swollen core during loading and that a dense core was surrounded by a swollen shell during release. The time evolution of the radius of single microgels was determined as functions of liquid flow rate, network size, and AMT concentration in the solution. Mass transport of AMT in the surrounding liquid, and in the dense shell, influenced the deswelling rate during loading. Mass transport in the swollen shell controlled the swelling rate during release. A steady-state kinetic model taking into account drug self-assembly, core-shell phase separation, and microgel volume changes was developed and found to be in semiquantitative agreement with the experimental loading and release data.

Microgels, such as polymeric hydrogels, are currently used as drug delivery devices (DDSs) for chemotherapeutics and/or unstable drugs. The clinical DDS DC bead® was studied with respect to loading and release, measured as relative bead-volume, of six amphiphilic molecules in a micropipette-assisted microscopy method. Theoretical models for loading and release was used to increase the mechanistic understanding of the DDS.

It was shown that equilibrium loading was independent of amphiphile concentration. The loading model showed that the rate-determining step was diffusion of the molecule from the bulk to the bead surface (‘film control’). Calculations with the developed and applied release model on the release kinetics were consistent with the observations, as the amphiphiles distribute unevenly in the bead. The rate determining step of the release was the diffusion of the amphiphile molecule through the developed amphiphile-free depletion layer. The release rate is determined by the diffusivity and the tendency for aggregation of the amphiphile where a weak tendency for aggregation (i.e. a large cac_b) lead to faster release. Salt was necessary for the release to happen, but at physiological concentrations the entry of salt was not rate-determining. This study provides valuable insights into the loading to and release from the DDS. Also, a novel release mechanism of the clinically used DDS is suggested.