Microfluidic preparation and optimization of (Kollicoat ® IR-b-PCL) polymersome for co-delivery of Nisin-Curcumin in breast cancer application.

This work aimed to develop amphiphilic nanocarriers such as polymersome based diblock copolymer of Kollicoat ® IR -block-poly(ε-caprolactone) (Kollicoat ® IR-b-PCL) for potential co-delivery of Nisin (Ni) and Curcumin (CUR) for treatment of breast cancer. To generate multi-layered nanocarriers of uniform size and morphology, microfluidics was used as a new technology. In order to characterise and optimize polymersome, design of experiments (Design-Expert) software with three levels full factorial design (3-FFD) method was used. Finally, the optimized polymersome was produced with a spherical morphology, small particle size (dH < 200 nm), uniform size distribution (PDI < 0.2), and high drug loading efficiency (Ni 78 % and CUR 93 %). Furthermore, the maximum release of Ni and CUR was found to be roughly 60 % and 80 % in PBS, respectively. Cytotoxicity assays showed a slight cytotoxicity of Ni and CUR -loaded polymersome (N- Ni /CUR) towards normal cells while demonstrating inhibitory activity against cancer cells compared to the free drugs. Also, the apoptosis assays and cellular uptake confirmed the obtained results from cytotoxic analysis. In general, this study demonstrated a microfluidic approach for preparation and optimization of polymersome for co-delivery of two drugs into cancer cells.

Dual-responsive nisin loaded chondroitin sulfate nanogel for treatment of bacterial infection in soft tissues.

Chondroitin sulfate-Nisin nanogels (CS-N NGs) were prepared by electrostatic interaction for nisin delivery as an antibacterial agent in the treatment of bacterial infections caused by some clinical strains of methicillin-resistant and methicillin-sensitive Staphylococcus aureus (S. aureus). The physical properties of CS-N NGs were evaluated using Fourier-transform infrared spectroscopy, dynamic light scattering, and field emission scanning electron microscopy. The average diameter of obtained CS-N NGs was about 65 nm and the stability of nanogels was assessed by zeta potential measurement. Enzyme and pH-responsibility of CS-N NGs due to the presence of susceptible bonds in chondroitin sulfate resulted in effective and controlled release of nisin in the simulated infectious medium. Also, the ability of prepared CS-N NGs for eradicating clinical methicillin resistance S. aureus strain was confirmed by Broth Microdilution Method (BMD) and the cytotoxicity analysis was carried out on Human Dermal Fibroblast (HDF) cells by MTT assay method. Based on the results, this versatile drug carrier could efficiently deliver the cationic antimicrobial peptides as a natural antibiotic for growth inhibition of methicillin-resistant S. aureus strains and further destroying the bacteria in the treatment of subcutaneous infections caused by methicillin-resistant S. aureus strains.

Thermoresponsive Nanogels Based on Different Polymeric Moieties for Biomedical Applications.

Nanogels, or nanostructured hydrogels, are one of the most interesting materials in biomedical engineering. Nanogels are widely used in medical applications, such as in cancer therapy, targeted delivery of proteins, genes and DNAs, and scaffolds in tissue regeneration. One salient feature of nanogels is their tunable responsiveness to external stimuli. In this review, thermosensitive nanogels are discussed, with a focus on moieties in their chemical structure which are responsible for thermosensitivity. These thermosensitive moieties can be classified into four groups, namely, polymers bearing amide groups, ether groups, vinyl ether groups and hydrophilic polymers bearing hydrophobic groups. These novel thermoresponsive nanogels provide effective drug delivery systems and tissue regeneration constructs for treating patients in many clinical applications, such as targeted, sustained and controlled release.

Self-assembled formation of chondroitin sulfate-based micellar nanogel for curcumin delivery to breast cancer cells.

Nanogel based drug delivery systems have been broadly used for cancer treatment. In this research, octadecylamine was grafted to chondroitin sulfate using three different mole ratios (10, 20, and 30) and named CS-ODA1, 2, and 3, respectively. The amide bond formation between chondroitin sulfate and octadecylamine was confirmed by 1H-nuclear magnetic resonance (HNMR) in the CS-ODA3 sample; therefore, further analysis was performed on this sample. Curcumin was loaded at defined Cur/CS-ODA ratios (5, 10 and 15%) and CS-ODA3 with 10% curcumin was selected for further experiments due to more entrapment efficiency (79.56% ± 5.56). In vitro release profile of the curcumin loaded nanogels showed >80% release after 70 h. In addition, the results of MTT analysis on the MCF-7 cell line showed almost no toxicity toward blank nanogels, while curcumin-loaded nanogels induced significant death after 24 h. In the end, analysis of the cell cycle using MCF-7 cells also confirmed the cytotoxicity of curcumin loaded nanogels. This study also showed that the presence of curcumin loaded chondroitin sulfate nanogels could successfully increase cellular uptake in comparison with free curcumin. The synthesized nanogels containing curcumin are expected to be effective for further studies in cancer treatment.

Dual responsive chondroitin sulfate based nanogel for antimicrobial peptide delivery.

Poly (l-lactide)-graft-chondroitin sulfate (PLLA-g-CS) copolymers were synthesized with different l-lactide contents via ring-opening polymerization. Chemical structure of the synthesized copolymers was confirmed by FTIR and HNMR analyses. The degree of polymerization and substitution of PLLA was found to be 0.56 and 2.98, respectively. Nisin was loaded in PLLA-g-CS nanogels at 37 and 42 °C. The hydrodynamic radius of the nanogels was 181 and 399 nm, respectively. The release profile was studied at two different temperatures and pHs over 7 days. The results indicated a variation of the cumulative release of nisin from 25 to 98% depending on the pH and temperature of release media. Cytotoxicity test of nisin loaded nanogels on human dermis fibroblast cells, confirmed no toxic effect. Finally, Antimicrobial activity of the nanogel was evaluated against Staphylococcus aureus and Escherichia coli bacteria. Overall, this study indicated that the dual responsive nanocarrier could potentially be used for infection therapeutic applications.

Design and optimization of kollicoat ® IR based mucoadhesive buccal film for co-delivery of rizatriptan benzoate and propranolol hydrochloride.

This work aimed to develop a mucoadhesive buccal film for potential co-delivery of rizatriptan benzoate (RB) and propranolol hydrochloride (PRH). Kollicoat ® IR, polyethylene oxide (PEO), hydroxypropyl methylcellulose (HPMC K4M), glycerol, stevia, and Aloe vera gel powder (AVgel powder) were used to prepare film by solvent casting method. In order to characterize and optimize formulations, Design-Expert software with central composite design (CCD) was used. The selected independent variables were concentrations of Kollicoat ® IR, PEO, glycerol, and AVgel powder. Five selected dependent variables were in vitro disintegration time, folding endurance, swelling ratio, and in vitro drugs release. Film with 50 mg PRH, 25 mg RB, 5 mg stevia, 63 mg HPMC K4M, 100 mg Kollicoat ® IR, 66.33 mg PEO, 0.22 ml glycerol, and 0.8 mg AVgel powder was selected as optimized formulation. The optimized film was characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) to evaluate morphology, chemical structure, and physical properties of the film. The mechanical properties of optimized film were measured by Santam instrument. Ex vivo permeation were studied by Franz diffusion cell, while rat buccal mucosa was used as a model membrane. The optimized film with incorporation of AVgel powder as a natural permeation enhancer could transport 73.22% of PRH and 96.11% of RB over 100 min through rat buccal mucosa and showed no buccal mucosal damage after histopathological evaluation. Overall, the optimized film could be a potential candidate for the effective treatment of migraine.

Synthesis and characterization of schizophyllan nanogels via inverse emulsion using biobased materials.

Oxidized sucrose cross-linked Schizophyllan nanogel was successfully synthesized via inverse emulsion method for the first time. The synthesis process was conducted in the absence of both toxic cross-linker and organic solvent. The nanogel crosslinking network was prepared using fractionated coconut oil as the continuous phase and oxidized sucrose as the cross-linker. The formation of ether linkage on schizophyllan cross-linked structure was confirmed by Fourier transform infrared microscopy (FTIR) analysis. Size and morphology were evaluated by DLS analysis and SEM. The results showed that increasing the concentration of surfactant causes the decrease in the size. By keeping surfactant amounts constant, the increase in the amount of crosslinker caused increase in the size and swelling degree. Nontoxicity of the nanogels was proved by in vitro MTT analysis. The obtained nanogels, possess special properties such as high water content, colloidal stability, bioactivity, functionality, and interior network for drug loading capacity offer great potential for the utilization of nanogels in biomedical applications.

Preparation and optimization of self-assembled chondroitin sulfate-nisin nanogel based on quality by design concept.

Self-assembled nanogel was prepared by electrostatic complexation of two oppositely charged biological macromolecules, which were cationic nisin and anionic chondroitin sulfate (ChS). The critical factors affected the physical properties of ChS-nisin nanogel was screened and optimized by Plackett-Burman design (PB) and central composite design (CCD). The independent factors selected were: concentration ratio of nisin to ChS, injection rate of nisin solution, buffer solvent type, magnetic stirring rate, pH of initial buffer solution, centrifuge-cooling temperature, and centrifuge rotation speed. Among these factors, concentration ratio changed the entrapment efficiency and loading capacity significantly. In addition, the hydrodynamic diameter and loading capacity were significantly influenced by injection rate and pH of initial buffer solution. The optimized nanogel structure was obtained by concentration ratio of 6.4mg/mL nisin to 1mg/mL ChS, pH of buffer solution at 4.6, and nisin solution injection rate of 0.2mL/min. The observed values of dependent responses were close to predicted values confirmed by model from response surface methodology. The results obviously showed that quality by design concept (QbD) could be effectively applied to optimize the developed ChS-nisin nanogel.

New formulation and approach for mucoadhesive buccal film of rizatriptan benzoate.

Mucoadhesive buccal film is developed as a promising dosage form, which has prominent advantages because of drug delivery through buccal mucosa. New formulation of buccal films containing rizatriptan benzoate (RB) was prepared by solvent casting method using various concentrations of hydroxypropyl methylcellulose (HPMC K4M), polyvinyl alcohol (PVA), polyethylene oxide (PEO), glycerol, stevia, and goat buccal mucosa used as a model membrane. In this work, the effect of polymers and plasticizer concentrations on drug release profile, disintegration and dissolution time, mechanical properties, and mucoadhesive characteristics of films was studied. Scanning electron microscopy analysis revealed uniform distribution of RB in film formulations. Chemical compounds and thermal analysis of the films were studied by Fourier transform infrared spectroscopy and differential scanning calorimetry, respectively. The buccal films produced were uniform in drug content and thickness. All formulations have in vitro release of 98-102% between 40 and 80 min. Also ex vivo mucoadhesion strength was in the range of 0.205 ± 0.035 to 0.790 ± 0.014 N for all formulations. A formulation consisting RB (50 mg), HPMC K4M, PVA, and PEO (63 mg), glycerol (1.5 ml), stevia (5 mg) was selected as our optimum composition. More satisfactory results were obtained in terms of disintegration and dissolution time, mechanical properties, and mucoadhesive characteristics. In addition, it showed about 99.89% RB released in 45 min. The results suggest that RB-loaded mucoadhesive buccal films could be a potential candidate to achieve optimum drug release for effective treatment of migraine.

Synthesis and characterization of enzyme-magnetic nanoparticle complexes: effect of size on activity and recovery.

The influence of particle size on the activity and recycling capabilities of enzyme conjugated magnetic nanoparticles was studied. Co-precipitation and oxidation of Fe(OH)(2) methods were used to fabricate three different sizes of magnetic nanoparticles (5 nm, 26 nm and 51 nm). Glucose oxidase was covalently bound to the magnetic nanoparticles by modifying the surfaces with 3-(aminopropyl)triethoxysilane (APTES) and a common protein crosslinking agent, glutaraldehyde. Analysis by Transmission Electron Microscopy (TEM) showed that the morphology of the magnetic nanoparticles to be spherical and sizes agreed with results of the Brunauer, Emmett, and Teller (BET) method. Magnetic strength of the nanoparticles was analyzed by magnetometry and found to be 49 emu g(-1) (5 nm), 73 emu g(-1) (26 nm), and 85 emu g(-1) (51 nm). X-ray photoelectron spectroscopy (XPS) confirmed each step of the magnetic nanoparticle surface modification and successful glucose oxidase binding. The immobilized enzymes retained 15-23% of the native GOx activity. Recycling stability studies showed approximately 20% of activity loss for the large (51 nm) and medium (26 nm) size glucose oxidase-magnetic nanoparticle (GOx-MNP) bioconjugate and about 96% activity loss for the smallest GOx-MNP bioconjugate (5 nm) after ten cycles. The bioconjugates demonstrated equivalent total product conversions as a single reaction of an equivalent amount of the native enzyme after the 5th cycle for the 26 nm nanoparticles and the 7th cycle for the 51 nm nanoparticles.

Engineering nanoassemblies of polysaccharides.

Polysaccharides offer a wealth of biochemical and biomechanical functionality that can be used to develop new biomaterials. In mammalian tissues, polysaccharides often exhibit a hierarchy of structure, which includes assembly at the nanometer length scale. Furthermore, their biochemical function is determined by their nanoscale organization. These biological nanostructures provide the inspiration for developing techniques to tune the assembly of polysaccharides at the nanoscale. These new polysaccharide nanostructures are being used for the stabilization and delivery of drugs, proteins, and genes, the engineering of cells and tissues, and as new platforms on which to study biochemistry. In biological systems polysaccharide nanostructures are assembled via bottom-up processes. Many biologically derived polysaccharides behave as polyelectrolytes, and their polyelectrolyte nature can be used to tune their bottom-up assembly. New techniques designed to tune the structure and composition of polysaccharides at the nanoscale are enabling researchers to study in detail the emergent biological properties that arise from the nanoassembly of these important biological macromolecules.

Layer-by-layer assembly of polysaccharide-based nanostructured surfaces containing polyelectrolyte complex nanoparticles.

Nanoscale chemical and topographical features have been demonstrated to influence a variety of significant responses of mammalian cells to biomaterials surfaces. Thus, an important goal for biomaterials scientists is the ability to engineer the nanoscale surface features of biologically active materials. The goal of the current work is to demonstrate that polyelectrolyte complex nanoparticles (PCNs) in polyelectrolyte multilayers (PEMs) can be combined to create surfaces with controlled nanoscale surface topography and nanoscale presentation of surface chemistry. The polysaccharides used in this work are the biomedically relevant chitosan, heparin, and hyaluronan. Nanostructured surface coatings were characterized on both modified gold substrates and tissue-culture polystyrene surfaces. PCNs were adsorbed to oppositely charged PEMs, and were also embedded within PEMs. The construction of the surface coatings was characterized by quartz crystal microbalance with dissipation (QCM-D). The surface morphology was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemistry of the coatings was confirmed by both X-ray photoelectron spectroscopy (XPS) and polarization modulation infra-red reflection absorption spectroscopy (PM-IRRAS). Morphologically, we found that PCNs were colloidally stable and homogeneously distributed when adsorbed on or in the PEMs. Chemical analysis confirms that the PCNs adsorbed to PEMs significantly altered the surface chemistry, indicating significant surface coverage. Furthermore, the position of the PCNs normal to the surface can be adjusted by adding PEMs on top of adsorbed PCNs. Thus, PCNs can be used to introduce discrete nanoscale surface topographical features and varying surface chemistry into PEM surface coatings in a controlled way.

Polysaccharide-based polyelectrolyte complex nanoparticles from chitosan, heparin, and hyaluronan.

The formation of polyelectrolyte complex nanoparticles (PCN) was investigated at different charge mixing ratios for the chitosan-heparin (chi-hep) and chitosan-hyaluronan (chi-ha) polycation-polyanion pairs. The range of 0.08-19.2 for charge mixing ratio (n(+)/n(-)) was examined. The one-shot addition of polycation and polyanion solutions used for the formation of the PCN permitted formation of both cationic and anionic particles from both polysaccharide pairs. The influence of the charge mixing ratio on the size and zeta potential of the particles was investigated. The morphology and stability of the particles when adsorbed to surfaces was studied by scanning electron microscopy (SEM). For most conditions studied, colloidally stable, nonstoichiometric PCN were formed in solution. However, PCN formation was inhibited by flocculation at charge mixing ratios near 1. When adsorbed to surfaces and dried, some formulations resulted in discrete nanoparticles, while others partially or completely aggregated or coalesced, leading to different surface morphologies.

Polyelectrolyte multilayer assembly as a function of pH and ionic strength using the polysaccharides chitosan and heparin.

The goal of this work is to explore the effects of solution ionic strength and pH on polyelectrolyte multilayer (PEM) assembly, using biologically derived polysaccharides as the polyelectrolytes. We used the layer-by-layer (LBL) technique to assemble PEM of the polysaccharides heparin (a strong polyanion) and chitosan (a weak polycation) and characterized the sensitivity of the PEM composition and layer thickness to changes in processing parameters. Fourier-transform surface plasmon resonance (FT-SPR) and spectroscopic ellipsometry provided in situ and ex situ measurements of the PEM thickness, respectively. Vibrational spectroscopy and X-ray photoelectron spectroscopy (XPS) provided details of the chemistry (i.e., composition, electrostatic interactions) of the PEM. We found that when PEM were assembled from 0.2 M buffer, the PEM thickness could be increased from less than 2 nm per bilayer to greater than 4 nm per bilayer by changing the solution pH; higher and lower ionic strength buffer solutions resulted in narrower ranges of accessible thickness. Molar composition of the PEM was not very sensitive to solution pH or ionic strength, but pH did affect the interactions between the sulfonates in heparin and amines in chitosan when PEM were assembled from 0.2 M buffer. Changes in the PEM thickness with pH and ionic strength can be interpreted through descriptions of the charge density and conformation of the polyelectrolyte chains in solution.