Engineering of inhalable nano-in-microparticles for co-delivery of small molecules and miRNAs.

In this study, novel Trojan particles were engineered for direct delivery of doxorubicin (DOX) and miR-34a as model drugs to the lungs to raise local drug concentration, decrease pulmonary clearance, increase lung drug deposition, reduce systemic side effects, and overcome multi-drug resistance. For this purpose, targeted polyelectrolyte nanoparticles (tPENs) developed with layer-by-layer polymers (i.e., chitosan, dextran sulfate, and mannose-g-polyethyleneimine) were spray dried into a multiple-excipient (i.e., chitosan, leucine, and mannitol). The resulting nanoparticles were first characterized in terms of size, morphology, in vitro DOX release, cellular internalization, and in vitro cytotoxicity. tPENs showed comparable cellular uptake levels to PENs in A549 cells and no significant cytotoxicity on their metabolic activity. Co-loaded DOX/miR-34a showed a greater cytotoxicity effect than DOX-loaded tPENs and free drugs, which was confirmed by Actin staining. Thereafter, nano-in-microparticles were studied through size, morphology, aerosolization efficiency, residual moisture content, and in vitro DOX release. It was demonstrated that tPENs were successfully incorporated into microspheres with adequate emitted dose and fine particle fraction but low mass median aerodynamic diameter for deposition into the deep lung. The dry powder formulations also demonstrated a sustained DOX release at both pH values of 6.8 and 7.4.

Whey Protein Isolate-Chitosan PolyElectrolyte Nanoparticles as a Drug Delivery System.

Whey protein isolate (WPI), employed as a carrier for a wide range of bioactive substances, suffers from a lack of colloidal stability in physiological conditions. Herein, we developed innovative stabilized PolyElectrolyte Nanoparticles (PENs) obtained by two techniques: polyelectrolyte complexation of negatively charged WPI and positively charged chitosan (CS), and ionic gelation in the presence of polyanion tripolyphosphate (TPP). Therefore, the WPI-based core was coated with a CS-based shell and then stabilized by TPP at pH 8. The nanostructures were characterized by physiochemical methods, and their encapsulation efficiency and in vitro release were evaluated. The spherical NPs with an average size of 248.57 ± 5.00 nm and surface charge of +10.80 ± 0.43 mV demonstrated high encapsulation efficiency (92.79 ± 0.69) and sustained release of a positively charged chemotherapeutic agent such as doxorubicin (DOX). Z-average size and size distribution also presented negligible increases in size and aggregates during the three weeks. The results obtained confirm the effectiveness of the simultaneous application of these methods to improve the colloidal stability of PEN.

Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction.

A novel polyelectrolyte nanocarrier was synthesized via layer-by-layer self-assembly of polycationic and polyanionic chains. The nanocarrier is composed of polyglutamate grafted chitosan core, dextran sulfate as a complexing agent, and polyethyleneimine shell decorated with folic acid. This polyelectrolyte complex has unique physicochemical properties so that the core is considered as an efficient carrier for LTX-315 and melittin peptides, and the shell is suitable for delivery of miR-34a. The spherical nanocarriers with an average size of 123 ± 5 nm and a zeta potential of -36 ± 1 mV demonstrated controlled-release of gene and peptides ensured a synergistic effect in establishing multiple cell death pathways on chemoresistance human breast adenocarcinoma cell line, MDA-MB-231. In vitro cell viability assays also revealed no cytotoxicity for the nanocarriers, and an IC50 of 15 µg/mL and 150 µg/mL for melittin and LTX-315, respectively, after 48 h, whereas co-delivery of melittin with miR-34a increased smart death induction by 54%.

Development of dual crosslinked mumio-based hydrogel dressing for wound healing application: Physico-chemistry and antimicrobial activity.

In this study, an antimicrobial mumio-based hydrogel dressing was developed for wound healing application. The mechanism of gel formation was achieved via a double crosslink network formation between gelatin (GT) and polyvinyl alcohol (PVA) using polyethylene glycol diglycidyl ether (PEGDE) and borax as crosslinking agents. To enhance the mechanical integrity of the hydrogel matrix, bacterial cellulose (BC) was integrated into the GT-PVA hydrogel to produce a composite gel dressing. The obtained hydrogel was characterized by FTIR, SEM, TGA, and XRD. Gel fraction, in vitro swelling and degradation as well as compressive modulus properties of the gel dressing were investigated as a function of change in PVA and BC ratios. By increasing the ratios of PVA and BC, the composite dressing showed lower swelling but higher mechanical strength. Comparing to other formulations, the gel with 4 %w/v PVA and 1 %w/v BC demonstrated to be most suitable in terms of stability and mechanical properties. In vitro cell cytotoxicity by MTT assay on human alveolar basal epithelial (A549) cell lines validated the gels as non-toxic. In addition, the mumio-based gel was compared to other formulations containing different bioactive agents of beeswax and cinnamon oil, which were tested for microbial growth inhibition effects against different bacteria (S. aureus and K. pneumoniae) and fungi (C. albicans and A. niger) strains. Results suggested that the gel dressing containing combinations of mumio, beeswax and cinnamon oil possess promising future in the inhibition of microbial infection supporting its application as a suitable dressing for wound healing.

Chemical stabilization of γ-polyglutamate by chitosan and the effect of co-solvents on the stability.

In protein-based formulations, conformational distortions and attractive interactions may cause insoluble and undesired aggregates. In the case of ionic peptides, including cationic or anionic, commonly electrostatic interactions are the main factors that control structure assembling. In this study, it was proposed that grafting of chitosan (CS) to γ-polyglutamic acid (γ-PGA) might exhibit much strong inhibiting effect on the formation of protein aggregates due to multiple amino groups and hydrophilic properties. To guarantee stable and safe biopharmaceutical formulation, the potency of a variety of stabilizers including sugars (glucose, sucrose), polyols (sorbitol, glycerol), surfactant (Tween 20), salting-out salt (PBS), and also different pH values have been evaluated on stabilizing or destabilizing the native state of CS-g-PGA copolymer using FTIR, CD, DLS, and SDS-PAGE. The comparable analysis revealed that the stability of CS-g-PGA was strongly dependent on pH owing to the polyelectrolyte characteristics of the polymers. Altogether these results implied that CS at optimized conditions might be an important precursor for the pharmaceutical industry and function as a new polymer for aggregation suppression and protein stabilization.

Nanoparticle-Based Rifampicin Delivery System Development.

The alkaline milieu of chronic wounds severely impairs the therapeutic effect of antibiotics, such as rifampicin; as such, the development of new drugs, or the smart delivery of existing drugs, is required. Herein, two innovative polyelectrolyte nanoparticles (PENs), composed of an amphiphilic chitosan core and a polycationic shell, were synthesized at alkaline pH, and in vitro performances were assessed by 1H NMR, elemental analysis, FT-IR, XRD, DSC, DLS, SEM, TEM, UV/Vis spectrophotometry, and HPLC. According to the results, the nanostructures exhibited different morphologies but similar physicochemical properties and release profiles. It was also hypothesized that the simultaneous use of the nanosystem and an antioxidant could be therapeutically beneficial. Therefore, the simultaneous effects of ascorbic acid and PENs were evaluated on the release profile and degradation of rifampicin, in which the results confirmed their synergistic protective effect at pH 8.5, as opposed to pH 7.4. Overall, this study highlighted the benefits of nanoparticulate development in the presence of antioxidants, at alkaline pH, as an efficient approach for decreasing rifampicin degradation.

Non-Invasive Human Embryo Metabolic Assessment as a Developmental Criterion.

The selection of a highly-viable single embryo in assisted reproductive technology requires an acceptable predictive method in order to reduce the multiple pregnancy rate and increase the success rate. In this study, the metabolomic profiling of growing and impaired embryos was assessed on the fifth day of fertilization using capillary electrophoresis in order to find a relationship between the profiling and embryo development, and then to provide a mechanistic insight into the appearance/depletion of the metabolites. This unique qualitative technique exhibited the appearance of most non-essential amino acids and lactate, and depleting the serine, alanyl-glutamine and pyruvate in such a manner that the embryos impaired in their development secreted a considerably higher level of lactate and consumed a significantly higher amount of alanyl-glutamine. The different significant ratios of metabolomic depletion/appearance between the embryos confirm their potential for the improvement of the prospective selection of the developed single embryos, and also suggest the fact that pyruvate and alanyl-glutamine are the most critical ATP suppliers on the fifth day of blastocyst development.

Redox-Sensitive and Hyaluronic Acid-Functionalized Nanoparticles for Improving Breast Cancer Treatment by Cytoplasmic 17α-Methyltestosterone Delivery.

Novel reduction-responsive hyaluronic acid-chitosan-lipoic acid nanoparticles (HACSLA-NPs) were designed and synthesized for effective treatment of breast cancer by targeting Cluster of Differentiation 44 (CD44)-overexpressing cells and reduction-triggered 17α-Methyltestosterone (MT) release for systemic delivery. The effectiveness of these nanoparticles was investigated by different assays, including release rate, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT), lactate dehydrogenase (LDH), caspase-3 activity, Rhodamine 123 (RH-123), and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In vitro experiments revealed that Methyltestosterone/Hyaluronic acid-chitosan-lipoic acid nanoparticles (MT/HACSLA-NPs) illustrated a sustained drug release in the absence of glutathione (GSH), while the presence of GSH led to fast MT release. HACSLA-NPs also showed high cellular internalization via CD44 receptors, quick drug release inside the cells, and amended cytotoxicity against positive CD44 BT-20 breast cancer cell line as opposed to negative CD44, Michigan Cancer Foundation-7 (MCF-7) cell line. These findings supported that these novel reduction-responsive NPs can be promising candidates for efficient targeted delivery of therapeutics in cancer therapy.

Stabilization of chitosan-based polyelectrolyte nanoparticle cargo delivery biomaterials by a multiple ionic cross-linking strategy.

PolyElectrolyte Nanoparticles (PENs) obtained by layer-by-layer self-assembly of polycations/polyanions suffer from a lack of colloidal stability in physiological conditions. We report a simple innovative approach for increasing their stability by multiple ionic cross-linkers. Herein, a chitosan-based core was stabilized by polyanions such as tripolyphosphate and dextran sulfate at pHs of 3 (aPENs) and 8 (bPENs) to improve the quality of electrostatic interactions in the core and manage self-assembly of polyethyleneimine shell onto the core. The physicochemical properties of the particles were characterized by DLS, SEM, TEM, FT-IR, and TGA. TEM micrographs showed visible core/shell structures of bPENs. From particle size and polydispersity indices, the bPENs stability was salt concentration-dependent. The release profiles of PENs using nicotinic acid demonstrated sustained release in a pH-independent manner with a good fit of Korsmeyer-Peppas model. These results suggest that multiple ionic cross-linkers can be an efficient approach to increase the colloidal stability of PENs.

A Comprehensive Physicochemical, In Vitro and Molecular Characterization of Letrozole Incorporated Chitosan-Lipid Nanocomplex.

PURPOSE: The aim of this study is to show a new mesomicroscopic insight into Letrozole (LTZ) loaded nanocomplexes and their ex vivo characteristics as a drug delivery system. METHODS: The LTZ loaded hybrid chitosan-based carrier was fabricated using a modified ionic crosslinking technique and characterized in more detail. To understand the mechanism of LTZ action encapsulated in the hybrid polymer-lipid carrier, all-atom molecular dynamics simulations were also used. RESULTS: The physicochemical properties of the carrier demonstrated the uniform morphology, but different drug loading ratios. In vitro cytotoxic activity of the optimized carrier demonstrated IC50 of 67.85 ± 0.55 nM against breast cancer cell line. The ex vivo study showed the positive effect of nanocomplex on LTZ permeability 7-10 fold greater than the free drug. The molecular dynamic study also confirmed the prsence of hydrophobic peak of lipids at a distance of 5 Å from the center of mass of LTZ which proved drug entrapment in the core of nanocomplex. CONCLUSIONS: The hybrid nanoparticle increased the cytotoxicity and tissue permeability of LTZ for oral delivery. This study also confirmed the atomic mesostructures and interaction of LTZ in the core of hybrid polymer-lipid nanoparticles.

Intrinsic parameters for the synthesis and tuned properties of amphiphilic chitosan drug delivery nanocarriers.

Chitosan (CS) is a material derived from chitin, the most abundant biopolymer on the planet. It has shown potential among a wide variety of biomedical applications especially within the context of self-assembling nanocarriers usable in biomedical applications such as drug delivery, macroscopic injectables, tissue-engineering scaffolds, and nano-imaging agents. To date, many reviews have been focused on the biomedical properties and applications of CS-based nanocarriers, but a review is lacking on the role and prospects of different factors such as formulation parameters and preparation conditions on the properties of amphiphilic chitosan nanocarriers (ACNs) that have shown critical value in advancing drug delivery.

Novel amphiphilic chitosan nanocarriers for sustained oral delivery of hydrophobic drugs.

Chitosan (CS) has been investigated for its ability to be modified hydrophobically and self-assembled into nanocarriers. They can encapsulate drugs and protect them in a promising approach. However, loss of carrier stability against biological environments induces low bioavailability of encapsulated drugs after oral administration. The objective of this work was to develop and characterize a novel self-assembled nanocarrier using cross-linker under acidic condition to enhance oral absorption of a hydrophobic model drug such as Letrozole (LTZ). Therefore, amphiphilic chitosan nanocarriers (ACNs) were prepared by oil-in-water emulsion/ionic gelation technique; self-assembled via electrostatic interactions between the negatively charged Palmitic acid (PL) and the positively charged CS and stabilized by cross linking with sodium tripolyphosphate solution (TPP) under ultrasonication. The results confirmed that the ACNs had sustained release at different pH values of the release medium depending on drug concentration. Increasing drug concentration led to prolonged release and increased pH values which were accompanied by higher release rate. Spherical nanocarriers with positive surface charge (+24mV) were formed with an average size of 186.6±32.95nm by Zetasizer and 84.7nm by scanning electron microscopy (SEM) at physiological pH. The structure of the carriers was determined by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) techniques. Eventually, the toxicity was measured by MTT assay and clearly proved that ACNs had no toxicity against the tumor MCF-7 and PC-12 cell lines.

Hydrophobic amino acids grafted onto chitosan: a novel amphiphilic chitosan nanocarrier for hydrophobic drugs.

OBJECTIVE: The objective of this study is to develop a novel biocompatible amphiphilic drug delivery for hydrophobic drugs, chitosan (CS) was grafted to a series of hydrophobic amino acids including l-alanine (A), l-proline (P), and l-tryptophan (W) by carbodiimide mediated coupling reaction. MATERIALS AND METHODS: Chemical characteristics of the modified polymers were determined and confirmed by FT-IR, 1H NMR, and UV-vis spectroscopy and the degree of substitution was quantified by elemental analysis. The modified polymers were used to form amphiphilic chitosan nanocarriers (ACNs) by the conventional self-assembly method using ultrasound technique. The morphology and the size of ACNs were analyzed by scanning electron microscope (SEM) and Dynamic light scattering (DLS). RESULTS AND DISCUSSION: The sizes of spherical ACNs analyzed by SEM were obviously smaller than those of determined by DLS. The ACNs effectively surrounded the hydrophobic model drug, letrozole (LTZ), and demonstrated different encapsulation efficiencies (EE), loading capacities (LC), and controlled drug release profiles. The characteristics of ACNs and the mechanism of drug encapsulation were confirmed by molecular modeling method. The modeling of the structures of LTZ, profiles of A, P, and W grafted onto CS and the wrapping process around LTZ was performed by quantum mechanics (QM) methods. There was a good agreement between the experimental and theoretical results. The cell viability was also evaluated in two cell lines compared with free drug by MTT assay. CONCLUSION: The hydrophobic portion effects on ACNs' characteristics and the proper selection of amino acid demonstrate a promising potential for drug delivery vector.