Development and In Vitro Evaluation of Surface Modified Poly(lactide-co-glycolide) Nanoparticles with Chitosan-4-Thiobutylamidine

ABSTRACT

The objective of this study was to develop a nanoparticulate drug delivery system based on the surface modification of poly(lactide-co-glycolide) (PLGA) nanoparticles with a thiolated chitosan. PLGA nanoparticles were prepared by the emulsification-solvent evaporation method. Immobilization of chitosan to the surface of PLGA nanoparticles via amide bonds was mediated by a carbodiimide. Thiol groups were covalently bound to the chitosan surface of particles by reaction with 2-iminothiolane. Obtained nanoparticles were characterized in vitro regarding size, zeta potential, thiol group content, stability at different pH values, mucoadhesion, and drug release. Results demonstrated that the surface modification of PLGA nanoparticles with thiolated chitosan (chitosan-TBA) leads to nanoparticles of a mean diameter of 889.5 ± 72 nm and positive zeta potential of + 24.74 mV. The modified nanoparticles contained 7.32 ± 0.24 μmol thiol groups per gram nanoparticles. The size of nanoparticles was strongly influenced by the pH of the surrounding medium, being 925.0 ± 76.3 nm at pH 2 and 577.8 ± 66.7 nm at pH 7.4. Thiolated nanoparticles showed a 3.3-fold prolonged residence time on the mucosa and an unchanged release profile in comparison to unmodified PLGA nanoparticles. These data suggest that surface modified chitosan-TBA conjugate PLGA nanoparticles have the potential to be used as mucoadhesive drug delivery system.     

Fabrication of a reticular poly(lactide-co-glycolide) cylindrical scaffold for the in vitro development of microvascular networks

Abstract

The microvascular network is a simple but critical system that is responsible for a range of important biological mechanisms in the bodies of all animals. The ability to generate a functional microvessel not only makes it possible to engineer vital tissue of considerable size but also serves as a platform for biomedical studies. However, most of the current methods for generating microvessel networks in vitro use rectangular channels which cannot represent real vessels in vivo and have dead zones at their corners, hence hindering the circulation of culture medium. We propose a scaffold-wrapping method which enables fabrication of a customized microvascular network in vitro in a more biomimetic way. By integrating microelectromechanical techniques with thermal reflow, we designed and fabricated a microscale hemi-cylindrical photoresist template. A replica mold of polydimethylsiloxane, produced by casting, was then used to generate cylindrical scaffolds with biodegradable poly(lactide-co-glycolide) (PLGA). Human umbilical vein endothelial cells were seeded on both sides of the PLGA scaffold and cultured using a traditional approach. The expression of endothelial cell marker CD31 and intercellular junction vascular endothelial cadherin on the cultured cell demonstrated the potential of generating a microvascular network with a degradable cylindrical scaffold. Our method allows cells to be cultured on a scaffold using a conventional culture approach and monitors cell conditions continuously. We hope our cell-covered scaffold can serve as a framework for building large tissues or can be used as the core of a vascular chip for in vitro circulation studies.     

Preparation and in vitro evaluation of meloxicam-loaded PLGA nanoparticles on HT-29 human colon adenocarcinoma cells

Context: The inhibitors of cyclooxygenase (COX)-2 play an important role in cancer chemoprevention. Certain COX-2 inhibitors exert antiproliferative and pro-apoptotic effects on cancer cells.

Objective: In this study, meloxicam, which is an enolic acid-type preferential COX-2 inhibitor, was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to maintain local high concentration, and its efficacy was determined.

Methods: NPs were prepared by using salting-out and emulsion-evaporation steps. Meloxicam-loaded NP formulations were evaluated with respect to the drug loading, particle size, polydispersity index, zeta potential, drug release rate, and residual poly(vinyl alcohol) (PVA) percentage. The effects of PLGA and PVA molecular weight variations on the physicochemical properties of NPs were investigated. Stability of meloxicam in NPs was assessed over 3 months. COX-2 expressing human colon adenocarcinoma cell line HT-29 was used in cellular uptake and viability assays.

Results: NPs had a spherical shape and a negative zeta potential, and their size ranged between 170–231?nm with a lower polydispersity index. NPs prepared with high molecular weight PLGA were shown to be physically stable over three months at 4°C. The increase in molecular weight of the polymer and emulsifier reduced the in vitro release rate of meloxicam from NPs. Meloxicam-loaded NPs showed cytotoxic effects on HT-29 cells markedly at 800 µM. Cancer cells had high uptake of coumarin-6-loaded NPs.

Conclusion: The PLGA NPs developed in this study can be a potentially effective drug delivery system of meloxicam for the treatment of colon cancer.     

Release of adriamycin from poly(lactide-co-glycolide)-polyethylene glycol nanoparticles

In order to develop a prolonged circulating drug carrier and to overcome p-glycoprotein-mediated multidrug resistance to adriamycin (ADR), which is a potent chemotherapeutic agent in the treatment of various cancers, poly(lactide-co-glycolide)-polyethylene glycol (PLGA-PEG) nanoparticles were prepared and characterized. ADR-loaded PLGA-PEG nanoparticles prepared by the emulsification-diffusion method were spherical and homogeneous with smooth surfaces when assessed by scanning electron microscopy. The nanoparticles were 200-230 nm in size and the encapsulation efficiency of ADR in the nanoparticles was 30 approximately 35%. The release of ADR from nanoparticles was extended compared to that from free ADR solution. After intravenous administration of adriamycin-loaded PLGA-PEG nanoparticles to rats, the plasma level of ADR from PLGA-PEG nanoparticle was extended until 24 hours and the mean residence time of ADR of nanoparticles was increased compared to that of ADR solution. And ADR-loaded nanoparticles showed a higher growth inhibitory effect than free ADR solution in an ADR resistant MCF-7 human breast carcinoma cell line. The prepared ADR-loaded PLGA-PEG nanoparticles can be used as a good delivery system for ADR.     

Paclitaxel-loaded poly(lactide-co-glycolide)/poly(ethylene vinyl acetate) composite for stent coating by ultrasonic atomizing spray

The mixture of poly(lactide-co-glycolide) (PLGA) and poly(ethylene vinyl acetate) (PEVA) forms a homogeneous liquid in an organic solvent such as tetrahydrofuran, and a phase-separated PLGA/PEVA composite can be prepared from it by evaporating the organic solvent. Exploiting this phenomenon, we designed a novel method of preparing a drug-loaded PLGA/PEVA composite and used it for coating drug-eluting stents (DESs). Paclitaxel (PTX), an anticancer drug, was chosen as a model drug. PLGA acts as a microdepot for PTX, and PEVA provides mechanical strength to the coating material. The presence of PLGA in the PLGA/PEVA composite suppressed PTX crystallization in the coating material, and PTX showed a sustained release rate over more than 30 days. The mechanical strength of the PLGA/PEVA composite was better than that of PEVA used as a control. After coating the stent with a PLGA/PEVA composite using ultrasonic atomizing spray, the morphology of the coated material was observed by scanning electron microscopy, and the release pattern of PTX was measured by high-performance liquid chromatography.     

Paclitaxel-loaded poly(lactide-co-glycolide)/poly(ethylene vinyl acetate) composite for stent coating by ultrasonic atomizing spray

Abstract

The mixture of poly(lactide-co-glycolide) (PLGA) and poly(ethylene vinyl acetate) (PEVA) forms a homogeneous liquid in an organic solvent such as tetrahydrofuran, and a phase-separated PLGA/PEVA composite can be prepared from it by evaporating the organic solvent. Exploiting this phenomenon, we designed a novel method of preparing a drug-loaded PLGA/PEVA composite and used it for coating drug-eluting stents (DESs). Paclitaxel (PTX), an anticancer drug, was chosen as a model drug. PLGA acts as a microdepot for PTX, and PEVA provides mechanical strength to the coating material. The presence of PLGA in the PLGA/PEVA composite suppressed PTX crystallization in the coating material, and PTX showed a sustained release rate over more than 30 days. The mechanical strength of the PLGA/PEVA composite was better than that of PEVA used as a control. After coating the stent with a PLGA/PEVA composite using ultrasonic atomizing spray, the morphology of the coated material was observed by scanning electron microscopy, and the release pattern of PTX was measured by high-performance liquid chromatography.     

Formulation and in vitro evaluation of bisphosphonate loaded microspheres for implantation in osteolysis

Chitosan and poly(lactide-co-glycolide) acid (PLGA) microspheres loaded with alendronate sodium (AS) were prepared for orthopedic as well as dental applications. In orthopedics the aim was to make the total joint prostheses stay in the body for a long time without causing bone tissue loss, while in dentistry it was aimed to treat the alveolar bone resorption caused by periodontitis and also to make the dental treatment using implants easier by reducing the bone loss in patients with osteoporosis. Solvent evaporation method was used to prepare AS loaded PLGA microspheres and emulsion polimerization method was used to prepare AS loaded chitosan microspheres. Particle size, loading efficacy, surface characteristics, and in vitro release characteristics were examined on prepared formulations. After the examination of the scanning electron microscopy photographs of microspheres, chitosan microspheres were observed to have spherical structure and smooth surface characteristics while PLGA microspheres were observed to have spherical porous surface structure. Loading efficacy was found to be 3.30% for chitosan microspheres and 7.70% for PLGA microspheres. It was observed that 85% of AS had been released at the end of the third day from chitosan microspheres whereas 58% was released at the end of the fifth day from PLGA microspheres. It was found that chitosan microspheres gave first order release while PLGA microspheres gave zero order release.     

Development and evaluation of chitosan and chitosan derivative nanoparticles containing insulin for oral administration

Abstract

Chitosan and chitosan derivative-based nanoparticles loaded with insulin were prepared by self-assembly, via electrostatic interactions between the negatively charged drug and the positively charged polymers. In the investigated chitosan derivatives, the amine groups were substituted to different extents (33, 52 or 99%) by 2-hydroxypropyl-3-trimethyl ammonium groups, rendering the polymers permanently positively charged, irrespective of the pH. This is an important property for this type of advanced drug delivery system, since the pH value changes throughout the gastrointestinal tract and electrostatic interactions are of crucial importance for the stability of the nanoparticles. Permanent positive charges are also in favor of mucoadhesion. In contrast, the electric charges of chitosan molecules depend on the pH of the surrounding medium. Since the solubility of the chitosan derivatives increased due to the introduction of quaternary ammonium groups, sodium tripolyphosphate (TPP) was added to the systems to create supplementary cross-links and stabilize the nanoparticles. The presence of TPP influenced both the dissolution of the polymer matrix as well as the resulting release kinetics. The underlying drug release mechanisms were found to be more complex than simple diffusion under constant conditions, likely involving also ionic interactions and matrix dissolution. The most promising formulation was based on a chitosan derivative with 33% substitution degree and characterized by a Z-average of 142?±?10?nm, a zeta potential of 29?±?1?mV, an encapsulation efficacy of 52?±?3% and, most importantly, the release of insulin was sustained for more than 210?min.     

In vitro evaluation of poly(caporlactone) grafted dextran (PGD) nanoparticles with cancer cell

This study dealt with the preparation and characterization of coumarin-6 loaded poly(caprolactone) grafted dextran (PGD) nanoparticles (NPs) and evaluation of cellular uptake by using human gastric cancer cell line (SNU-638), in vitro. The potential application of these PGD NPs for sustained drug delivery was evaluated by the quantification and localization of the cellular uptake of fluorescent PGD NPs. Coumarin-6 loaded PGD NPs were prepared by a modified oil/water emulsion technique and characterized by various physico-chemical methods such as, laser light scattering for particle size and size distribution, atomic force microscopy (AFM), zeta-potential and spectrofluorometry to identify the release of fluorescent molecules from the NPs. SNU-638 was used to measure the cellular uptake of fluorescent PGD NPs. Confocal laser scanning microscopic images clearly showed the internalization of NPs by the SNU-638 cells. Cell viability was assessed by treating the SNU-638 cells with PGD NPs for 48 h. The results reveal, that these biodegradable polymeric NPs holds promise in biomedical field as a carrier.     

Folate-mediated solid–liquid lipid nanoparticles for paclitaxel-coated poly(ethylene glycol)

Background: As a promising anticancer drug, severe side-effects of current clinical formulations for paclitaxel have restricted its use, developing a better technical-economical formulation for paclitaxel delivery is needed. Method: In this study, the compound of folate-poly(ethylene glycol) (PEG)-phosphatidylethanolamine was synthesized and characterized with Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. The solid-liquid lipid nanoparticle (SLLN) for paclitaxel modified with folate and poly(ethylene glycol) (folate-PEG-SLLN) was prepared and characterized. Morphology of folate-PEG-SLLN was examined by transmission electron microscopy. The particle size and zeta potential were performed by Zetapals. Encapsulation efficiency was analyzed by HPLC. The in vitro drug release of paclitaxel was investigated via membrane dialysis. The in vivo pharmacokinetics was measured with male Sprague-Dawley rats. Treatment efficiency was investigated with the mouse with sarcoma180 ascites tumor. Results: Paclitaxel loaded on the newly designed binary SLLN showed a longer and sustained in vitro releasing property. More importantly, S180 tumor-bearing mice treated with paclitaxel-loaded SLLN exhibited higher tumor inhibition rate, comparing with animals administered with paclitaxel injection alone (45.3% and 37.3%, respectively). Conclusion: The newly developed paclitaxel delivery system may have improved in vivo antitumor activity. The results demonstrated a great interest to use folate-mediated SLLN as a prospective drug delivery system for paclitaxel.     

Preparation of thiomer microparticles and in vitro evaluation of parameters influencing their mucoadhesive properties

It was the aim of this study to develop mucoadhesive microparticulate delivery systems based on thiomers and to investigate parameters influencing their mucoadhesive properties. Microparticles were prepared via coazervation of thiolated or unmodified polycarbophil with fluorescein-diacetate as marker. The protective effect of the polymers toward enzymatic hydrolysis by intestinal enzymes was investigated. Mucoadhesion studies with microparticles, applied in dry and prehydrated form, were performed by ascertaining their residence time on intestinal mucosa. Furthermore, the influence of the amount of thiol groups on mucoadhesion was studied in vitro. Results showed that in comparison to unmodified polycarbophil, thiolated polycarbophil provided a more than 3-fold higher protective effect for the incorporated marker fluorescein-diacetate toward hydrolysis. When being applied in dry form 23.4 ± 4.8% of the fluorescence marker being embedded in thiomer microparticles remained adhering to the intestinal mucosa within 3 h. In contrast, only 11.6 ± 2.0% of the marker remained on the mucosa, when the thiomer microparticles were applied in prehydrated form. In addition, tests performed to assess the impact of the amount of thiol groups pointed out that a high amount of thiol groups is advantageous in order to further improve mucoadhesive properties. This knowledge should contribute to the design of highly efficient drug delivery systems being based on thiomer microparticles.     

In vitro evaluation of betamethasone-loaded nanoparticles

The aim of the present work was to investigate the preparation of nanoparticles as a potential drug carrier in the treatment of various inflammatory diseases. A nanoprecipitation method was used to entrap betamethasone in a poly[ε-caprolactone] matrix. Process parameters such as the initial drug load, the surfactants (polyvinyl alcohol, PVA; sodium cholate, SC), and their concentration in the aqueous phase were analyzed for their influences on particle properties. Particle size changed with increasing surfactant concentrations (PVA: 250 to 400 nm; sodium cholate: 330 to 150 nm) due to changes in interface stability and viscosity of the aqueous phase. The zeta potential was around neutrality with PVA and between - 28 and - 42 mV with SC. Betamethasone encapsulation rates of about 75% and 90% slightly increased with higher surfactant concentration. Drug release profiles exhibited an initial burst release with both surfactants, PVA (8-18%) or SC (25-35%) followed by a sustained release delivering 15% to 40% of the entrapped drug within 48 hours. The present nanoparticulate formulations exhibit promising properties of a colloidal drug carrier for betamethasone. Although SC seems to be advantageous due to its biocompatibility, in terms of sustained drug release pattern, the use of PVA is favorable.     

Preparation of poly(ε-lysine)-cyclodextrin coated Fe3O4 nanoparticles for selective separation of natural medicine: Scutellarin

In the present study, poly(ε-lysine)-cyclodextrin coated Fe₃O₄ nanoparticles (PL-CD@Fe₃O₄) were prepared by co-precipitation method. Then the PL-CD@Fe₃O₄ nanoparticles were studied of the characterization and the selective separation performance for natural medicine (scutellarin). The experimental results showed that PL-CD@Fe₃O₄ nanoparticles were successfully prepared with a particle size of about 30 nm and a saturation magnetization of 40 emu/g. When the nanoparticles were in the environment of pH = 7, the stability was the best, the adsorption capacity was the strongest, and the number of recyclable times was the most. Langmuir model deduced that the maximum adsorption capacity of the nanoparticles was 76.3 mg/g. In addition, the experimental results of using nanoparticles to separate SCU in the crude extract showed that the purity of SCU could be increased from 72.2% to 93.5% after repeated cyclic separation three times. Two dimensional NMR analysis showed that the selective separation of SCU by PL-CD@Fe₃O₄ was mainly caused by the host–guest interaction between cyclodextrin and SCU. In conclusion, the PL-CD@Fe₃O₄ nanoparticles are a very promising material for the extraction and separation of other similar natural drugs.     

Preparation and evaluation of poly(lactic-co-glycolic acid) microparticles as a carrier for pulmonary delivery of recombinant human interleukin-2: II. In vitro studies on aerodynamic properties of dry powder inhaler formulations

Objective: The aim of this study was the preparation and evaluation of dry powder formulations of recombinant human interleukin-2 (rhIL-2)-loaded microparticles to be administered to the lung by inhalation.

Methods: As indicated in our previous study, the microparticles were prepared by modified water-in-oil-in-water (w₁/o/w₃) double emulsion solvent extraction method using poly(lactic-co-glycolic acid) (PLGA) polymers. The dry powder formulations were prepared with blending of microparticles and mannitol as a coarse carrier. The actual aerodynamic characteristics of the microparticles alone and prepared mixtures with mannitol are evaluated by using the eight-stage Andersen cascade impactor.

Results: Due to the low tapped density of microparticles (<0.4?g/cm³), the theoretical aerodynamic diameter (MMADt) values were calculated (<5 μm) on the basis of the geometrical particle diameter and tapped density values. The lowest tapped density value (0.17?g/cm³) belongs to the cyclodextrin-containing formulation. According to the results obtained using the cascade impactor, the emitted doses for all microparticle formulations were found to be rather high and during the aerosolization for all the formulations except F3 and F5, >90% of the capsule content was determined to be released. However, the actual aerodynamic diameter (MMADa) values were seen to be higher than the MMADt values. The blending of the microparticles with mannitol allowed their aerodynamic diameters to decrease and their fine particle fraction values to increase.

Conclusion: The obtained results have shown that the mixing of rhIL-2-loaded microparticles with mannitol possess suitable aerodynamic characteristics to be administered to the lungs by inhalation.     

Poly(ethylene glycol) decorated poly(methylmethacrylate) nanoparticles for protein adsorption

Poly(ethylene glycol) decorated poly(methyl methacrylate) particles were synthesized by means of emulsion polymerization using poly(ethylene glycol) sorbitan monolaurate (Tween-20) as surfactant. PMMA/PEG particles presented mean diameter (195 ± 15) nm, indicating narrow size distribution. The adsorption behavior of bovine serum albumin (BSA) and concanavalin A (ConA) onto PMMA/PEG particles was investigated by means of spectrophotometry. Adsorption isotherms obtained for BSA onto PMMA/PEG particles fitted well sigmoidal function, which is typical for multilayer adsorption. Con A adsorbed irreversibly onto PMMA/PEG particles. The efficiency of ConA covered particles to induce dengue virus quick agglutination was evaluated.     

Dramatic improvements in toughness in poly(lactide-co-glycolide) nanocomposites

Poly(lactide-co-glycolide) (PLG), a biocompatible and biodegradable polymer, is dramatically toughened by adding small amounts of surface modified clay nanoparticles. The elongation during tensile tests increases from 7% for the pure polymer to 210% for the nanocomposite, accompanied with a modest increase in modulus. In contrast, PLG nanocomposites based on fumed silica treated with hexamethyldisilazane show only modest improvements in toughness. Electron microscopy, X-ray scattering, rheometry, and dielectric relaxation spectroscopy are used to investigate the toughening mechanism. Multiple crazing occurs in the clay nanocomposite after yielding. Small angle X-ray scattering studies show significant orientation of the clay nanoparticles along the tensile stress direction during deformation. The clay nanocomposites show a new, slow relaxation mode, most likely due to interfacial adsorbption of PLG chains on the surface of the clay nanoparticles. The dramatic increase in toughness is attributed to physical crosslinks introduced by the clay nanoparticles, a mechanism absent in the PLG/silica nanocomposites. The physical crosslinks increase the brittle fracture strength of the polymer and, consequently, trigger a toughening mechanism via multiple crazing and shear yielding.     

Photovoltaic characterization of hybrid solar cells using surface modified TiO(2) nanoparticles and poly(3-hexyl)thiophene

We report on the photovoltaic performance of bulk heterojunction solar cells using novel nanoparticles of 6-palmitate ascorbic acid surface modified TiO(2) as an electron acceptor embedded into the donor poly(3-hexyl)thiophene (P3HT) matrix. Devices were fabricated by using P3HT with varying amounts of red TiO(2) nanoparticles (1:1, 1:2, 1:3?w-w ratio). The devices were characterized by measuring current-voltage characteristics under simulated AM 1.5 conditions. Incident photon to current efficiency (IPCE) was spectrally resolved. The nanoscale morphology of such organic/inorganic hybrid blends was also investigated using atomic force microscopy (AFM).     

Encapsulation and release of rifampicin using poly(vinyl pyrrolidone)-poly(methacrylic acid) polyelectrolyte capsules

Poly(vinyl pyrrolidone) and poly(methacrylic acid) multilayer capsules based on hydrogen bonding have been prepared by the layer-by-layer approach and used to encapsulate and release rifampicin, an anti-tuberculosis drug. Removal of silica core using a buffer of ammonium fluoride and hydrofluoric acid at about pH 3 was found to produce better capsules than hydrofluoric acid alone. An eight-layered capsule had a wall thickness of 20 nm. Maximum encapsulation was found to be about 86 μg at 40 °C with 1 ± 0.2 × 10⁶ capsules. Release studies showed a burst kind of release and maximum release was obtained above pH 7 where the capsules disintegrate rapidly thereby releasing the drug in a short period. Interactions studies with Mycobacterium smegmatis showed that the capsules were cytocompatible and the released drug functioned with the same efficacy as the free drug.     

Preparation,characterization and in vitro anticancer activity of paclitaxel conjugated magnetic nanoparticles

In this study, magnetic nanoparticles (MNPs) coated with L-aspartic acid (F-Asp NPs) were synthesized through a co-precipitation method and conjugated with paclitaxel (PTX) (F-Asp-PTX NPs) by esterification reaction between the carboxylic acid end groups on MNPs surface and the hydroxyl groups of the PTX and studied its cytotoxic effect in vitro. The successful conjugating of PTX onto the nanoparticles (NPs) was confirmed by X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and transmission electron microscopy (TEM) techniques. The results showed that the average size was 46.11?±?7.8 (mean?±?SD (n?=?25)) nm. The cytotoxicity of void of PTX and F-Asp-PTX NPs were compared to each other by MTT assay of the treated MCF-7 cell line. The F-Asp-PTX NPs showed pH-dependent drug release behavior. These studies specify that F-Asp-PTX NPs have a very remarkable anticancer effect, for breast cancer cell line.