A microparticulate based formulation to protect therapeutic enzymes from proteolytic digestion: phenylalanine ammonia lyase as case study.

Phenylketonuria is a genetic disorder affecting the metabolism of phenylalanine (phe) due to a deficiency in the enzyme phenylalanine hydroxylase. This disorder is characterized by an elevated phe blood level, which can lead to severe intellectual disabilities in newborns. The current strategy to prevent these devastating consequences is limited to a life-long phe-free diet, which implies major lifestyle changes and restrictions. Recently, an injectable enzyme replacement therapy, Pegvaliase, has been approved for treating phenylketonuria, but is associated with significant side-effects. In this study a phe-metabolizing system suitable for oral delivery is designed to overcome the need for daily injections. Active phenylalanine ammonia-lyase (PAL), an enzyme that catalyses phe metabolism, is loaded into mesoporous silica microparticles (MSP) with pore sizes ranging from 10 to 35 nm. The surface of the MSP is lined with a semipermeable barrier to allow permeation of phe while blocking digestive enzymes that degrade PAL. The enzymatic activity can be partially preserved in vitro by coating the MSP with poly(allylamine) and poly(acrylic acid)-bowman birk (protease inhibitor) conjugate. The carrier system presented herein may provide a general approach to overcome gastro-intestinal proteolytic digestion and to deliver active enzymes to the intestinal lumen for prolonged local action.

Preactivated silicone oil as potential long-term vitreous replacement with nonemulsifying properties.

AIM: Assessment of preactivated and thiolated silicone oils as potential long-term vitreous replacement. METHODS: Thioglycolic acid (TGA) and 3-mercaptopropionic acid (MPA) were covalently coupled to amino-modified silicone oil and subsequently preactivated with 2-mercaptonicotinic acid (2-MNA). Each silicone thiomer was evaluated in view of oxidation, reductive reliquefaction, emulsification, and cytotoxicity. RESULTS: Both thiol ligands were coupled in almost quantitative yield to the silicone oils' backbone with a total amount of thiol groups of 223 ± 25 and 219 ± 11 µmol/g for silicone-TGA and silicone-MPA, respectively. A following preactivation with 2-MNA of around 50% of all thiol groups could be achieved. Preactivated silicones showed a protection against oxidation as the viscosity of silicone-TGA and silicone-MPA after iodine treatment were two- and fourfold higher than the preactivated counterparts, respectively. The percentage of emulsification was below 8% for both preactivated silicones in comparison to control with 100% emulsification. Silicone-TGA-MNA and silicone-MPA-MNA could be aspired via a 20 G needle within 35 s and showed a reversible sol-gel transition. CONCLUSION: Preactivated silicone oils tackle the shortcomings of currently available silicone oils as they unite suitable handling qualities with a resistance against emulsification, which renders them promising for long-term vitreous replacement. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 551-559, 2017.

Development, in vitro and in vivo evaluation of a self-emulsifying drug delivery system (SEDDS) for oral enoxaparin administration.

AIM: The aim of this study was to develop SEDDS for oral enoxaparin administration and evaluate it in vitro and in vivo. METHODS: The emulsifying properties of SEDDS composed of long chain lipids (LC-SEDDS), medium chain lipids (MC-SEDDS), short chain lipids (SC-SEDDS) and no lipids (NL-SEDDS) were evaluated. Thereafter, enoxaparin was incorporated via hydrophobic ion pairing in the chosen SEDDS, which were evaluated regarding their mucus permeating properties, stability towards pancreatic lipase, drug release profile and cytotoxicity. Finally, in vivo performance of SEDDS was evaluated. RESULTS: The average droplet size of chosen LC-SEDDS, MC-SEDDS and NL-SEDDS ranged between 30 and 40nm. MC-SEEDS containing 30% Captex 8000, 30% Capmul MCM, 30% Cremophor EL and 10% propylene glycol and NL-SEDDS containing 31.5% Labrafil 1944, 22.5% Capmul PG-8, 9% propylene glycol, 27% Cremophor EL and 10% DMSO exhibited 2-fold higher mucus diffusion than LC-SEDDS and were therefore chosen for further studies. The enoxaparin-dodecylamine complex (ENOX/DOA) was incorporated in a payload of 2% (w/w) into MC-SEDDS and NL-SEDDS. After 90min 97% of MC-SEDDS and 5% of NL-SEDDS were degraded by pancreatic lipase. Both MC-SEDDS and NL-SEDDS showed sustained in vitro enoxaparin release. Furthermore, orally administrated MC-SEDDS and NL-SEDDS yielded an absolute enoxaparin bioavailability of 2.02% and 2.25%, respectively. CONCLUSION: According to the abovementioned findings, SEDDS could be considered as a potential oral LMWH delivery system.

Totally S-protected hyaluronic acid: Evaluation of stability and mucoadhesive properties as liquid dosage form.

AIM: It is the aim of this study to synthesize hyaluronic acid (HA) derivatives bearing mucoadhesive properties and showing prolonged stability at pH 7.4 and under oxidative condition as liquid dosage form. METHODS: HA was modified by thiolation with l-cysteine (HA-SH) and by conjugation with 2-mercaptonicotinic acid-l-cysteine ligand to obtain an S-protected derivative (HA-MNA). The polymers were characterized by determination of thiol group content and mercaptonicotinic acid content. Cytotoxicity, stability and mucoadhesive properties (rheological evaluation and tensile test) of the polymers were evaluated. RESULTS: HA-SH and HA-MNA could be successfully synthesized with a degree of modification of 5% and 9% of the total moles of carboxylic acid groups, respectively. MTT assay revealed no toxicity for the polymers. HA-SH resulted to be unstable both at pH 7.4 and under oxidative conditions, whereas HA-MNA was stable under both conditions. Rheological assessment showed a 52-fold and a 3-fold increase in viscosity for HA-MNA incubated with mucus compared to unmodified HA and HA-SH, respectively. Tensile evaluation carried out with intestinal and conjunctival mucosa confirmed the higher mucoadhesive properties of HA-MNA compared to HA-SH. CONCLUSIONS: According to the presented results, HA-MNA appears to be a potent excipient for the formulation of stable liquid dosage forms showing comparatively high mucodhesive properties.

Thiolated graphene oxide as promising mucoadhesive carrier for hydrophobic drugs.

The aim of this study was to improve the mucoadhesive properties of graphene by conjugating thiol ligands, in order to formulate an oral delivery system for hydrophobic drugs showing long mucus residence time. Graphene oxide was obtained by oxidation of graphite and then was thiolated following two synthetic paths. On the one hand, the hydroxyl groups were conjugated with thiourea passing through the formation of a brominated intermediate. On the other hand, the carboxylic acid groups were conjugated with cysteamine via carbodiimide chemistry. The mucoadhesive properties of thiolated graphene were evaluated by rheological measurements and by residence time assay. Then, valsartan was loaded on thiolated graphene and the release profile was evaluated in simulated intestinal fluid. Following both synthetic paths it was possible to obtain thiolated graphene bearing 215-302µmol SH/g product. Both products induced after 1h incubation an increase of mucus viscosity of about 22-33-fold compared to unmodified graphite. The residence time assay confirmed that 60% of thiolated graphene could be retained on intestinal mucosa after 4h incubation, whereas just 20% of unmodified graphite could be retained. Valsartan could be loaded with a drug loading of about 31±0.3% and a sustained release profile was observed for both formulations. According to the presented data, the thiolation of graphene could improve its mucoadhesive properties. Therefore, thiolated graphene represents a promising platform for oral delivery of hydrophobic drugs, possessing a long residence time on intestinal mucosa which allows the release of the loaded drug close to the adsorptive epithelium.

Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation.

The aim of this study was the development of zeta potential changing self-emulsifying drug delivery systems (SEDDS). Various cationic surfactants were incorporated into a formulation consisting of 30% Cremophor EL, 30% Capmul MCM, 30% Captex 355 and 10% propylene glycol (w/w). A substrate of intestinal alkaline phosphatase (IAP), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium (PA), was thereafter incorporated into SEDDS. Size, zeta potential and polydispersity index were determined. Phosphate release studies were performed using three different models, namely, isolated IAP, Caco-2 cell monolayer and rat intestinal mucosa and the amount of released phosphate was quantified by malachite green assay. Interaction of SEDDS and mucus was investigated regarding surface charges and mucus diffusion studies were performed using rotating tube technique. SEDDS were diluted 1:100 in 100mM HEPES buffer and a negative zeta potential was obtained. By addition of isolated IAP, 15% to 20% phosphate was liberated from SEDDS within 3h and a shift of zeta potential from negative to positive was observed. On Caco-2 cell monolayer and rat intestinal mucosa, 12% and 23% phosphate were released, respectively, from SEDDS diluted 1:1000 in glucose-HEPES buffer. Positively charged droplets were bound to negatively charged mucus resulting in a decrease of zeta potential, whereas negatively charged SEDDS showed no interaction. Furthermore, negatively charged SEDDS diffused faster through mucus layer as higher extent of incorporated Lumogen was present in deeper mucus segments in comparison to positively charged ones. Accordingly, zeta potential changing SEDDS provide an effective mucus permeation combined with higher cellular uptake when droplets reach absorptive epithelium membrane.

Mucoadhesive polymers: Synthesis and in vitro characterization of thiolated poly(vinyl alcohol).

The aim of this study was to synthesize thiolated poly(vinyl alcohol) (PVA) and to evaluate its mucoadhesive properties. Thiourea and 3-mercaptopropionic acid were utilized in order to obtain thiolated PVAs, namely, TPVA1 and TPVA2, respectively. TPVA1 and TPVA2 displayed 130.44 ± 14.99 and 958.35 ± 155.27 µmol immobilized thiol groups per gram polymer, respectively, which were then evaluated regarding reactivity of thiol groups, swelling behavior and mucoadhesive properties. Both thiolated PVAs exhibited the highest reactivity at pH 8.0 whereas more than 95% of free thiol groups were preserved at pH 5.0. Thiolation of PVA decelerated water uptake and prolonged disintegration time of test discs compared to unmodified PVA. Contact time of TPVA1- and TPVA2-based test discs on porcine intestinal mucosa was 3.2- and 15.8-fold prolonged, respectively, in comparison to non-thiolated PVA as measured by rotating cylinder method. According to tensile studies on mucosa, the total work of adhesion (TWA) and the maximum detachment force (MDF) were increased when compared to PVA. Furthermore, thiolated PVAs preserved higher percentage of viable cells compared to unmodified PVA within 24h as evaluated by MTT assay. Accordingly, thiolated PVA represents a novel excipient that can likely improve the mucoadhesive properties of various pharmaceutical formulations.

Novel bioadhesive polymers as intra-articular agents: Chondroitin sulfate-cysteine conjugates.

The aim of this study was to generate and characterize a chondroitin sulfate-cysteine conjugate (CS-cys) as a novel bioadhesive agent for intra-articular use. Mucoadhesive properties of synthesized CS-cys were investigated by rheological measurement of polymer-mucus mixture and rotating cylinder method, while bioadhesive features of CS-cys on porcine articular cartilage were evaluated via tensile studies. Thiolation was achieved by attachment of l-cysteine to CS via amide bond formation mediated by carbodiimide as a coupling reagent. The conjugate exhibited 421.17±35.14 µmol free thiol groups per gram polymer. The reduced CS-cys displayed 675.09±39.67 µmol free thiol groups per gram polymer after disulfide bonds reduction using tris(2-carboxyethyl)phosphine hydrochloride. The increase in dynamic viscosity of thiolated CS due to oxidative disulfide bond formation was demonstrated using capillary viscometer. The combination of CS-cys and mucus led to 4.57-fold increase in dynamic viscosity in comparison with mucus control. Furthermore, adhesion time to porcine mucosa of CS-cys-based test disk was enhanced by 2.48-fold compared to unmodified CS as measured by rotating cylinder method suggesting the interaction between thiomers and mucus gel layer via disulfide bonds formation. Tensile studies of thiolated CS on porcine articular cartilage showed 5.37- and 1.76-fold increase in the total work of adhesion and the maximum detachment force, respectively, in comparison with unmodified CS indicating bioadhesive features of CS-cys. Cytotoxicity of CS-cys was assessed in Caco-2 cells and rat primary articular chondrocytes using MTT and LDH release assay, thereby showing the safety of CS-cys at a concentration of 0.25% (w/v) in Caco-2 cells. Furthermore, 0.1% of CS-cys was found non-toxic to rat primary articular chondrocytes. According to these results, CS-cys provides improved bioadhesive properties that might be useful as an intra-articular agent for treatment of osteoarthritis.

Insulin loaded mucus permeating nanoparticles: Addressing the surface characteristics as feature to improve mucus permeation.

AIM: It was the aim of this study to combine two strategies - namely the virus-mimicking strategy and the surface PEGylation strategy - in order to generate highly mucus permeating nanocarriers for oral insulin delivery. METHODS: Chondroitin sulphate was covalently conjugated with poly(ethylene glycol) 5 kDa at different degree of modification and with the functionalized polymers NPs were formulated via complexation with chitosan. NPs were characterized by particle size, zeta potential, surface hydrophilicity and permeation ability in porcine mucus and on excised mucosa. RESULTS: The NPs presented a size between 510 and 670 nm and a zeta potential between -1 and -5 mV when dispersed in simulated intestinal fluid. The mucus permeation test revealed a correlation between the NPs hydrophilicity and their ability to move through mucus. A 5-fold higher amount of NPs with the higher degree of PEGylation could permeate through fresh mucus compared to non-PEGylated NPs. Moreover, highly PEGylated NPs showed a 3.7-fold greater ability to be retained in intestinal mucosa against buffer flow compared to unmodified NPs. Finally, insulin was incorporated with a payload of 2.18% and the release profile showed a 65% release within 4h. CONCLUSIONS: Results of this study provide strong evidence for the potential of combining different surface modification strategies in order to improve the mucus permeating properties of NPs for oral peptide delivery.

Preactivated thiolated nanoparticles: A novel mucoadhesive dosage form.

Within this study a novel form of mucoadhesive nanoparticles (NPs) exhibiting a prolonged residence time on mucosal tissues was developed. In order to achieve this goal a new thiomer was synthesized by the covalent attachment of the amino acid l-cysteine ethyl ester to poly(acrylic acid) (100 kDa). The free thiol groups were in the following preactivated with the aromatic thiol bearing ligand 2-mercaptonicotinic acid (2-MNA) and the amount of coupled l-cysteine ethyl ester as well as the amount of attached 2-MNA was determined. Based on this, preactivated thiomer NPs were prepared by ionic gelation with polyethylenimine (PEI). The resulting NPs were characterized regarding size and zeta potential. Furthermore their mucoadhesive properties were investigated via rheological measurements with porcine intestinal mucus and via determination of the particles' mucosal residence time. Results showed that 1666.74 µmol l-cysteine ethyl ester and 603.07 µmol 2-MNA could be attached per gram polymer. NPs were in a size range of 112.67-252.84 nm exhibiting a zeta potential of -29 mV. Thiolated NPs only led to a 2-fold increase in mucus viscosity whereas preactivated NPs showed a 6-fold higher mucus viscosity than unmodified NPs. The mucosal residence time of thiolated NPs was 1.6-fold prolonged and that of preactivated NPs even 4.4-fold higher compared to unmodified particles. Accordingly, preactivated thiolated NPs providing a prolonged residence time on mucosal membranes could be a promising dosage form for various applications.

Methods to determine the interactions of micro- and nanoparticles with mucus.

The present review provides an overview of methods and techniques for studying interactions of micro- and nanoparticulate drug delivery system with mucus. Nanocarriers trapped by mucus are featuring a change in particle size and zeta potential that can be utilized to predict their mucus permeation behavior. Furthermore, interactions between nanoparticulate drug delivery systems and mucus layer modify the viscoelasticity of mucus which can be detected via rheological studies and quartz crystal microbalance with dissipation monitoring (QCM-D) analysis. Having a closer look at molecular interactions between drug carrier and mucus small-angle neutron scattering (SANS) is an appropriate analysis technique. Moreover, different methods to determine particle diffusion in mucus such as the newly established Transwell diffusion system, rotating silicone tube technique, multiple-particle tracking (MPT) and diffusion NMR are summarized within this review. The explanations and discussed pros and cons of collated methods and techniques should provide a good starting point for all those looking forward to move in this interesting field.

Nanoparticles decorated with proteolytic enzymes, a promising strategy to overcome the mucus barrier.

The intestinal mucus gel layer represents a stumbling block for drug adsorption. This study is aimed to formulate a nanoparticulate system able to overcome this barrier by cleaving locally the glycoprotein substructures of the mucus. Mucolytic enzymes such as papain (PAP) and bromelain (BRO) were covalently conjugated to poly(acrylic acid) (PAA). Nanoparticles (NPs) were then formulated via ionic gelation method and characterized by particle size, zeta potential, enzyme content and enzymatic activity. The NPs permeation quantified by rotating tube studies was correlated with changes in the mucus gel layer structure determined by pulsed-gradient-spin-echo NMR (PGSE-NMR), small-angle neutron scattering (SANS) and spin-echo SANS (SESANS). PAP and BRO functionalized NPs had an average size in the range of 250 and 285 nm and a zeta potential that ranged between -6 and -5 mV. The enzyme content was 242 µg enzyme/mg for PAP modified NPs and 253 µg enzyme/mg for BRO modified NPs. The maintained enzymatic activity was 43% for PAP decorated NPs and 76% for BRO decorated NPs. The rotating tube technique revealed a better performance of BRO decorated NPs compared to PAA decorated NPs, with a 4.8-fold higher concentration of NPs in the inner slice of mucus. Addition of 0.5 wt% of enzyme functionalized NPs to 5 wt% intestinal mucin led to c.a. 2-fold increase in the mobility of the mucin as measured by PGSE-NMR indicative of a significant break-up of the structure of the mucin. SANS and SESANS measurements further revealed a change in structure of the intestinal mucus induced by the incorporation of the functionalized NPs mostly occurring at a length scale longer than 0.5 µm. Accordingly, BRO decorated NPs show higher potential than PAP functionalized NPs as mucus permeating drug delivery systems.

Mucus permeating carriers: formulation and characterization of highly densely charged nanoparticles.

The GI mucus layer represents a significant block to drug carriers absorption. Taking an example from nature, virus-mimicking nanoparticles (NPs) with highly densely charged surface were designed with the aim to improve their mucus permeation ability. NPs were formulated by combining chitosan with chondroitin sulfate and were characterized by particle size, ζ-potential and hydrophobicity. The interaction occurring between NPs and diluted porcine intestinal mucus was investigated by a new method. Furthermore, the rotating tube technique was exploited to evaluate the NPs permeation ability in fresh undiluted porcine intestinal mucus. NPs (400-500 nm) presenting a slightly positive (4.02 mV) and slightly negative (-3.55 mV) ζ-potential resulted to be hydrophobic and hydrophilic, respectively. On the one hand the hydrophobic NPs undergo physico-chemical changes when incubated with mucus, namely the size increased and the ζ-potential decreased. On the other hand, the hydrophilic NPs did not significantly change size and net charge during incubation with mucus. Both types of NPs showed a 3-fold higher diffusion ability compared to the reference 50/50 DL-lactide/glycolide copolymer NPs (136 nm, -23 mV, hydrophilic). Based on these results, this work gives valuable information for the further design of mucus-penetrating NPs.

Pre-systemic metabolism of orally administered drugs and strategies to overcome it.

The oral bioavailability of numerous drugs is not only limited by poor solubility and/or poor membrane permeability as addressed by the biopharmaceutical classification system (BCS) but also by a pre-systemic metabolism taking place to a high extent in the intestine. Enzymes responsible for metabolic reactions in the intestine include cytochromes P450 (CYP450), transferases, peptidases and proteases. Furthermore, in the gut nucleases, lipases as well as glycosidases influence the metabolic pathway of drugs and nutrients. A crucial role is also played by the intestinal microflora able to metabolize a wide broad of pharmaceutical compounds. Strategies to provide a protective effect towards an intestinal pre-systemic metabolism are based on the co-administration of enzyme inhibitor being optimally immobilized on unabsorbable and undegradable polymeric excipients in order to keep them concentrated there where an inhibitory effect is needed. Furthermore, certain polymeric excipients such as polyacrylates exhibit per se enzyme inhibitory properties. In addition, by incorporating drugs in cyclodextrines, in self-emulsifying drug delivery systems (SEDDS) or liposomes a protective effect towards an intestinal enzymatic attack can be achieved. Being aware of the important role of this pre-systemic metabolism by integrating it in the BCS as third dimension and keeping strategies to overcome this enzymatic barrier in mind, the therapeutic efficacy of many orally given drugs can certainly be substantially improved.