Cationic Polyelectrolyte Nanocapsules of Moxifloxacin for Microbial Keratitis Therapy: Development, Characterization, and Pharmacodynamic Study.

Microbial keratitis (MK) is a vision-threatening disease and the fourth leading cause of blindness worldwide. In this work, we aim to develop moxifloxacin (MXN)-loaded chitosan-based cationic mucoadhesive polyelectrolyte nanocapsules (PENs) for the effective treatment of MK. PENs were formulated by polyelectrolyte complex coacervation method and characterized for their particle size, surface charge, morphology, mucoadhesive property, in-vitro and ex-vivo release, ocular tolerance, and antimicrobial efficacy studies. The pharmacodynamic study was conducted on rabbit eye model of induced keratitis and it is compared with marketed formulation (MF). Developed PENs showed the size range from 230.7 ± 0.64 to 249.0 ± 0.49 nm and positive surface charge, spherical shape along with appropriate physico-chemical parameters. Both in-vitro and ex-vivo examination concludes that PENs having more efficiency in sustained release of MXN compared to MF. Ocular irritation studies demonstrated that no corneal damage or ocular irritation. The in-vivo study proved that the anti-bacterial efficacy of PENs was improved when compared with MF. These results suggested that PENs are a feasible choice for MK therapy because of their ability to enhance ocular retention of loaded MXN through interaction with the corneal surface of the mucous membrane.

Sodium alginate/poly(4-vinylpyridine) polyelectrolyte multilayer films: Preparation, characterization and ciprofloxacin HCl release.

Polyelectrolyte multilayer (PEC) films of sodium alginate (Na-Alg) and poly(4-vinylpyridine) (P4VP) were prepared and were loaded with an antibacterial agent, ciprofloxacin HCl (CIP.HCl) aiming to design new hydrophilic films with controlled physicochemical properties and drug release behaviour that may find application as components of transdermal drug delivery systems. The PEC films were characterized by SEM, XRD, TGA, AFM and FTIR spectroscopy. The hydrophilicity of the PEC films was examined by using contact angle measurement. The number of layers and the nature of the outer layer affect the physicochemical characteristics, CIP.HCl loading and release behaviour of the films. The three layer film PEC-3, which is composed of Na-Alg outer layer deposited on a P4VP/Na-Alg double layer, is characterized by the lowest roughness (Rq = 16.3 nm) and the most hydrophilic surface with a contact angle value of 38.1° among all other films. Its crystallinity index is 0.36, and starts to degrade at 195 °C. It exhibits 130-135% equilibrium swelling capacity in acid buffer and water respectively. PEC-3 is the film with the highest drug loading capacity and drug loading efficiency values of 3.51% and 87% respectively. A cumulative drug release of 65% is obtained from PEC-3 within 24 h in pH = 1.2 buffer solution.

Phosphonium Polyelectrolyte Complexes for the Encapsulation and Slow Release of Ionic Cargo.

Polyelectrolyte complexation, the combination of anionically and cationically charged polymers through ionic interactions, can be used to form hydrogel networks. These networks can be used to encapsulate and release cargo, but the release of cargo is typically rapid, occurring over a period of hours to a few days and they often exhibit weak, fluid-like mechanical properties. Here we report the preparation and study of polyelectrolyte complexes (PECs) from sodium hyaluronate (HA) and poly[tris(hydroxypropyl)(4-vinylbenzyl)phosphonium chloride], poly[triphenyl(4-vinylbenzyl)phosphonium chloride], poly[tri(n-butyl)(4-vinylbenzyl)phosphonium chloride], or poly[triethyl(4-vinylbenzyl)phosphonium chloride]. The networks were compacted by ultracentrifugation, then their composition, swelling, rheological, and self-healing properties were studied. Their properties depended on the structure of the phosphonium polymer and the salt concentration, but in general, they exhibited predominantly gel-like behavior with relaxation times greater than 40 s and self-healing over 2-18 h. Anionic molecules, including fluorescein, diclofenac, and adenosine-5'-triphosphate, were encapsulated into the PECs with high loading capacities of up to 16 wt %. Fluorescein and diclofenac were slowly released over 60 days, which was attributed to a combination of hydrophobic and ionic interactions with the dense PEC network. The cytotoxicities of the polymers and their corresponding networks with HA to C2C12 mouse myoblast cells was investigated and found to depend on the structure of the polymer and the properties of the network. Overall, this work demonstrates the utility of polyphosphonium-HA networks for the loading and slow release of ionic drugs and that their physical and biological properties can be readily tuned according to the structure of the phosphonium polymer.

In vivo Studies on Pharmacokinetics, Toxicity and Immunogenicity of Polyelectrolyte Nanocapsules Functionalized with Two Different Polymers: Poly-L-Glutamic Acid or PEG.

BACKGROUND: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different "stealth" polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG). METHODS: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay. RESULTS: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines. CONCLUSION: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system.

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Matrix metalloproteinase-cleavable nanocapsules for tumor-activated drug release.

In the war against cancer, nanotechnology-based drug delivery systems may play a significant role by enhancing the efficacy of conventional therapies. Here, we tried to address some major limitations plaguing anticancer drugs, namely, poor water solubility and off-target toxicity. The systems we propose are cross-linked polyelectrolyte nanocapsules based on an oil-core and a matrix metalloproteinase-2 (MMP-2)-cleavable shell. They can load hydrophobic drugs, prevent their systemic leakage, and release their payload upon an endogenous stimulus. Both the stability enhancement and the stimuli-responsive drug release mechanisms were achieved by cross-linking the nanocapsule shell with an MMP-2-sensitive peptide. On the basis of this strategy, the system maintained its stability in PBS up to one month. Further, when tested on 3D tumor and healthy spheroid models, the nanocapsules were able to disrupt their integrity preferentially in the tumor-like microenvironment. The high level of MMP-2 enzymes expressed by tumor spheroids, indeed, catalyzed the disassembly of the nanocapsules, which ultimately leads to drug release. Therefore, this device holds great potential as a smart system that allows for the safe transport of hydrophobic drugs and for a spatially controlled release upon an endogenous stimulus coming from the very nature of the tumor itself. STATEMENT OF SIGNIFICANCE: The performance of nanoparticle-based therapeutic approaches is often hindered by some intrinsic limitations typically including laborious drug loading methods, synthetic routes of preparation and stability issues. In this work, we implemented for the first time a smart drug delivery strategy into oil-core multilayer nanocapsules, which represent an ideal family of nanocarriers. To this aim, we developed a robust method enabling the use of soft matters like oil-core nanocapsules combined with a microenvironmentally triggered release mechanism. The efficacy of nanocapsules was tested on tumor and healthy spheroids. Results clearly demonstrate their selective drug release, triggered by a stimulus intrinsically present in tumor microenvironment. We believe this study is of particular interest for cancer research and paves the way for the use of these robust stimuli-responsive nanocapsules in vivo.

Chitosan based copolymer-drug conjugate and its protein targeted polyelectrolyte complex nanoparticles to enhance the efficiency and specificity of low potency anticancer agent.

The effective delivery of low potency anticancer drug is a major challenge. The present study introduces the novel chitosan-polylactic acid (CS-PLA)-drug conjugate and its transferrin receptor targeted polyelectrolyte complex nanoparticles (PEC Nps), encapsulating free drug to increase its potency and specificity. The model drug curcumin (CR) was used and incorporated in this system in both conjugated and encapsulated form. The synthesis of CS-PLA-CR copolymer was confirmed by 1H NMR, FTIR, UV-visible spectrum, DSC thermogram and zeta potential. Further, the nanoparticles engulfing free CR, with average 340 nm particle sizes, were prepared through simple ionic gelation technique utilizing positive charges on copolymer by polyanion sodium alginate (CS-PLA-CR/SA PEC Nps). The prepared Nps showed the high CR content of over 92% with extended period of CR release (60% and 85% at pH 7.4 and 5 respectively even after 8 days). The results were compared with the unmodified CS (without PLA) as a control to understand the effect of PLA side chain. Transferrin (Tf) conjugation on PEC Nps displayed superior cytotoxicity and cellular uptake compared to non-targeted Nps on MCF-7 cell line. Thus, CR loaded Tf-CS-PLA-CR/SA PEC Nps may provide an efficient and targeted delivery for cancer treatment.

Drug transport mechanisms and in vitro release kinetics of vancomycin encapsulated chitosan-alginate polyelectrolyte microparticles as a controlled drug delivery system.

In this study, chitosan-alginate polyelectrolyte microparticles containing the antibiotic, vancomycin chloride were prepared using the ionotropic gelation (coacervation) technique. In vitro release and drug transport mechanisms were studied concerning the chitosan only and alginate only microparticles as a control group. Further, the effect of porosity on the drug transport mechanism was also studied for chitosan-alginate mixed particles produced by lyophilizing in contrast to the air-dried non-porous particles. According to the in vitro release data, alginate only and chitosan only microparticles showed burst release and prolonged release respectively. Chitosan-alginate lyophilized microparticles showed the best-controlled release of vancomycin with the average release of 22µg per day for 14days. Also, when increasing alginate concentration there was no increase in the release rate of vancomycin. The release data of all the microparticles were treated with Ritger-Peppas, Higuchi, Peppas-Sahlin, zero-order, and first-order kinetic models. The best fit was observed with Peppas-Sahlin model, indicating the drug transport mechanism was controlled by both Fickian diffusion and case II relaxations. Also, Fickian diffusion dominates the drug transport mechanism of all air-dried samples during the study period. However, the Fickian contribution was gradually reducing with time. Porosity significantly effects the drug transport mechanism as case II relaxation dominates after day 10 of the lyophilized microparticles.

Urinary excretion of ciprofloxacin after administration of extended release tablets in healthy volunteers. Swellable drug-polyelectrolyte matrix versus bilayer tablets.

This paper builds on a previous paper in which new ciprofloxacin extended-release tablets were developed based on a ciprofloxacin-based swellable drug polyelectrolyte matrix (SDPM-CIP). The matrix contains a molecular dispersion of ciprofloxacin ionically bonded to the acidic groups of carbomer, forming the polyelectrolyte-drug complex CB-CIP. This formulation showed that the release profile of the ciprofloxacin bilayer tablets currently commercialised can be achieved with a simpler strategy. Thus, since ciprofloxacin urine concentrations are associated with the clinical cure of urinary tract infections, the goal of this work was to compare the urinary excretion of SDPM-CIP tablets with those of the CIPRO XR® bilayer tablets. A batch of SDPM-CIP tablets was manufactured by the wet granulation method and the CB-CIP ionic complex was obtained in situ. Fasted healthy volunteers received a single oral dose of 500 mg ciprofloxacin of either formulation in a randomised crossover study. Urinary concentrations were assessed by HPLC at intervals up to 36 h. Pharmacokinetic parameters (rate of urinary excretion, maximum urine excretion rate, tmax, area under the curve, amount and percentage of the ciprofloxacin dose excreted in urine) showed no statistical differences between both formulations at any of the time intervals of collection. The processing conditions to obtain SDPM-CIP tablets are easy to scale up since they involve technology currently employed in the pharmaceutical industry and the process is less challenging to implement. In addition, SDPM-CIP tablets met pharmacopoeial quality specifications.

Noninvasive measurement of transdermal drug delivery by impedance spectroscopy.

The effectiveness in transdermal delivery of skin permeation strategies (e.g., chemical enhancers, vesicular carrier systems, sonophoresis, iontophoresis, and electroporation) is poorly investigated outside of laboratory. In therapeutic application, the lack of recognized techniques for measuring the actually-released drug affects the scientific concept itself of dosage for topically- and transdermally-delivered drugs. Here we prove the suitability of impedance measurement for assessing the amount of drug penetrated into the skin after transdermal delivery. In particular, the measured amount of drug depends linearly on the impedance magnitude variation normalized to the pre-treated value. Three experimental campaigns, based on the electrical analysis of the biological tissue behavior due to the drug delivery, are reported: (i) laboratory emulation on eggplants, (ii) ex-vivo tests on pig ears, and finally (iii) in-vivo tests on human volunteers. Results point out that the amount of delivered drug can be assessed by reasonable metrological performance through a unique measurement of the impedance magnitude at one single frequency. In particular, in-vivo results point out sensitivity of 23 ml-1, repeatability of 0.3%, non-linearity of 3.3%, and accuracy of 5.7%. Finally, the measurement resolution of 0.20 ml is compatible with clinical administration standards.

Comprehensive and Systematic Analysis of the Immunocompatibility of Polyelectrolyte Capsules.

The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.

Biomimetic calcium phosphate/polyelectrolyte multilayer coatings for sequential delivery of multiple biological factors.

Combinations of growth factors synergistically enhance tissue regeneration, but typically require sequential, rather than co-delivery from biomaterials for maximum efficacy. Polyelectrolyte multilayer (PEM) coatings can deliver multiple factors without loss of activity; however, sequential delivery from PEM has been limited due to interlayer diffusion that results in co-delivery of the factors. This study shows that addition of a biomimetic calcium phosphate (bCaP) barrier layer to a PEM coating effectively prevents interlayer diffusion and enables sequential delivery of two different biomolecules via direct cell access. A simulated body fluid method was used to deposit a layer of bCaP followed by 30 bilayers of PEM made with poly-l-Lysine (+) and poly l-Glutamic acid (-) (bCaP-PEM). Measurements of MC3T3-E1 proliferation and viability over time on bCaP-PEM were used to demonstrate the sequential delivery kinetics of a proliferative factor [fibroblast growth factor-2 (FGF-2)] followed by a cytotoxic factor (antimycin A, AntiA). FGF-2 and AntiA both retained their bioactivity within bCaP-PEM, yet no release of FGF-2 or AntiA from bCaP-PEM was observed when cells were absent indicating a cell-mediated, local delivery process. This coating technique is useful for a variety of applications that would benefit from highly localized, sequential delivery of multiple biomolecules governed by cell initiated degradation that avoids off-target effects associated with diffusion-based release. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1500-1509, 2017.

Nasal inserts containing ondansetron hydrochloride based on Chitosan-gellan gum polyelectrolyte complex: In vitro-in vivo studies.

The aim of this study was the production of ondansetron hydrochloride loaded lyophilized insert for nasal delivery. The nasal insert was prepared by the lyophilisation technique using Chitosan-gellan gum polyelectrolyte complex as the polymer matrix. The ondansetron loaded inserts were evaluated with respect to water uptake, bioadhesion, drug release kinetic study, ex vivo permeation study, and in vivo study. Lyophilised nasal inserts were characterized by differential scanning calorimetry, scanning electron microscopy and X-ray diffraction study. Scanning electron microscopy confirmed the porous sponge like structure of inserts whereas release kinetic model revealed that drug release followed non-fickian case II diffusion. The nasal delivery showed improved bioavailability as compared to oral delivery. In conclusion, the ondansetron containing nasal inserts based on Chitosan-gellan gum complex with potential muco-adhesive potential is suitable for nasal delivery.