PEG-Coated Large Mesoporous Silicas as Smart Platform for Protein Delivery and Their Use in a Collagen-Based Formulation for 3D Printing.

Silica-based mesoporous systems have gained great interest in drug delivery applications due to their excellent biocompatibility and high loading capability. However, these materials face challenges in terms of pore-size limitations since they are characterized by nanopores ranging between 6-8 nm and thus unsuitable to host large molecular weight molecules such as proteins, enzymes and growth factors (GFs). In this work, for an application in the field of bone regeneration, large-pore mesoporous silicas (LPMSs) were developed to vehicle large biomolecules and release them under a pH stimulus. Considering bone remodeling, the proposed pH-triggered mechanism aims to mimic the release of GFs encased in the bone matrix due to bone resorption by osteoclasts (OCs) and the associated pH drop. To this aim, LPMSs were prepared by using 1,3,5-trimethyl benzene (TMB) as a swelling agent and the synthesis solution was hydrothermally treated and the influence of different process temperatures and durations on the resulting mesostructure was investigated. The synthesized particles exhibited a cage-like mesoporous structure with accessible pores of diameter up to 23 nm. LPMSs produced at 140 °C for 24 h showed the best compromise in terms of specific surface area, pores size and shape and hence, were selected for further experiments. Horseradish peroxidase (HRP) was used as model protein to evaluate the ability of the LPMSs to adsorb and release large biomolecules. After HRP-loading, LPMSs were coated with a pH-responsive polymer, poly(ethylene glycol) (PEG), allowing the release of the incorporated biomolecules in response to a pH decrease, in an attempt to mimic GFs release in bone under the acidic pH generated by the resorption activity of OCs. The reported results proved that PEG-coated carriers released HRP more quickly in an acidic environment, due to the protonation of PEG at low pH that catalyzes polymer hydrolysis reaction. Our findings indicate that LPMSs could be used as carriers to deliver large biomolecules and prove the effectiveness of PEG as pH-responsive coating. Finally, as proof of concept, a collagen-based suspension was obtained by incorporating PEG-coated LPMS carriers into a type I collagen matrix with the aim of designing a hybrid formulation for 3D-printing of bone scaffolds.

Oral self-emulsifying delivery systems for systemic administration of therapeutic proteins: science fiction?

Objective: The aim of this study was to develop self-emulsifying drug delivery systems (SEDDS) for oral delivery of therapeutic proteins through hydrophobic ion pairing. Method: Horseradish peroxidase (HRP), a model protein, was ion paired with sodium docusate to increase its hydrophobicity. The formed enzyme - surfactant complex was incorporated into SEDDS, followed by permeation studies across Caco-2 cell monolayer and freshly excised rat intestine. Results: Hydrophobic ion pairs (HIP) were formed between HRP and sodium docusate with the efficiency of 87.49 ± 1.35%. The formed complex maintained 60.97 ± 1.48% of the original enzyme activity. The ion pair was subsequently loaded into SEDDS with a payload of 0.1% (mass per cent, m/m). The obtained emulsion formed by SEDDS had a droplet size in the range from 20 to 200 nm with negative zeta potential. Permeation mechanism of the enzyme was energy-dependent and the encapsulation of the HIP complex in SEDDS enhanced the permeation of the enzyme through the Caco-2 cell monolayer and freshly excised rat intestine by 4 times and 2.5 times compared to the free enzyme, respectively. Conclusion: According to these findings, hydrophobic ion pairing followed by incorporation to SEDDS might be considered as a potential strategy for oral delivery of therapeutic proteins.

Effects of formulation development methods on the stability of model protein pharmaceuticals embedded in solid lipid matrices.

This study was conducted to investigate the influence of formulation development methods on the stability (secondary structure, aggregation, and biological activity) of protein drugs embedded in lipid matrices. Catalase, horseradish peroxidase, and α-chymotrypsin were employed as model proteins, while Precirol® AT05 (glyceryl palmitostearate) was used as lipid matrix. Protein-loaded lipid matrices were prepared using melting and mixing and wet granulation methods. Attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, size exclusion chromatography (SEC) and biological activity analyses were performed. ATR FT-IR analysis indicated significant interference of the lipid with the protein amide-I band, which was eliminated using spectral subtraction. Wet granulation method induced more changes in protein secondary structure compared to melting and mixing method. SEC analysis gave evidence of protein aggregation for catalase upon adopting the wet granulation method. The biological activity of catalase was found to reduce significantly than other two proteins upon using wet granulation method, which might be ascribed to both secondary structure alterations and the formation of aggregates. Horseradish peroxidase and α-chymotrypsin did not form any soluble aggregates. In conclusion, melting and mixing method emerged as a better incorporation method compared to wet granulation because of better stability shown by the formulated proteins.

Beyond the Roles in Biomimetic Chemistry: An Insight into the Intrinsic Catalytic Activity of an Enzyme for Tumor-Selective Phototheranostics.

Protein-assisted biomimetic synthesis has been an emerging offshoot of nanofabrication in recent years owing to its features of green chemistry, facile process, and ease of multi-integration. As a result, many proteins have been used for biomimetic synthesis of varying kinds of nanostructures. Although the efforts on exploring new proteins and investigating their roles in biomimetic chemistry are increasing, the most essential intrinsic properties of proteins are largely neglected. Herein we report a frequently used enzyme (horseradish peroxidase, HRP) to demonstrate the possibility of enzymatic activity retaining after accomplishing the roles in biomimetic synthesis of ultrasmall gadolinium (Gd) nanodots and stowing its substrate 2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), denoted as Gd@HRPABTS. It was found that ca. 70% of the enzymatic activity of HRP was preserved. The associated changes of protein structure with chemical treatments were studied by spectroscopic analysis. Leveraging on the highly retained catalytic activity, Gd@HRPABTS exerts strong catalytic oxidation of peroxidase substrate ABTS into photoactive counterparts in the presence of intrinsic H2O2 inside the tumor, therefore enabling tumor-selective catalytic photoacoustic (PA) imaging and photothermal therapy (PTT). In addition, the MR moiety of Gd@HRPABTS provides guidance for PTT and further diagrams that Gd@HRPABTS is clearable from the body via kidneys. Preliminary toxicity studies show no observed adverse effects by administration of them. This study demonstrates beyond the well-known roles in biomimetic chemistry that HRP can also preserve its enzymatic activity for tumor catalytic theranostics.

Photo-crosslinkable, injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage.

Millions of patients worldwide suffer from cartilage injury and age/disease-related cartilage degeneration. However, cartilage, such as articular cartilage, is poor at self-regeneration. Current treatments are often invasive with limited efficacy. Developing minimal invasive strategies for effective cartilage repair is highly desired. Here, we report an injectable, photo-crosslinkable sericin hydrogel as a biomimetic extracellular matrix for minimal invasively repairing cartilage. Sericin was functionalized to be sericin methacryloyl (SerMA), which formed an in situ hydrogel upon UV light irradiation via photo-crosslinking. Possessing excellent biocompatibility, SerMA hydrogels were adhesive to chondrocytes, and promoted the proliferation of attached chondrocytes even in a nutrition-lacking condition. SerMA hydrogels exhibited photoluminescent property allowing real-time monitoring hydrogels' status. The mechanical properties and degradation rates (73% for SMH-1, 47% for SMH-2 and 37% for SMH-3 after 45 days) of SerMA hydrogels were readily tunable by varying methacryloyl modification degrees to meet various repair requirements. Notably, the in vivo implantation of chondrocyte-laden SerMA hydrogels effectively formed artificial cartilages after 8 weeks. Most importantly, the artificial cartilages molecularly resembled native cartilage as evidenced by high accumulation of cartilage-specific ECM components and upregulated expression of cartilage-critical genes. Together, this sericin hydrogel is a promising tissue engineering scaffold for generating artificial cartilage in vivo towards effective, minimal invasive cartilage repair.

Horseradish and radish peroxidases eaten with fish could help explain observed associations between fish consumption and protection from age-related dementia.

A juxtaposition of regional cuisines and recent prospective studies of fish consumption in China and Japan points to fresh horseradish and/or radish (HRR) as possible contributors to delaying age-related dementia. The hypothesis is that the inverse association found sometimes between fish intake and cognitive decline is partially due to exposure of the oral cavity to active peroxidases from HRR served in conjunction with fish. This hypothesis can be tested by specifically looking at whether HRR is consumed with fish and whether such HRR is prepared in a way that preserves activity of HRR peroxidases. It is possible that by putting active HRR peroxidases in their mouths, elderly people supplement their age-diminished salivary antioxidant capacity and break down additional hydrogen peroxide (H2O2) in the oral cavity before it can migrate into the brain, thus decreasing the incidence of brain cell death induction by chronically-elevated H2O2. Intentional exposure of the oral cavity to active HRR peroxidases could be a prophylactic for delaying dementia. Because vegetable peroxidases are inactivated by gastric juices, it will be difficult to obtain benefit from HRR peroxidases' antioxidant effect via ingestion in encapsulated dietary supplements.

Biomimetic Strategy To Reversibly Trigger Functionality of Catalytic Nanocompartments by the Insertion of pH-Responsive Biovalves.

We describe an innovative strategy to generate catalytic compartments with triggered functionality at the nanoscale level by combining pH-reversible biovalves and enzyme-loaded synthetic compartments. The biovalve has been engineered by the attachment of stimuli-responsive peptides to a genetically modified channel porin, enabling a reversible change of the molecular flow through the pores of the porin in response to a pH change in the local environment. The biovalve functionality triggers the reaction inside the cavity of the enzyme-loaded compartments by switching the in situ activity of the enzymes on/off based on a reversible change of the permeability of the membrane, which blocks or allows the passage of substrates and products. The complex functionality of our catalytic compartments is based on the preservation of the integrity of the compartments to protect encapsulated enzymes. An increase of the in situ activity compared to that of the free enzyme and a reversible on/off switch of the activity upon the presence of a specific stimulus is achieved. This strategy provides straightforward solutions for the development of catalytic nanocompartments efficiently producing desired molecules in a controlled, stimuli-responsive manner with high potential in areas, such as medicine, analytical chemistry, and catalysis.

Design of Block Copolymer Micellar Aggregates for Co-Delivery of Enzyme and Anticancer Prodrug.

Traditional enzyme-prodrug therapy (EPT) is a two-step strategy, which has many serious deficiencies, so having a one-step EPT treatment becomes a problem of immediate interest. This study aims to achieve an effective co-delivery of horseradish peroxidase (HRP) as a kind of enzyme for prodrug activation and ethyl 3-indoleacetate (EIA) as anticancer prodrug. A ternary block copolymer PEG-PAsp(AED)-CA consisting of poly(ethylene glycol) (PEG), reduction-sensitive poly (N-(2,2'-dithiobis(ethylamine)) aspartamide) PAsp(AED), and cholic acid (CA) was synthesized and assembled into spherical micelles which encapsulated EIA in its hydrophobic core and HRP in a reduction-sensitive interlayer. TEM photographs show that the polymer micelle is around 40 nm, and the cell survival rate test shows that the EIA/HRP polymer micelle is highly lethal to human lung adenocarcinoma cells. Thus, co-delivery of EIA and HRP demonstrates great potential in cancer therapy, offering a structurally simple and highly tunable platform for the synchronous delivery of enzymes and prodrugs in EPT.

Intra- and trans-cellular delivery of enzymes by direct conjugation with non-multivalent anti-ICAM molecules.

Intercellular adhesion molecule 1 (ICAM-1) is a cell-surface protein overexpressed in many diseases and explored for endocytosis and transcytosis of drug delivery systems. All previous evidence demonstrating ICAM-1-mediated transport of therapeutics into or across cells was obtained using nanocarriers or conjugates coupled to multiple copies of anti-ICAM antibodies or peptides. Yet, transport of therapeutics linked to non-multivalent anti-ICAM ligands has never been shown, since multivalency was believed to be necessary to induce transport. Our goal was to explore whether non-multivalent binding to ICAM-1 could drive endocytosis and/or transcytosis of model cargo in different cell types. We found that anti-ICAM was specifically internalized by all tested ICAM-1-expressing cells, including epithelial, fibroblast and neuroblastoma cells, primary or established cell lines. Uptake was inhibited at 4°C and in the presence of an inhibitor of the ICAM-1-associated pathway, rather than inhibitors of the clathrin or caveolar routes. We observed minimal transport of anti-ICAM to lysosomes, yet prominent and specific transcytosis across epithelial monolayers. Finally, we coupled a model cargo (the enzyme horseradish peroxidase (HRP)) to anti-ICAM and separated a 1:2 antibody:enzyme conjugate for non-multivalent ICAM-1 targeting. Similar to anti-ICAM, anti-ICAM-HRP was specifically internalized and transported across cells, which rendered intra- and trans-cellular enzyme activity. Therefore, non-multivalent ICAM-1 targeting also provides transport of cargoes into and across cells, representing a new alternative for future therapeutic applications via this route.

Feedback from visual cortical area 7 to areas 17 and 18 in cats: How neural web is woven during feedback.

To investigate the feedback effect from area 7 to areas 17 and 18, intrinsic signal optical imaging combined with pharmacological, morphological methods and functional magnetic resonance imaging (fMRI) was employed. A spatial frequency-dependent decrease in response amplitude of orientation maps was observed in areas 17 and 18 when area 7 was inactivated by a local injection of GABA, or by a lesion induced by liquid nitrogen freezing. The pattern of orientation maps of areas 17 and 18 after the inactivation of area 7, if they were not totally blurred, paralleled the normal one. In morphological experiments, after one point at the shallow layers within the center of the cat's orientation column of area 17 was injected electrophoretically with HRP (horseradish peroxidase), three sequential patches in layers 1, 2 and 3 of area 7 were observed. Employing fMRI it was found that area 7 feedbacks mainly to areas 17 and 18 on ipsilateral hemisphere. Therefore, our conclusions are: (1) feedback from area 7 to areas 17 and 18 is spatial frequency modulated; (2) feedback from area 7 to areas 17 and 18 occurs mainly ipsilaterally; (3) histological feedback pattern from area 7 to area 17 is weblike.

A glucose-powered antimicrobial system using organic-inorganic assembled network materials.

A new glucose-driven photodynamic antimicrobial system was developed to efficiently kill bacteria and fungi, taking advantage of organic-inorganic network materials encapsulating glucose oxidase and horseradish peroxidase and bioluminescence resonance energy transfer (BRET).

Food allergens are transferred intact across the rat blood-placental barrier in vivo.

We investigated the mechanism of transplacental macromolecular transport in rats on the nineteenth day of pregnancy using tracers, transmission electron microscopy and immunohistochemistry. The blood-placental barrier of full-term rat placentas was composed of a trilaminar layer of trophoblast cells that separates the fetal capillaries from the maternal blood spaces: a layer of cytotrophoblasts lining the maternal blood space and a bilayer of syncytiotrophoblast surrounding the fetal capillaries. Horseradish peroxidase, intravenously injected into the maternal circulation, was found in the maternal blood spaces, the interspaces between the cytotrophoblasts and the syncytiotrophoblast I, many pits and small vesicles in the syncytiotrophoblast I, vesicles of the syncytiotrophoblast II, fetal connective tissue and fetal capillaries. Intravenously injected ovalbumin was detected in the maternal blood spaces, a trilaminar layer and the fetal capillaries. Neonatal Fc receptor (FcRn), a receptor for IgG, was localized at the maternal side of the blood-placental barrier. These results show that the structure of the rat blood-placental barrier is quite similar to the human blood-placental barrier, and non-specific macromolecules and food allergens may penetrate through the blood-placental barrier of the full-term placenta from the maternal to fetal circulation mediated by FcRn.

Human erythrocytes as drug carriers: loading efficiency and side effects of hypotonic dialysis, chlorpromazine treatment and fusion with liposomes.

Human red blood cells (RBCs) are emerging as a highly biocompatible microparticulate drug delivery system. So far, drugs have commonly been loaded into freshly isolated RBCs using rather disruptive methods based on hypotonic shock, and assessment of damage was restricted to hemolysis. Here, we investigated loading of RBCs from blood bank units with enzymes of various molecular weights using hypotonic dialysis (HD), pretreatment with chlorpromazine (CPZ) and fusion with liposomes. The latter two techniques have received little attention in RBC loading so far. Along with loading efficiency, all methods were tested for the induction of side effects. Very importantly, next to hemolysis, we also addressed morphological changes and phosphatidyl serine (PS) exposure, which has been recognized as a critical parameter associated with premature RBC removal and induction of transfusion-related pathologies. The efficiency of loading using hypotonic dialysis decreased with the molecular weight of the enzyme. For liposomes and chlorpromazine, loading efficiencies were higher and independent of enzyme molecular weights. While hypotonic dialysis always induced a high degree of hemolysis, irreversible modifications in the morphology of the cells and PS exposure, the side effects that were induced by loading using CPZ and liposomes were limited. In particular, PS exposure, although high immediately after treatment, returned to physiological levels after recovery. Retention and deformability studies using a spleen-mimicking device showed that RBCs treated with CPZ and liposomes behave like physiological RBCs, while HD led to very fragile and poorly deformable RBCs.

[The influence of strabismus and monocular deprivation on the size of callosal cells in cortical areas 17 and 18 of the cat].

To reveal the changes in visual cortex structure following impaired early binocular experience, the size (somatic area) of callosal cells in areas 17, 18 ofmonocularly deprived and convergent strabismic cats was measured. Horseradish peroxidase was injected into the single ocular dominance columns of areas 17, 18 and the transition zone 17/18. In both groups of impaired cats the mean size of callosal cells in area 17 was increased in comparison to intact cats. In area 18, the similar difference was found in monocularly deprived cats only. It was shown that the differences in the mean sizes of cells are due to the increase of the number of large cells. In strabismic cats, the portion of large cells (soma > 200 mkm2) in area 17 was 58% and in area 18 was 8%. The relative share of large cells in areas 17 and 18 of monocularly deprived cats was similar (28 and 26 % correspondingly). These data show that early binocular vision impairments may lead to the changes in cytoarchitecture of cortical layers where the interhemispheric connections originate.

A novel double-coating carrier produced by solid-in-oil and solid-in-water nanodispersion technology for delivery of genes and proteins into cells.

A novel intracellular delivery method both for genes and proteins is one of the most coveted systems in the drug delivery field. In the present study, we developed a double-coating carrier loaded with gene and protein produced by solid-in-oil and solid-in-water nanodispersion technology. The double-coating carriers did not require electrostatic interactions during the preparation so were able to encapsulate plasmid DNA, ovalbumin (pI 4.5), horseradish peroxidase (pI 7.2), and cytochrome-c (pI 10.5) in a consistent manner. The carriers had practical encapsulation efficiencies and release profiles for genes and proteins. Furthermore, effective gene expression and cellular uptakes of both anionic and cationic proteins were achieved by modification of carriers with functional molecules. These findings indicate that the double-coating carrier has high potential for cellular delivery of various drugs and is a novel, superior method for both gene and protein delivery into cells.

Direct quantitation of peptide-mediated protein transport across a droplet-interface bilayer.

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.

Protein diffusion in photopolymerized poly(ethylene glycol) hydrogel networks.

In this study, protein diffusion through swollen hydrogel networks prepared from end-linked poly(ethylene glycol)-diacrylate (PEG-DA) was investigated. Hydrogels were prepared via photopolymerization from PEG-DA macromonomer solutions of two molecular weights, 4600 Da and 8000 Da, with three initial solid contents: 20, 33 and 50 wt/wt% PEG. Diffusion coefficients for myoglobin traveling across the hydrogel membrane were determined for all PEG network compositions. The diffusion coefficient depended on PEG molecular weight and initial solid content, with the slowest diffusion occurring through lower molecular weight, high-solid-content networks (D(gel) = 0.16 ± 0.02 × 10(-8) cm(2) s(-1)) and the fastest diffusion occurring through higher molecular weight, low-solid-content networks (D(gel) = 11.05 ± 0.43 × 10(-8) cm(2) s(-1)). Myoglobin diffusion coefficients increased linearly with the increase of water content within the hydrogels. The permeability of three larger model proteins (horseradish peroxidase, bovine serum albumin and immunoglobulin G) through PEG(8000) hydrogel membranes was also examined, with the observation that globular molecules as large as 10.7 nm in hydrodynamic diameter can diffuse through the PEG network. Protein diffusion coefficients within the PEG hydrogels ranged from one to two orders of magnitude lower than the diffusion coefficients in free water. Network defects were determined to be a significant contributing factor to the observed protein diffusion.

Cortical layer V neurons in the auditory and visual cortices of normal, reeler, and yotari mice.

Both in the Reelin-deficient reeler and Dab1-deficient yotari mice, layer V corticospinal tract neurons in the sensory-motor cortex are radially spread instead of being confined to a single cortical layer. In the present study, we examined distribution pattern of cortical layer V neurons in the visual and auditory cortices of reeler and yotari mice with the injection of HRP into the superior and inferior colliculi of the adult animals, respectively. After the injection of HRP into the superior colliculus of the normal mouse, retrogradely labeled cells were distributed in layer V of the visual cortex, while the similar injection of HRP in the reeler and yotari mice produced radial dispersion of retrograde labeling through all of the depths of the visual cortex of these mutant mice. Next, we injected HRP into the inferior colliculus of the normal, reeler and yotari mice. Retrogradely labeled neurons were distributed in layer V of the normal auditory cortex, whereas they were again radially scattered in the auditory cortex of the reeler and yotari mice. Taken together with the previous and present findings, layer V cortical efferent neurons are radially scattered in the sensory-motor, visual and auditory cortices of the reeler and yotari mice.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Preparation and evaluation of thiomer nanoparticles via high pressure homogenization.

The aim of this study was to establish and evaluate a high pressure homogenization method for the preparation of thiomer nanoparticles. Particles were formulated by incorporation of the model protein horseradish peroxidase in chitosan-glutathione (Ch-GSH) and poly(acrylic acid)-glutathione (PAA-GSH) via co-precipitation followed by air jet milling. The resulting microparticles were suspended in distilled water using an Ultraturax and subsequently micronized by high pressure homogenization. Finally, resulting particles were evaluated regarding size distribution, shape, zeta potential, drug load, protein activity and release behaviour. The mean particle size after 30 cycles with a pressure of 1500 bar was 538 +/- 94 nm for particles consisting of Ch-GSH and 638 +/- 94 nm for particles consisting of PAA-GSH. Nanoparticles of Ch-GSH had a positive zeta-potential of +1.03 mv, whereas nanoparticles from PAA-GSH had a negative zeta potential of -6.21 mv. The maximum protein load for nanoparticles based on Ch-GSH and based on PAA-GSH was 45 +/- 2% and 37 +/- %, respectively. The release profile of nanoparticles followed a first order release kinetic. Thiolated nanoparticles prepared by a high pressure homogenization technique were shown to be stable and provide controlled drug release characteristics. The preparation method described here might be a useful tool for a more upscaled production of nanoparticulate drug delivery systems.