Comparison of Protein-like Model Particles Fabricated by Micro 3D Printing to Established Standard Particles.

Innovative analytical instruments and development of new methods has provided a better understanding of protein particle formation in biopharmaceuticals but have also challenged the ability to obtain reproducible and reliable measurements. The need for protein-like particle standards mimicking the irregular shape, translucent nature and near-to-neutral buoyancy of protein particles remained one of the hot topics in the field of particle detection and characterization in biopharmaceutical formulations. An innovative protein-like particle model has been developed using two photo polymerization (2PP) printing allowing to fabricate irregularly shaped particles with similar properties as protein particles at precise size of 50 µm and 150 µm, representative of subvisible particles and visible particles, respectively. A study was conducted to compare the morphological, physical, and optical properties of artificially generated protein particles, polystyrene spheres, ETFE, and SU-8 particle standards, along with newly developed protein-like model particles manufactured using 2PP printing. Our results suggest that 2PP printing can be used to produce protein-like particle standards that might facilitate harmonization and standardization of subvisible and visible protein particle characterization across laboratories and organizations.

Amphiphilic Copolymers Shuttle Drugs Across the Blood-Brain Barrier.

Medical treatment of diseases of the central nervous system requires transport of drugs across the blood-brain barrier (BBB). Here, it is extended previously in vitro experiments with a model compound to show that the non-water-soluble and brain-impermeable drug domperidone (DOM) itself can be enriched in the brain by use of an amphiphilic copolymer as a carrier. This carrier consists of poly(N-(2-hydroxypropyl)-methacrylamide), statistically copolymerized with 10 mol% hydrophobic lauryl methacrylate, into whose micellar aggregates DOM is noncovalently absorbed. As tested in a BBB model efficient transport of DOM across, the BBB is achievable over a wide range of formulations, containing 0.8 to 35.5 wt% domperidone per copolymer. In neither case, the polymer itself is translocated across the BBB model. In vivo experiments in mice show that already 10 min after intraperitoneal injection of the polymer/domperidone (PolyDOM) formulation, domperidone can be detected in blood and in the brain. Highest serum and brain levels of domperidone are detected 40 min after injection. At that time point serum domperidone is increased 48-fold. Most importantly, domperidone is exclusively detectable in high amounts in the brain of PolyDOM injected mice and not in mice injected with bare domperidone.

Interaction of N-(2-hydroxypropyl)methacrylamide based homo, random and block copolymers with primary immune cells.

Polymer nanoparticles (NP), e.g., polymeric micelles, represent a promising platform for drug delivery including the field of immune modulation. In respect to this potential application, identification of chemical and structural properties that affect interaction of polymers with immune cells is an important step in their preclinical characterization. A series of well-defined, fluorescently labeled homopolymers, random as well as block copolymers based on the clinically approved N-(2-hydroxypropyl)methacrylamide (HPMA) were prepared to study the influence of polymer architecture on the interaction of polymers with primary human und murine immune cells systematically. The number average of the molar mass (M(n)) for all polymers was set to the range of 4-14 kDa with a varying ratio of hydrophilic and hydrophobic units and dispersities (D) in the range of 1.17-1.29. Cell uptake greatly depended on the polymer molecular weight and micro structure: Comparison of polymers of the same molar mass but varying ratio of hydrophilic and hydrophobic units revealed a strict dependency of cellular uptake on the size of the hydrophobic block. HPMA-ran-LMA copolymers with high amounts of lauryl side chains (15 and 20% LMA content) had highest internalization rates into human and mouse immune cells (monocytes, granulocytes, B and T cells). Our findings underline the role of particle size and composition of polymeric carriers in the field of nanomedicine.

A minimal hydrophobicity is needed to employ amphiphilic p(HPMA)-co-p(LMA) random copolymers in membrane research.

Because a polymer environment might be milder than a detergent micelle, amphiphilic polymers have attracted attention as alternatives to detergents in membrane biochemistry. The polymer poly[N-(2-hydroxypropyl)-methacrylamid] [p(HPMA)] has recently been modified with hydrophobic lauryl methacrylate (LMA) moieties, resulting in the synthesis of amphiphilic p(HPMA)-co-p(LMA) polymers. p(HPMA)-co-p(LMA) polymers with a LMA content of 5 or 15% have unstable hydrophobic cores. This, on one hand, promotes interactions of the hydrophobic LMA moieties with membranes, resulting in membrane rupture, but at the same time prevents formation of a hydrophobic, membrane mimetic environment that is sufficiently stable for the incorporation of transmembrane proteins. On the other hand, the p(HPMA)-co-p(LMA) polymer with a LMA content of 25% forms a stable hydrophobic core structure, which prevents hydrophobic interactions with membrane lipids but allows stable incorporation of membrane proteins. On the basis of our data, it becomes obvious that amphiphilic polymers have to have threshold hydrophobicities should an application in membrane protein research be anticipated.

Interaction of pHPMA-pLMA copolymers with human blood serum and its components.

Immediately after administration, polymer therapeutics are exposed to complex biological media like blood which may influence and alter their physicochemical properties due to interactions with proteins or serum components. Among such interactions those leading to larger sized aggregates can be sensitively detected by dynamic light scattering (DLS) as a pre in vivo screening method. Random copolymers from N-(2-hydroxypropyl)methacrylamide and lauryl methacrylate p(HPMA-co-LMA) and copolymers loaded with the model drug domperidone were characterized by DLS in isotonic salt solution and in blood serum. The bare amphiphilic copolymer micelles (Rh=30 nm in isotonic salt solution) formed large aggregates in serum of over 100 nm radius which were shown to originate from interactions with very low density lipoproteins (VLDLs). Encapsulation of the hydrophobic drug domperidone resulted, at first, in drug-copolymer formulations with larger hydrodynamic radii (39 nm

Aggregation behavior of amphiphilic p(HPMA)-co-p(LMA) copolymers studied by FCS and EPR spectroscopy.

A combined study of fluorescence correlation spectroscopy and electron paramagnetic resonance spectroscopy gave a unique picture of p(HPMA)-co-p(LMA) copolymers in aqueous solutions, ranging from the size of micelles and aggregates to the composition of the interior of these self-assembled systems. P(HPMA)-co-p(LMA) copolymers have shown high potential as brain drug delivery systems, and a detailed study of their physicochemical properties can help to elucidate their mechanism of action. Applying two complementary techniques, we found that the self-assembly behavior as well as the strength of hydrophobic attraction of the amphiphilic copolymers can be tuned by the hydrophobic LMA content or the presence of hydrophobic molecules or domains. Studies on the dependence of the hydrophobic lauryl side chain content on the aggregation behavior revealed that above 5 mol % laury side-chain copolymers self-assemble into intrachain micelles and larger aggregates. Above this critical alkyl chain content, p(HPMA)-co-p(LMA) copolymers can solubilize the model drug domperidone and exhibit the tendency to interact with model cell membranes.

Amphiphilic HPMA-LMA copolymers increase the transport of Rhodamine 123 across a BBB model without harming its barrier integrity.

The successful non-invasive treatment of diseases associated with the central nervous system (CNS) is generally limited by poor brain permeability of various developed drugs. The blood-brain barrier (BBB) prevents the passage of therapeutics to their site of action. Polymeric drug delivery systems are promising solutions to effectively transport drugs into the brain. We recently showed that amphiphilic random copolymers based on the hydrophilic p(N-(2-hydroxypropyl)-methacrylamide), pHPMA, possessing randomly distributed hydrophobic p(laurylmethacrylate), pLMA, are able to mediate delivery of domperidone into the brain of mice in vivo. To gain further insight into structure-property relations, a library of carefully designed polymers based on p(HPMA) and p(LMA) was synthesized and tested applying an in vitro BBB model which consisted of human brain microvascular endothelial cells (HBMEC). Our model drug Rhodamine 123 (Rh123) exhibits, like domperidone, a low brain permeability since both substances are recognized by efflux transporters at the BBB. Transport studies investigating the impact of the polymer architecture in relation to the content of hydrophobic LMA revealed that random p(HPMA)-co-p(LMA) having 10mol% LMA is the most auspicious system. The copolymer significantly increased the permeability of Rh123 across the HBMEC monolayer whereas transcytosis of the polymer was very low. Further investigations on the mechanism of transport showed that integrity and barrier function of the BBB model were not harmed by the polymer. According to our results, p(HPMA)-co-p(LMA) copolymers are a promising delivery system for neurological therapeutics and their application might open alternative treatment strategies.

HPMA based amphiphilic copolymers mediate central nervous effects of domperidone.

In this study we give evidence that domperidone encapsulated into amphiphilic p(HPMA)-co-p(laurylmethacrylate) (LMA) copolymer aggregates is able to cross the blood-brain barrier, since it affected motor behaviour in animals, which is a sensitive measure for CNS actions. Carefully designed copolymers based on the clinically approved p(HPMA) were selected and synthesized by a combination of controlled radical polymerization and post-polymerization modification. The hydrodynamic radii (R(h) ) of amphiphilic p(HPMA)-co-p(LMA) alone and loaded with domperidone were determined by fluorescence correlation spectroscopy.

Langmuir-Blodgett films of biocompatible poly(HPMA)-block-poly(lauryl methacrylate) and poly(HPMA)-random-poly(lauryl methacrylate): influence of polymer structure on membrane formation and stability.

Membranes based on functional biocompatible polymers can be regarded as a useful model system to study biological interactions, e.g. antibody-antigen interactions or protein polymer interactions. These model systems may give a better insight into these processes and may help to find suitable polymeric structures offering biocompatibility as well as reduced polymer protein interaction. In this respect, Langmuir-Blodgett (LB) layer formation at the air/water (A/W) interface is studied in respect to polymer architecture in this article. For this purpose, narrowly distributed N-(2-hydroxypropyl)-methacrylamide (HPMA) random and block copolymers have been prepared by the RAFT polymerization method. For random copolymers different molecular weights were prepared. As for the block copolymers also the ratio of hydrophilic and hydrophobic units was varied in order to study the influence of hydrophobic block length on collapse pressure and area. The molecular weights of all polymers were around 15 kDa and 30 kDa. In the case of block copolymers we found a direct correlation of the length of the hydrophobic block to the collapse area. Furthermore, hysteresis experiments clearly point out that block copolymers form stable LB layers. No remarkable changes in collapse pressure or area could be observed. In contrast the area occupied by random copolymers changes at each hysteresis cycle indicating a loss of polymer to the aqueous subphase. In addition the LB layers were transferred onto mica substrates. The block copolymers formed stable and defect free membranes over an area of 100 microm(2) with a roughness (rms) 1.3-1.4 A. On the contrary, membranes based on random copolymers turned out to have a higher surface roughness. Our findings clearly underline the influence of polymer structure on the LB layer formation at the A/W interface.