1-Benzylindoles as inhibitors of cytosolic phospholipase A2α: synthesis, biological activity, aqueous solubility, and cell permeability.

Cytosolic phospholipase A2α (cPLA2α) is considered an interesting target for the development of new anti-inflammatory drugs, as it is significantly involved in the formation of pro-inflammatory lipid mediators. Recently, in a ligand-based virtual screening approach, 2,4-dichlorobenzyl-substituted 4-[2-(indol-3-ylmethylene)hydrazineyl]benzoic acid 7 was found to be an inhibitor of cPLA2α with micromolar activity. This compound has now been systematically varied to increase inhibitory potency. The studies performed led to 5-(1-benzylindol-3-ylmethyl)-2H-tetrazol-2-yl)pentanoic acid derivatives that exhibited submicromolar activity against the enzyme. The most potent compounds were also tested for their water solubility and for permeability in a Caco-2 model. Among other things, it was found that in Caco-2 cells, the pentanoic acid chain of the molecules can be metabolised to a considerable extent to propionic acid by ß-oxidation.

Challenges of nanoparticle albumin bound (nab™) technology: Comparative study of Abraxane® with a newly developed albumin-stabilized itraconazole nanosuspension.

Nanoparticle albumin bound™ (nab™) technology is an established delivery platform for development of albumin stabilized nanoparticles as drug delivery systems for poorly water-soluble drugs. By using albumin for particle stabilization, nab™ technology does not require solubilizers or emulsifiers for the formulation of poorly water-soluble drugs for intravenous use. Despite the great potential, however, to date only two products based on nab™ technology have been approved by the Food and Drug Administration: Abraxane® (nab™ paclitaxel) and Fyarro® (nab™ rapamycin). In this study, the commercially available product Abraxane® was characterized in comparison to an albumin stabilized nanosuspension for the poorly water-soluble drug itraconazole. The aim of this study was to identify critical product parameters of the nanosuspensions depending on the manufacturing process in order to assess the transferability of nab™ technology to other drugs. The colloidal properties, stabilizing protein composition and particle disintegration behavior were analyzed. In addition, studies were carried out on the impact of the key process step, the high-pressure homogenization, using a design of experiments (DoE) approach. A nanosuspension comprising spherical, stable drug nanoparticles stabilized by a large fraction of dissolved albumin around the nanoparticles were identified. During the manufacturing process, the drug core was coated with a layer of albumin, which was cross-linked to a certain level. The Abraxane® and itraconazole suspensions differed in the analyzed protein fraction, with stronger cross-linking at the particle surface for Abraxane®. Both active pharmaceutical ingredients were present in the amorphous state as nanoparticles. In vitro disintegration studies performed to mimic a strong dilution during intravenous application showed the disintegration of the nanoparticles. All in all, the analysis underlined the transferability of the nab™ technology to selected other poorly water-soluble drugs with the great advantage of eliminating solubilizers and emulsifiers for intravenous applications.

Improved Reversion of Calcifications in Porcine Aortic Heart Valves Using Elastin-Targeted Nanoparticles.

Calcified aortic valve disease in its final stage leads to aortic valve stenosis, limiting cardiac function. To date, surgical intervention is the only option for treating calcific aortic valve stenosis. This study combined controlled drug delivery by nanoparticles (NPs) and active targeting by antibody conjugation. The chelating agent diethylenetriaminepentaacetic acid (DTPA) was covalently bound to human serum albumin (HSA)-based NP, and the NP surface was modified using conjugating antibodies (anti-elastin or isotype IgG control). Calcification was induced ex vivo in porcine aortic valves by preincubation in an osteogenic medium containing 2.5 mM sodium phosphate for five days. Valve calcifications mainly consisted of basic calcium phosphate crystals. Calcifications were effectively resolved by adding 1-5 mg DTPA/mL medium. Incubation with pure DTPA, however, was associated with a loss of cellular viability. Reversal of calcifications was also achieved with DTPA-coupled anti-elastin-targeted NPs containing 1 mg DTPA equivalent. The addition of these NPs to the conditioned media resulted in significant regression of the valve calcifications compared to that in the IgG-NP control without affecting cellular viability. These results represent a step further toward the development of targeted nanoparticular formulations to dissolve aortic valve calcifications.

Triglyceride-filled albumin-based nanocapsules: A promising new system to avoid discarding poorly water-soluble drug candidates.

Even though current drug discovery provides a variety of potential drug candidates, many of those substances are difficult to formulate due to their poor water-solubility. To overcome this obstacle a technological formulation is crucial. Albumin-based nanocarriers are a possible intravenous delivery system which is already approved and commercially available. However, no universal carrier for poorly water-soluble substances is found yet. In the present study, new preparation processes for nanocapsules consisting of a medium-chain triglyceride (MCT) core and a human serum albumin (HSA) shell were developed. The nanocarrier system exhibits desirable physicochemical properties with a hydrodynamic diameter of 150 nm and a polydispersity index of 0.1. Furthermore, the nanocapsules were stable towards the addition of electrolytes and also in basic to neutral pH range. The nanocapsules were storage stable for at least 7 months at 4 °C and could also be lyophilized to reach an even longer shelf life of at least 21 months. In addition, the nanocapsule system showed no cytotoxicity in cell culture. The developed system represents a suitable carrier for a variety of different poorly water-soluble drug substances (e.g., fenofibrate, naproxen, indomethacin) showing a high potential for a universal formulation platform for further lipophilic active pharmaceutical ingredients (APIs).

Synthesis, activity, metabolic stability and cell permeability of new cytosolic phospholipase A2α inhibitors with 1-indolyl-3-phenoxypropan-2-one structure.

Cytosolic phospholipase A2α (cPLA2α), the key enzyme of the arachidonic acid cascade, is considered to be an interesting target for the development of new anti-inflammatory drugs. Potent inhibitors of the enzyme include indole-5-carboxylic acids with propan-2-one residues in position 1 of the indole. Previously, it was found that central pharmacophoric elements of these compounds are their ketone and carboxylic acid groups, which unfortunately are subject to pronounced metabolism by carbonyl reductases and glucuronosyltransferases, respectively. Here we show that the metabolic stability of these inhibitors can be improved by introducing alkyl substituents in the vicinity of the ketone group or by increasing their rigidity. Furthermore, permeability tests with Caco-2 cells revealed that the indole derivatives have only low permeability, which can be attributed to their affinity to efflux transporters. Among other things, the polar ketone group in the center of the molecules seems to be a decisive factor for their reverse transport. After its removal, the permeability increased significantly. The enhancement in metabolic stability and permeability achieved by the structural variations carried out was accompanied by a more or less pronounced decrease in the inhibitory potency of the compounds against cPLA2α.

Synthesis and pharmacokinetic properties of novel cPLA2α inhibitors with 1-(carboxyalkylpyrrolyl)-3-aryloxypropan-2-one structure.

Indole-5-carboxylic acids with 3-aryloxy-2­oxopropyl residues in position 1 have been shown to be potent inhibitors of cytosolic phospholipase A2α (cPLA2α), an enzyme involved in the formation of pro-inflammatory lipid mediators. Unfortunately, in animal experiments, only very low plasma concentrations could be achieved after peroral administration of this type of compound. Since insufficient metabolic stability was suspected as the cause, structural modifications were made to optimize this property. These included the conversion of the aromatic into an aliphatic carboxylic acid function as well as the rigidification of the lipophilic structural elements. A selected pyrrole-3-propionic acid was tested for its peroral in vivo bioavailability in mice. However, higher plasma concentrations could not be achieved also with this compound. Using the Caco2 cell permeation assay, substances investigated were found to be very good substrates for gastrointestinal efflux transporters, which explains their poor peroral absorption.

Permeability of dopamine D2 receptor agonist hordenine across the intestinal and blood-brain barrier in vitro.

Hordenine, a bioactive food compound, has several pharmacological properties and has recently been identified as a dopamine D2 receptor (D2R) agonist. Since the pharmacokinetic profile of hordenine has been described to a limited extent, the present study focused on the transfer and transport of hordenine across the intestinal epithelium and the blood-brain barrier (BBB) in vitro. Hordenine was quickly transferred through the Caco-2 monolayer in only a few hours, indicating a rapid oral uptake. However, the high bioavailability may be reduced by the observed efflux transport of hordenine from the bloodstream back into the intestinal lumen and by first pass metabolism in intestinal epithelial cells. To determine the biotransformation rate of hordenine, the metabolite hordenine sulfate was synthesized as reference standard for analytical purposes. In addition, transfer studies using primary porcine brain capillary endothelial cells (PBCEC) showed that hordenine is able to rapidly penetrate the BBB and potentially accumulate in the brain. Thus, a D2R interaction of hordenine and activation of dopaminergic signaling is conceivable, assuming that the intestinal barrier can be circumvented by a route of administration alternative to oral uptake.

Hexafluoroisopropyl Carbamates as Selective MAGL and Dual MAGL/FAAH Inhibitors: Biochemical and Physicochemical Properties.

A series of hexafluoroisopropyl carbamates with indolylalkyl- and azaindolylalkyl-substituents at the carbamate nitrogen was synthesized and evaluated for inhibition of the endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The synthesized derivatives with butyl to heptyl spacers between the heteroaryl and the carbamate moiety were inhibitors of both enzymes. For investigated compounds in which the alkyl chain was partially incorporated into a piperidine ring, different results were obtained. Compounds with a methylene spacer between the piperidine ring and the heteroaromatic system were found to be selective MAGL inhibitors, while an extension of the alkyl spacer to two to four atoms resulted in dual inhibition of FAAH/MAGL. The only small change in enzyme inhibitory activity with variation of the heteroaromatic system indicates that the reactive hexafluoroisopropyl carbamate group is mainly responsible for the strength of the inhibitory effect of the compounds. Selected derivatives were also tested for hydrolytic stability in aqueous solution, liver homogenate and blood plasma as well as for aqueous solubility and for permeability in a Caco-2 cell model. Some compounds showed a slightly higher MAGL inhibitory effect than the known selective MAGL inhibitor ABX-1431 and also partly surpassed this substance with regard to certain physicochemical and biochemical properties such as water solubility and cell permeability.

Spacer length and serum protein adsorption affect active targeting of trastuzumab-modified nanoparticles.

Receptor-mediated active targeting of nanocarriers is a widely investigated approach to specifically address cancerous cells and tissues in the human body. The idea is to use these formulations as drug carriers with enhanced specificity and therefore reduced systemic side effects. Until today a big obstacle to reach this goal remains the adsorption of serum proteins to the nanocarrier's surface after contact with biological fluids. In this context different nanoparticle characteristics could be beneficial for effective active targeting after formation of a protein corona which need to be identified. In this study trastuzumab was used as an active targeting ligand which was covalently attached to human serum albumin nanoparticles. For coupling reaction different molecular weight spacers were used and resulting physicochemical nanoparticle characteristics were evaluated. The in vitro cell association of the different nanoparticle formulations was tested in cell culture experiments with or without fetal bovine serum. For specific receptor-mediated cell interaction SK-BR-3 breast cancer cells with human epidermal growth factor receptor 2 (HER2) overexpression were used. MCF-7 breast cancer cells with normal HER2 expression served as control. Furthermore, serum protein adsorption on respective nanoparticles was characterized. The qualitative and quantitative composition of the protein corona was analyzed by SDS-PAGE and LC-MS/MS and the influence of protein adsorption on active targeting capability was determined.

Erratum to 'Spacer length and serum protein adsorption affect active targeting of trastuzumab-modified nanoparticles' [Biomaterials and Biosystems 5 (2022) 100032].

[This corrects the article DOI: 10.1016/j.bbiosy.2021.100032.].

Identification of main influencing factors on the protein corona composition of PLGA and PLA nanoparticles.

Poly(DL-lactic-co-glycolic acid) and poly(DL-lactic acid) are widely used for the preparation of nanoparticles due to favorable characteristics for medical use like biodegradability and controllable degradation behavior. The contact with different media like human plasma or serum leads to the formation of a protein corona that determines the NP's in vivo processing. In this study, the impact of surface end group identity, matrix polymer hydrophobicity, molecular weight, and incubation medium on the protein corona composition was evaluated. Corona proteins were quantified using Bradford assay, separated by SDS-PAGE, and identified via LC-MS/MS. The acquired data revealed that surface end group identity had the most profound effect on corona composition in both quantitative and qualitative terms. Regarding matrix polymer hydrophobicity, adsorption profiles on NP systems with similar physicochemical characteristics resembled each other. The molecular weight of the matrix polymers proved to impact quantity, but not quality of corona bound proteins. The corona of plasma incubated NP showed adsorption of incubation medium-specific proteins but resembled those of serum incubated NP in terms of protein function, average mass and isoelectric point. Overall, the NP physicochemical properties proved to be easily adjustable determining factors of protein corona formation in physiological environments.

Light-responsive polymeric nanoparticles based on a novel nitropiperonal based polyester as drug delivery systems for photosensitizers in PDT.

Although nanoparticles (NPs) bear a great potential in tumour therapy, just a few nanosized drug delivery systems are commercially available. Besides their advantages like passive drug targeting and stable embedment of lipophilic active pharmaceutical ingredients, targeted drug release is a major challenge for a safe therapy. While drug release of commonly used materials depends on physiological factors, nanoparticles prepared by using stimuli responsive polymers offer a promising approach. External irradiation of light-sensitive nanoparticles enables local drug release, resulting in selective accumulation and consequently more effective treatment with less side effects. In this study light-responsive nanoparticles based on a new innovative light-responsive polyester (Nip-SLrPE) combined with poly(DL-lactide-co-glycolide) (PLGA) were prepared and examined for their physicochemical characteristics and light-triggered properties. As model drug the photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorine (mTHPC) was incorporated and light-depending drug release was investigated. Furthermore, cytotoxic potential of selected formulations for PDT and intracellular accumulation of mTHPC were evaluated. In conclusion, nanoparticles based on the new light-sensitive Nip-SLrPE showed auspicious light-responsive properties, resulting in promising results for a smart drug delivery system.

Nanoparticle albumin-bound mTHPC for photodynamic therapy: Preparation and comprehensive characterization of a promising drug delivery system.

Nanoparticle albumin-bound (nab)-technology is an industrial applicable manufacturing method for the preparation of albumin-based drug carriers of poorly water-soluble drugs. In the present study the advantages of nanotechnology, albumin as an endogenous protein with the capability of high tumor enrichment and the selective light activation of the photosensitizer Temoporfin (mTHPC) were combined to a new delivery system for oncological use. The herewith provided well-established photodynamic therapy may enable a beneficial alternative for the treatment of solid tumors. In the present study a reproducible method for the preparation of stable mTHPC-albumin nanoparticles via nab-technology was established. The nanoparticles were physicochemically characterized with regard to particle size and size distribution and the impact of this preparation method on nanoparticle as well as mTHPC stability was investigated. Nanoparticles with improved colloidal stability over a broad pH range and in the presence of physiological NaCl concentrations were achieved in high yield. Due to high pressure homogenization a certain oxidative decay of mTHPC was observed. Cell culture experiments revealed an effective cellular uptake of mTHPC in a cholangiocarcinoma cell line (TFK-1). After light-activation high cytotoxicity was shown for photosensitizer loaded nanoparticles enabling the application of the proposed formulation in photodynamic therapy.

Reversion of arterial calcification by elastin-targeted DTPA-HSA nanoparticles.

Generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE) are characterized by pathologic calcifications in the media of large- and medium sized arteries. GACI is associated with biallelic mutations in ENPP1 in the majority of cases, whereas mutations in ABCC6 are known to cause PXE. Different treatment approaches including bisphosphonates and orally administered pyrophosphate (PPi) were investigated in recent years, but reversion of calcification could not be achieved. With this study, we pursued the idea of a combination of controlled drug delivery through nanoparticles and active targeting via antibody conjugation to develop a treatment for GACI and PXE. To establish a suitable drug delivery system, the chelating drug diethylenetriamine pentaacetic acid (DTPA) was conjugated to nanoparticles composed of human serum albumin (HSA) as biodegradable and non-toxic particle matrix. To accomplish an active targeting of the elastic fibers exposed through calcification of the affected areas, the nanoparticle surface was functionalized with an anti-elastin antibody. Cytotoxicity and cell interaction studies revealed favorable preconditions for the intended i.v. application. The chelating ability was evaluated in vitro and ex vivo on aortic ring culture isolated from two mouse models of GACI and PXE. The positive results led to the conclusion that the produced nanoparticles might be a promising therapy in the treatment of GACI and PXE.

Nrf2 Activation Protects Mouse Beta Cells from Glucolipotoxicity by Restoring Mitochondrial Function and Physiological Redox Balance.

Influencing the redox balance of pancreatic beta cells could be a promising strategy for the treatment of diabetes. Nuclear factor erythroid 2p45-related factor 2 (Nrf2) is present in beta cells and regulates numerous genes involved in antioxidant defense. As reactive oxygen species (ROS) are important for beta cell signaling but induce oxidative stress when present in excess, this study elucidates the influence of Nrf2-activating compounds on different kinds of ROS and correlates changes in redox balance to effects on mitochondrial function, insulin release, and cell viability. Acute glucose stimulation (15 mmol/L) of murine islet cells of C57Bl/6N mice affects ROS and redox status of the cells differently. Those ROS monitored by dihydroethidium, which detects superoxide radical anions, decrease. By contrast, oxidant status, monitored by dichlorodihydrofluorescein, as well as intracellular H2O2, increases. Glucolipotoxicity completely prevents these fast, glucose-mediated alterations and inhibits glucose-induced NAD(P)H production, mitochondrial hyperpolarization, and ATP synthesis. Oltipraz (10 µmol/L) or dimethyl fumarate (DMF, 50 µmol/L) leads to nuclear accumulation of Nrf2, restores mitochondrial activity and glucose-dependent ROS turnover, and antagonizes glucolipotoxicity-induced inhibition of insulin release and apoptosis. Importantly, these beneficial effects only occur when beta cells are challenged and damaged by high lipid and carbohydrate supply. At physiological conditions, insulin release is markedly reduced in response to both Nrf2 activators. This is not associated with severe impairment of glucose-induced mitochondrial hyperpolarization or a rise in apoptosis but coincides with altered ROS handling. In conclusion, Nrf2 activators protect beta cells against glucolipotoxicity by preserving mitochondrial function and redox balance. As our data show that this maintains glucose-stimulated insulin secretion, targeting Nrf2 might be suited to ameliorate progression of type 2 diabetes mellitus. By contrast, nonstressed beta cells do not benefit from Nrf2 activation, thus underlining the importance of physiological shifts in ROS homeostasis for the regulation of beta cell function.

Incorporation of doxorubicin in different polymer nanoparticles and their anticancer activity.

Background: Nanoparticles are under investigation as carrier systems for anticancer drugs. The expression of efflux transporters such as the ATP-binding cassette (ABC) transporter ABCB1 is an important resistance mechanism in therapy-refractory cancer cells. Drug encapsulation into nanoparticles has been shown to bypass efflux-mediated drug resistance, but there are also conflicting results. To investigate whether easy-to-prepare nanoparticles made of well-tolerated polymers may circumvent transporter-mediated drug efflux, we prepared poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) nanoparticles loaded with the ABCB1 substrate doxorubicin by solvent displacement and emulsion diffusion approaches and assessed their anticancer efficiency in neuroblastoma cells, including ABCB1-expressing cell lines, in comparison to doxorubicin solution. Results: The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement led to the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH 7 reaching 53 µg doxorubicin/mg nanoparticle. These PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH 7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anticancer effects. However, nanoparticle-encapsulated doxorubicin did not display increased efficacy in ABCB1-expressing cells relative to doxorubicin solution. Conclusion: Doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anticancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anticancer efficacy. The design of drug-loaded nanoparticles with optimised anticancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anticancer therapy. Further research is required to understand under which circumstances nanoparticles can be used to overcome efflux-mediated resistance in cancer cells.

Doxorubicin-loaded human serum albumin nanoparticles overcome transporter-mediated drug resistance in drug-adapted cancer cells.

Resistance to systemic drug therapy is a major reason for the failure of anticancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3rVCR1) and doxorubicin (UKF-NB-3rDOX20). Doxorubicin-loaded nanoparticles displayed increased anticancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were re-sensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles successfully circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.

Serum type and concentration both affect the protein-corona composition of PLGA nanoparticles.

Background: When nanoparticles (NPs) are applied into a biological fluid, such as blood, proteins bind rapidly to their surface forming a so-called "protein corona". These proteins are strongly attached to the NP surface and confers them a new biological identity that is crucial for the biological response in terms of body biodistribution, cellular uptake, and toxicity. The corona is dynamic in nature and it is well known that the composition varies in dependence of the physicochemical properties of the NPs. In the present study we investigated the protein corona that forms around poly(lactide-co-glycolide) (PLGA) NPs at different serum concentrations using two substantially different serum types, namely fetal bovine serum (FBS) and human serum. The corona was characterized by means of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), Bradford protein assay, zeta potential measurements, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Additionally, the time-dependent cell interaction of PLGA NPs in the absence or presence of a preformed protein corona was assessed by in vitro incubation experiments with the human liver cancer cell line HepG2. Results: Our data revealed that the physiological environment critically affects the protein adsorption on PLGA NPs with significant impact on the NP-cell interaction. Under comparable conditions the protein amount forming the protein corona depends on the serum type used and the serum concentration. On PLGA NPs incubated with either FBS or human serum a clear difference in qualitative corona protein composition was identified by SDS-PAGE and LC-MS/MS in combination with bioinformatic protein classification. In the case of human serum a considerable change in corona composition was observed leading to a concentration-dependent desorption of abundant proteins in conjunction with an adsorption of high-affinity proteins with lower abundance. Cell incubation experiments revealed that the respective corona composition showed significant influence on the resulting nanoparticle-cell interaction. Conclusion: Controlling protein corona formation is still a challenging task and our data highlight the need for a rational future experimental design in order to enable a prediction of the corona formation on nanoparticle surfaces and, therefore, the resulting biodistribution in the body.

In vitro evaluation of innovative light-responsive nanoparticles for controlled drug release in intestinal PDT.

Nanoparticles (NP) have gained importance as drug delivery systems for pharmaceutical challenging drugs. Their size properties allow passive targeting of cancer tissue by exploiting the enhanced permeability and retention (EPR) effect. Furthermore, surface modifications enable an active drug targeting for diseased regions in the human body. Besides the advantages, the drug release from commonly used biodegradable NP is mostly depending on physiological circumstances. Hence, there is a need for a more controllable drug release. The use of light-responsive polymers is an innovative conception enabling a more distinct drug release by an external light stimulus. The idea provides potential for an increase in efficiency and safety of local therapies. In this study, innovative light-sensitive NP were investigated for a photodynamic therapy (PDT) of gastrointestinal tumors. Nanoparticles based on a newly developed light-responsive polycarbonate (LrPC) and poly(lactic-co-glycolic-acid) (PLGA) were loaded with the approved photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (mTHPC). Mucus penetrating properties were obtained by surface PEGylation of the nanoparticles either by using LrPC in combination with a PEGylated PLA (PEG-PLA) or by a combination with PEGylated LrPC (LrPC-PEG). Cytotoxic potential in dependency of a light-induced drug release was investigated in different cytotoxicity assays. Intracellular accumulation in mucus producing colon-carcinoma cell line HT-29-MTX was analysed by HPLC and confocal laser microscopy.

Effect of nanoparticle size and PEGylation on the protein corona of PLGA nanoparticles.

Upon intravenous administration of nanoparticles (NP) into the bloodstream, proteins bind rapidly on their surface resulting in a formation of a so-called 'Protein Corona'. These proteins are strongly attached to the NP surface and provide a new biological identity which is crucial for the reaction at the nano-biointerface. The structure and composition of the protein corona is greatly determined by the physico-chemical properties of the NP and the characteristics of the biological environment. The overall objective of this study was to characterize the role of NP size/surface curvature and PEGylation on the formation of the protein corona. Therefore, we prepared NP in a size of 100 and 200 nm using the biodegradable polymers poly(DL-lactide-co-glycolide) (PLGA) and poly(DL-lactide-co-glycolide)-co-polyethylene glycol diblock (PLGA-PEG) and subsequently incubated them with fetal bovine serum (FBS) to induce the formation of a protein corona. After removal of unbound protein, we employed different analytical approaches to study the corona in detail. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to gain a first impression about amount and composition of the corona proteins. Identification was carried out after tryptic in-solution digestion and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). In addition, we successfully established the Bradford protein assay as a suitable colorimetric method to quantify total adsorbed protein amount after alkaline hydrolysis of PLGA based NP. Our results revealed that protein adsorption on PLGA- and PLGA-PEG-NP didn't depend on NP size within the range of 100 and 200 nm. PEGylation led to a significant reduced amount of bound proteins. The depletion of proteins which are involved in immune response was remarkable and indicated a prolonged circulation time in body.