Is hyaluronic acid the perfect excipient for the pharmaceutical need?

Hyaluronic acid has become an interesting and important polymer as an excipient for pharmaceutical products due to its beneficial properties, like solubility, biocompatibility and biodegradation. To improve the properties of hyaluronic acid, different possibilities for chemical modifications are presented, and the opportunities as novel systems for drug delivery are discussed. This review gives an overview over the production of hyaluronic acid, the possibilities of its chemical modification and the current state of in vitro and in vivo research. Furthermore, market approved and commercially available products are reviewed and derivatives undergoing clinical trials and applying for market approval are shown. In particular, hyaluronic acid has been studied for different administrations in rheumatology, ophthalmology, local anesthetics, cancer treatment and bioengineering of tissues. The present work concludes with perspectives for future administration of pharmaceuticals based on hyaluronic acid.

A gellan gum derivative as in-situ gelling cationic polymer for nasal drug delivery.

The aim of the present study was the development of a novel gellan gum derivative exhibiting mucoadhesive properties for nasal application. Accomplishing this, amino groups have been introduced to the polymeric backbone. The resulting synthesis products were characterized in terms of the amount of attached amino groups, regarding hydration, zeta potential and gel characteristics. Mucoadhesiveness was assessed studying rheological synergism, by rotating cylinder and regarding tensile studies. Next to erythrocyte-/cytotoxicity evaluation, the impact on ciliary beat frequency of nasal epithelial cells was investigated. Results revealed coupling rates up to 1259.50 ± 75.98 µmol/g polymer as well as accelerated hydration of the derivatives. Comparing aminated with unmodified gellan, enhanced mucoadhesion was verified by a 32-fold increase in viscosity of polymer/mucus mixtures and by a 14-fold extended mucosal adhesion time. Tensile studies demonstrated a 9-fold higher total work of adhesion and a 3.75-fold elevated maximum detachment force. Cellular membrane was not seriously impaired. CBF studies proved a reversible inhibition due to the application of the novel derivative. According to the outlined findings, aminated gellan gum can be considered as a promising excipient for nasal dosage forms improving drug bioavailability by superior adhesive features.

Zeta potential changing nanoemulsions: Impact of PEG-corona on phosphate cleavage.

The aim of this study was to evaluate the impact of a PEG-corona on oily droplets of a nanoemulsion on phosphate cleavage on their surface. A PEG-free nanoemulsion composed of 60% oleic acid, 30% Capmul MCM EP and 10% Span 85 being additionally stabilized by 1% cetyltrimethylammonium bromide (CTAB) and 3% phosphatidic acid (PA) was evaluated regarding phosphate release, zeta potential change and mucus permeation properties. In order to evaluate the impact of PEG-corona on phosphate release 10%, 20% and 30% of polyethoxylated-35 castor oil were incorporated in the nanoemulsion. The developed PEG-free nanoemulsion exhibited the droplet size of 123 nm with PDI of 0.24, whereas the droplet size of the nanoemulsions containing PEG ranged from 166 nm to 128 nm with PDI about 0.26. In case of the PEG-free formulation enzymatically induced phosphate cleavage was 3-fold and 7-fold higher than that from formulations containing 20% and 30% PEG-surfactant, respectively. Accordingly, the zeta potential shift of PEG-free formulation reached ~Δ 40 mV within 4 h, whereas zeta potential of PEG-containing formulations did not show any significant changes remaining constant at ~-30 mV. In contrast, PEG-containing formulations exhibited a 3.3-fold to 4-fold higher mucus permeation than the PEG-free formulation. According to the results, a PEG-corona has a great impact on phosphate cleavage and zeta potential change, which has to be taken into consideration for the development of highly efficient zeta potential changing nanocarriers, as zeta potential constitutes one of the crucial parameter regarding the permeation properties through physiological barriers.

Diaminated Starch: A Competitor of Chitosan with Highly Mucoadhesive Properties due to Increased Local Cationic Charge Density.

The purpose of this study was to synthesize diaminated starch as a novel mucoadhesive polymer. Starch was tosylated and then reacted with ethylenediamine. The degree of amination was determined by 2,4,6-trinitrobenzene sulfonic acid assay. Properties of diaminated starch including solubility, cytotoxicity, swelling behavior, and mucoadhesion were compared to chitosan. Diaminated starch displayed 2083 ± 121.6 µmol of diamine substructures/g of polymer. At pH 6, diaminated starch exhibited a ζ potential of 6 mV, whereas it was close to zero in the case of unmodified starch. In addition, diaminated starch displayed water solubility over the entire pH range and minor cytotoxicity. The novel polymer showed pronounced swelling behavior in water increasing its initial weight 18- and 6-fold at pH 5 and 6, respectively. Moreover, diaminated starch exhibited 92-fold higher-mucoadhesivity properties than those of chitosan. According to these results, diaminated starch might be a promising novel excipient for the design of mucoadhesive formulations.

Intestinal enzyme delivery: Chitosan/tripolyphosphate nanoparticles providing a targeted release behind the mucus gel barrier.

AIM: The aim of this study was to evaluate the potential of chitosan/tripolyphosphate (TPP) nanoparticles to provide a targeted release of ß-galactosidase behind the intestinal mucus gel barrier. METHODS: Nanoparticles were prepared by ionic gelation of chitosan and TPP in the presence of ß-galactosidase. Particles were characterized regarding size, polydispersity index and drug load. Target mediated hydrolysis of the TPP cross-linker followed by particle degradation and release of ß-galactosidase was investigated during incubation with isolated as well as cell and tissue associated intestinal alkaline phosphatase (IAP). Phosphate content in the media was quantified via malachite assay, whereas particle disintegration was monitored in parallel by measuring the decrease in particle size as well as in optical density at 600 nm. The released amount of ß-galactosidase was either determined utilizing bicinchoninic acid (BCA) protein detection or via an enzymatic activity assay with 2-nitrophenyl ß-D-galactopyranoside (ONPG) as substrate. Protection towards tryptic degradation was verified by ONPG assay. RESULTS: The size of nanoparticles was 573 ±â€¯34 nm and a payload of 376 ±â€¯18 µg ß-galactosidase per mg particles was achieved. Degradation studies with isolated IAP revealed a maximum phosphate cleavage of 118 ±â€¯1 µg/mg particles, a size decrease up to 38 ±â€¯7 % and a release of 58 ±â€¯0.5 % ß-galactosidase. Release of 94 ±â€¯6 % of the incorporated initial amount of ß-galactosidase was proven after 3 h incubation on porcine mucosa. Furthermore a protection against tryptic degradation was attained resulting in a 3-fold higher residual enzymatic activity of encapsulated ß-galactosidase compared to a control of free enzyme. CONCLUSION: Chitosan/TPP nanoparticles seem to be qualified as a suitable carrier for a targeted delivery of active ingredients to mucosal tissues expressing alkaline phosphatase.

Cationic starch derivatives as mucoadhesive and soluble excipients in drug delivery.

The aim of this study was to develop a novel mucoadhesive cationic polymer by introducing primary amino groups to the polymeric backbone of starch. This newly synthesized polymer should exhibit superior properties over chitosan regarding solubility, mucoadhesiveness and cytotoxicity. Increasing amounts of sodium periodate were used to cleave and oxidize vicinal diols under aldehyde formation obtaining three different degrees of modification. In a subsequent step, primary amines were introduced via reductive amination with ammonia. Degree of amination was examined with TNBS-assay and zeta potential measurements. Mucoadhesiveness was investigated by rotating cylinder, tensile studies and rheological measurements. Primary amino groups were successfully attached to the polymer, proven by zeta potential measurements and UV-spectroscopy. Depending on the amount of periodate used in the reaction, coupling rates of up to 514 µmol/g polymer were achieved. All synthesized derivatives showed 100% solubility in a pH range of 1-9. Aminated starch with the highest coupling rate of 514 µmol/g showed a 9.5-fold prolonged retention time on intestinal mucosa and a 2.7-fold higher total work of adhesion on the mucosal tissue compared to chitosan. Furthermore, cytotoxic examinations of all tested polymers showed only a low impact on cell viability after 24 h, whereby starch derivatives possessed even less cell toxic effects than chitosan. Summarizing these results, cationic starch derivatives seem to be promising excipients for mucosal drug delivery with superior properties compared to chitosan, the most examined cationic polymer.

Self-emulsifying drug delivery systems: In vivo evaluation of their potential for oral vaccination.

Oral Immunization remains a challenge as antigens are rapidly metabolized in the gastrointestinal tract. In numerous previous studies, Self-emulsifying drug delivery systems (SEDDS) have demonstrated to be a promising tool for oral delivery of biologics. In this study, the potential of SEDDS as vehicle for oral vaccination has been evaluated. At this purpose, the model antigen Bovine serum albumin (BSA) has been incorporated in SEDDS after ion pairing. Squalane and monophosphoryl lipid A (MPLA) were chosen as adjuvants and dissolved in SEDDS containing BSA (SEDDS-BSA-squalane and SEDDS-BSA-MPLA). Formulations were administered orally to BALB/c mice. As control unformulated BSA was administrated orally (BSA-oral) and subcutaneously (BSA-sc). Systemic (anti BSA IgG titre) and mucosal (anti BSA IgA titre) immugenicity of BSA loaded in SEDDS and of unformulated BSA administered orally and subcutaneously was assessed and compared with each other. SEDDS-BSA-squalane and SEDDS-BSA-MPLA induced both higher anti BSA-IgG titre and anti BSA-IgA titre than orally administered unformulated BSA. BSA-sc induced the highest systemic immune response, however, the highest mucosal immune response was achieved via oral administration of SEDDS-BSA-squalane and SEDDS-BSA-MPLA. In general, SEDDS-BSA-MPLA showed the most promising systemic and mucosal immune response. According to these results, SEDDS seems to be a promising carrier for oral delivery of vaccines. STATEMENT OF SIGNIFICANCE: Oral vaccination is still a great challenge, as orally administered antigens are easily degraded in the gastrointestinal (GI) tract by peptidases and proteases. During the last years, self-emulsifying drug delivery systems (SEDDS) consisting of a mixture of oils and surfactants have been developed for the oral administration of hydrophilic macromolecular drugs. In this study, Bovine serum albumin (BSA) was chosen as model antigen and incorporated into self-emulsifying drug delivery systems (SEDDS) after hydrophobic ion pairing. Lipid A from Salmonella Minnesota R595 (MPLA) and squalane were chosen as adjuvants. SEDDS-BSA-MPLA and SEDDS-BSA-squalane were administered orally to mice. SEDDS-BSA-MPLA induced the strongest systemic (anti BSA-IgG titre) and mucosal (anti BSA-IgA titre) immune response. Based on these results, SEDDS are a promising alternative carrier for oral vaccine delivery.

In Vitro-in Vivo Correlation of Mucoadhesion Studies on Buccal Mucosa.

BACKGROUND: For the development of novel buccoadhesive formulations, their physicochemical properties, strength of the interfacial joint, and residence time on the buccal mucosa are considered as a measure for their in vivo mucoadhesive properties. Focusing on these parameters, the predictive power of established in vitro systems was assessed for mucoadhesive properties in humans using discs as the model solid dosage form. METHODS: Compressed into discs, hydroxyethyl cellulose, carboxymethyl cellulose, carbopol, polycarbophil, alginate, and xanthan gum were used as model polymers. Mucosal residence time, maximum detachment force (MDF), and total work of adhesion (TWA) were determined ex vivo on the porcine buccal mucosa and in vivo on healthy volunteers. The impact of detachment velocity, humidification, and experimental set-up employed for tensile studies was examined and correlated to in vivo studies. RESULTS: Ex vivo results for mucosal residence time showed a very high correlation ( r = 0.997) with data obtained in vivo. For tensile studies, a set-up optimized for moistening the interface, speed, and alignment of the tensile force provided ex vivo results with very high correlation to in vivo experiments with r = 0.983 obtained for MDF and r = 0.973 for TWA, respectively. CONCLUSIONS: Experimental set-ups for the determination of mucosal residence time and tensile studies could be identified as valid methods for the development of intraoral solid dosage forms.

Thiolated polymers: Bioinspired polymers utilizing one of the most important bridging structures in nature.

Thiolated polymers designated "thiomers" are obtained by covalent attachment of thiol functionalities on the polymeric backbone of polymers. In 1998 these polymers were first described as mucoadhesive and in situ gelling compounds forming disulfide bonds with cysteine-rich substructures of mucus glycoproteins and crosslinking through inter- and intrachain disulfide bond formation. In the following, it was shown that thiomers are able to form disulfides with keratins and membrane-associated proteins exhibiting also cysteine-rich substructures. Furthermore, permeation enhancing, enzyme inhibiting and efflux pump inhibiting properties were demonstrated. Because of these capabilities thiomers are promising tools for drug delivery guaranteeing a strongly prolonged residence time as well as sustained release on mucosal membranes. Apart from that, thiomers are used as drugs per se. In particular, for treatment of dry eye syndrome various thiolated polymers are in development and a first product has already reached the market. Within this review an overview about the thiomer-technology and its potential for different applications is provided discussing especially the outcome of studies in non-rodent animal models and that of numerous clinical trials. Moreover, an overview on product developments is given.

Covalently binding mucoadhesive polymers: N-hydroxysuccinimide grafted polyacrylates.

AIM: The aim of the study was to establish a novel type of covalently mucus-binding polymers by targeting selectively amino groups within mucus glycoproteins. METHODS: N-Hydroxysuccinimide (NHS) was attached to carboxylic groups of polyacrylic acid (PAA). The reaction was mediated by the coupling reagent N,N'-dicyclohexylcarbodiimide (DCC) achieving polymeric NHS esters being able to form amide bonds with free amino groups. The chemical structure of the obtained conjugates was characterized via FTIR- and UV spectroscopy. Reactivity towards mucosal amino groups was evaluated UV spectrometrically upon addition of L-glycine. Furthermore, tensile force evaluations on intestinal mucosa as well as rheological experiments with mucus were performed in order to prove mucoadhesive potential. RESULTS: Depending on the amount of NHS added to the synthesis, coupling rates of 876 to 1820 µmol NHS per gram polymer were obtained. Kinetic studies of amide bond formation showed a substrate dependent reaction velocity. Rheological synergism of PAA-NHS was proven by a 7.9-fold increased mucus viscosity compared to the control polymer. In further mucoadhesion studies PAA-NHS showed a 5.5-fold improved adhesion time compared to unmodified PAA. Tensile force evaluation confirmed these results with a 1.7-fold higher maximum detachment force (MDF) and 2.7-fold increased total work adhesion (TWA) for PAA-NHS compared to the unmodified control polymer. CONCLUSION: The results of the present study provide strong evidence that coupling NHS to polymers could be a promising tool for the development of novel mucoadhesive excipients.

In vitro evaluation of a self-emulsifying drug delivery system (SEDDS) for nasal administration of dimenhydrinate.

The objective of the study was the development and in vitro characterization of a self-emulsifying drug delivery system (SEDDS) for the nasal application of dimenhydrinate. Final composition of SEDDS was established based on drug solubility and stability studies. Dimenhydrinate was loaded into the SEDDS pre-concentrates to 7.5% (m/v). The droplet size of the final SEDDS formulations was in a range between 60 and 220 nm. Permeability, as well as tissue toxicity, of the formulations was investigated using bovine nasal mucosa. Enhancement in permeation up to 2.8-fold compared to pure dimenhydrinate was confirmed. Furthermore, toxicity studies did not reveal any serious tissue damages related to the SEDDS. Additionally, irritation potential of SEDDS was evaluated in ciliary beat frequency measurements. Incorporation of dimenhydrinate into SEDDS might therefore be considered as a promising approach within the field of nasal delivery of antiemetics by utilizing permeation enhancement strategy.

Chitosan: The One and Only? Aminated Cellulose as an Innovative Option for Primary Amino Groups Containing Polymers.

The aim of this study was the synthesis and in vitro characterization of aminated cellulose as alternative excipient to chitosan. The aldehyde form of cellulose was generated via the oxidative cleavage of vicinal diols by the addition of increasing concentrations of sodium periodate. The insertion of primary amines was achieved by reductive amination with ammonia. The degree of substitution was calculated via primary amino group quantification using a 2,4,6-trinitrobenzenesulfonic acid assay. Mucoadhesiveness was examined by adopting the rotating-cylinder method and tensile studies using porcine intestinal mucosa. Hydration was evaluated at pH 2-11. The successful formation of aldehydes as well as a subsequent introduction of up to 311.61 micromoles per gram of primary amines were proven to correlate with the amount of added periodate. There was a 3- to 14-fold prolongation in the mucosal residence time of the new polymer in comparison to chitosan, as measured by the rotating-cylinder method. Although cationic cellulose did not reach the maximum detachment force of chitosan, the total work of adhesion of the newly synthesized cellulose derivate was higher than that of chitosan. The higher the degree of amination, the higher the degree of hydration in neutral and alkaline aqueous media was. Compared to chitosan, the novel cationic cellulose derivative displays improved mucoadhesive properties as well as sufficient hydration at physiological pH. Therefore, aminated cellulose is a promising alternative to the cationic polymers, such as chitosan, used thus far.

Non-ionic thiolated cyclodextrins - the next generation.

INTRODUCTION: The current study was aimed at developing a novel mucoadhesive thiolated cyclodextrin (CD) without ionizable groups and an intact ring backbone for drug delivery. MATERIALS AND METHODS: Thiolated beta CD (ß-CD) was prepared through bromine substitution of its hydroxyl groups followed by replacement to thiol groups using thiourea. The thiolated ß-CD was characterized in vitro via dissolution studies, cytotoxicity studies, mucoadhesion studies on freshly excised porcine intestinal mucosa, and inclusion complex formation with miconazole nitrate. RESULTS: Thiolated ß-CDs namely ß-CD-SH600 and ß-CD-SH1200 displayed 558.66 ± 78 and 1,163.45 ± 96 µmol thiol groups per gram of polymer, respectively. Stability constant (Kc) of 190 M-1 confirmed the inclusion complex formation of miconazole nitrate with ß-CD-SH. Inclusion complexes of ß-CD-SH600 and ß-CD-SH1200 resulted in 157- and 257-fold increased solubility of miconazole nitrate, respectively. In addition, more than 80% of thiol groups were stable even after 6 hours at pH 5. Both ß-CD-SH compounds showed at least 1.3-fold improved solubility in water. In contrast to cationic thiolated CDs of the first generation, both thiomers showed no significant cytotoxicity. The mucoadhesive properties of the new thiolated CDs were 39.73- and 46.37-fold improved, respectively. CONCLUSION: These results indicate that ß-CD-SH might provide a new favorable tool for delivery of poorly soluble drugs providing a prolonged residence time on mucosal surfaces.

New perspectives of starch: Synthesis and in vitro assessment of novel thiolated mucoadhesive derivatives.

AIM: The purpose of this study was to develop a novel thiolated starch polymer with improved mucoadhesive properties by conjugation of cysteamine to starch as a natural polymer of restricted mucoadhesive properties. METHODS: Aldehyde substructures were integrated into starch via oxidative cleavage of vicinal diols by increasing amounts of sodium periodate followed by covalent attachment of cysteamine to oxidized starch via reductive amination. Thiol groups were quantified via Ellman's reaction and their impact on mucoadhesion was analyzed by rheological investigations, the rotating cylinder method and tensile studies on porcine mucosa. RESULTS: The total amount of immobilized thiol groups revealed a correlation between degree of oxidation and thiolation. Modified starch demonstrated an up to 1.66-fold increase in water uptake in comparison to native starch. Modification of starch resulted in greatly improved cohesive properties and improvement in mucoadhesion. Rheological investigations revealed a 2- to 4-fold rise in viscosity of mucus. Tensile studies revealed a linear correlation between degree of oxidation/thiolation and enhancement of maximum detachment force and total work adhesion. CONCLUSION: In terms of these results, thiolated starch is a new, promising, polymer in the field of mucoadhesive drug delivery systems.

Impact of different hydrophobic ion pairs of octreotide on its oral bioavailability in pigs.

The objective of this study was to investigate the impact of different hydrophobic ion pairs (HIP) on the oral bioavailability of the model drug octreotide in pigs. Octreotide was ion paired with the anionic surfactants deoxycholate, decanoate and docusate differing in lipophilicity. These hydrophobic ion pairs were incorporated in self-emulsifying drug delivery systems (SEDDS) based on BrijO10, octyldodecanol, propylene glycol and ethanol in a concentration of 5mg/ml. SEDDS were characterized regarding size distribution, zeta potential, stability towards lipase, log DSEDDS/release medium and mucus diffusion behavior. The oral bioavailability of octreotide was evaluated in pigs via LC-MS/MS analyses. Most efficient ion pairing was achieved at a molar ratio of 1:3 (peptide: surfactant). SEDDS containing the octreotide-deoxycholate, -decanoate and -docusate ion pair exhibited a mean droplet size of 152nm, 112nm and 191nm and a zeta potential of -3.7, -4.6 and -5.7mV, respectively. They were completely stable towards degradation by lipase and showed a log DSEDDS/release medium of 1.7, 1.8 and 2.7, respectively. The diffusion coefficient of these SEDDS was in the range of 0.03, 0.11 and 0.17×10-9cm2/s, respectively. In vivo studies with these HIPs showed no improvement in the oral bioavailability in case of octreotide-decanoate. In contrast, octreotide-deoxycholate and octreotide-docusate SEDDS resulted in a 17.9-fold and 4.2-fold higher bioavailability vs. CONTROL: According to these results, hydrophobic ion pairing could be identified as a key parameter for SEDDS to achieve high oral bioavailability.

Nasal drug delivery: Design of a novel mucoadhesive and in situ gelling polymer.

The aim of the present study was to establish a novel polymeric excipient for liquid nasal dosage forms exhibiting viscosity increasing properties, improved mucoadhesion and stability towards oxidation in solution. In order to achieve this goal, 2-mercaptonicotinic acid was first coupled to l-cysteine by disulfide exchange reaction and after purification directly attached to the polymeric backbone of xanthan gum by carbodiimide mediated amide bond formation. The resulting conjugate was characterized with respect to the amount of coupled ligand, the in situ gelling behavior, mucoadhesive properties and stability towards oxidation. Furthermore, the influence of preactivated polymers on ciliary beat frequency (CBF) of porcine nasal epithelial cells was investigated. Results showed, that 252.52±20.54µmol of the ligand was attached per gram polymer. No free thiol groups could be detected on the polymeric backbone indicating entire preactivation. Rheological investigations of polymer mucus mixtures revealed a 1.7-fold and 2.5-fold enhanced mucoadhesion of entirely preactivated xanthan (Xan-Cys-MNA) compared to thiolated xanthan (Xan-Cys) and unmodified xanthan (Xan). Tensile force evaluation reported a 2.87 and 5.11-fold higher total work of adhesion (TWA) as well as a 1.63 and 2.41-fold higher maximum detachement force of Xan-Cys-MNA compared to Xan-Cys and Xan. In the presence of H2O2 as an oxidizing agent Xan-Cys-MNA showed unlike Xan-Cys no increase in viscosity, indicating high stability towards oxidation. Addition of CaCl2 to Xan-Cys-MNA solutions caused a decrease in viscosity at nevertheless higher total viscosity. Results from CBF studies proved nasal safety for the novel conjugate. According to these results, entirely preactivated thiolated xanthan gum seems to be a promising excipient for nasal dosage forms in order to improve drug bioavailability.

Charge changing phosphorylated polymers: Proof of in situ mucoadhesive properties.

The objective of this study was to design a novel polyethylene glycol (PEG) derivative exhibiting mucus permeating and mucoadhesive properties. Therefore, the enzymatically degradable phosphate ester, phosphotyrosine (Ptyr) was covalently attached to PEG-diamine. The synthesized PEG-Ptyr was studied in terms of enzymatic degradability on Caco 2 cells and by isolated intestinal alkaline phosphatase (IAP). Furthermore, the influence of enzymatic degradation on charge distribution of the polymer as well as on mucus diffusion and mucoadhesion was investigated. Within this study, the phosphate ester in PEG-Ptyr could be cleaved on the cell monolayer and by the isolated IAP, whereby the degradation rate was 10-fold higher utilizing the isolated enzyme. Implementation of negative charges on PEG due to modification with Ptyr led to an increased electrophoretic mobility, which was reduced after enzymatic degradation of the phosphate ester, most likely due to the alterations in charge distribution on the polymeric backbone. Interactions with mucus components were determined within mucus diffusion studies and rheological investigations. Herein, PEG-Ptyr showed a 3-fold lower mucus diffusion, after incubation with IAP. Within rheological investigations, dynamic viscosities increased by the factor of 3, after the phosphate ester in PEG-Ptyr was degraded by IAP. Results obtained within these experiments provided evidence for the in situ mucoadhesive properties of charge changing phosphorylated polymers. The combination of mucus permeating and mucoadhesive features of phosphorylated PEGs could be a highly interesting tool for future applications, such as for coating nanoparticles.

2,2'Dithiodinicotinyl ligands: Key to more reactive thiomers.

The aim of this study was to establish a novel type of preactivated thiomers exhibiting a comparatively higher reactivity with mucus and consequently improved mucoadhesive properties. In order to achieve this goal, the dimeric form of 2-mercaptonicotinic acid (MNA-MNA) was directly attached to the polymeric backbone of chitosan (CHI) via amide bond formation mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) used as a coupling reagent. The remaining free amino groups were in the following reacted with succinic anhydride (Succ) in order to obtain a uniformly anionically charged polymer (CHI-Succ-MNA-MNA). Within this study, different coupling rates of up to 170 µmol MNA-MNA per gram polymer were achieved. The attachment of the dimeric ligand resulted in a preactivated thiomer with a comparatively more reactive disulfide substructure due to the additional nitrogen atom in conjugation over the aromatic moieties. Furthermore, the obtained polymer is entirely preactivated and thus prevented against undesired oxidation reactions. Kinetic studies of disulfide exchange reactions showed a 3.8-fold higher reactivity of CHI-Succ-MNA-MNA in comparison to a state-of-the-art preactivated thiomer. Within rheological measurements, CHI-Succ-MNA-MNA with a coupling rate of 170 µmol (CHI-Succ-MNA-MNA 170) led to a 5.7-fold higher mucus viscosity than the non-thiolated control polymer (CHI-Succ) indicating a rheological synergism due to mucoadhesive properties. These results were confirmed by a second mucoadhesion study, which showed a significantly prolonged retention time of CHI-Succ-MNA-MNA on the small intestinal mucosa compared to CHI-Succ (P<0.02). Accordingly, the double preactivation seems to be a promising strategy in order to obtain entirely preactivated polymers with enhanced mucoadhesive properties.

Zeta potential changing phosphorylated nanocomplexes for pDNA delivery.

The objective of this study was to evaluate the suitability of a zeta potential changing system as gene delivery system. The phosphate ester bearing ligand 6-phosphogluconic acid (6-PGA) was attached to linear and branched polyethyleneimine (PEI) via a carbodiimide-mediated reaction whereby 287 µmol and 413 µmol 6-PGA could be coupled per gram polymer. Nanocomplexes of these modified polymers with pDNA showed a zeta potential of +12 mV for nanocomplexes with the linear PEI-6PGA and +16 mV in case of the branched derivative. By the addition of carboxymethylcellulose (CMC), zeta potentials of the complexes were reduced to +2.86 and +3.25, respectively. Phosphate release studies on Caco 2 cells and HEK-293 cells demonstrated the ability to cleave the phosphate ester. Compared to HEK-293 cells, enzymatic degradation of the phosphate ester in Caco 2 cells was 2.3-fold higher from nanocomplexes comprising linear PEI and 4.3-fold higher from those with branched PEI. Furthermore, incubation with alkaline phosphatase led to an increase in the zeta potential of nanocomplexes based on linear PEI-6PGA to +6.96mV and +8.26 mV in nanocomplexes comprising branched PEI-6PGA. Studying transfection efficiency in Caco 2 cells and HEK-293 cells, a higher expression of the green fluorescent protein (GFP) could be detected in HEK-293 cells. In presence of a phosphate inhibitor, transfection efficiencies were decreased in both cells lines, due to a lacking shift of the zeta potential of the tested pDNA complexes. According to these results, zeta potential changing systems seem to be a promising strategy for future gene delivery systems, as this concept allows the in situ generation of positive charges in close proximity to the cellular surface.