In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

In vitro macrophage uptake and in vivo biodistribution of PLA–PEG nanoparticles loaded with hemoglobin as blood substitutes: effect of PEG content

The aim of the present work is to investigate the effect of PEG content in copolymer on physicochemical properties, in vitro macrophage uptake, in vivo pharmacokinetics and biodistribution of poly(lactic acid) (PLA)–poly(ethylene glycol) (PEG) hemoglobin (Hb)-loaded nanoparticles (HbP) used as blood substitutes. The HbP were prepared from PLA and PLA–PEG copolymer of varying PEG contents (5, 10, and 20 wt%) by a modified w/o/w method and characterized with regard to their morphology, size, surface charge, drug loading, surface hydrophilicity, and PEG coating efficiency. The in vitro macrophage uptake, in vivo pharmacokinetics, and biodistribution following intravenous administration in mice of HbP labeled with 6-coumarin, were evaluated. The HbP prepared were all in the range of 100–200 nm with highest encapsulation efficiency 87.89%, surface charge −10 to −33 mV, static contact angle from 54.25° to 68.27°, and PEG coating efficiency higher than 80%. Compared with PLA HbP, PEGylation could notably avoid the macrophage uptake of HbP, in particular when the PEG content was 10 wt%, a minimum uptake (6.76%) was achieved after 1 h cultivation. In vivo, besides plasma, the major cumulative organ was the liver. All PLA–PEG HbP exhibited dramatically prolonged blood circulation and reduced liver accumulation, compared with the corresponding PLA HbP. The PEG content in copolymer affected significantly the survival time in blood. Optimum PEG coating (10 wt%) appeared to exist leading to the most prolonged blood circulation of PLA–PEG HbP, with a half-life of 34.3 h, much longer than that obtained by others (24.2 h). These results demonstrated that PEG 10 wt% modified PLA HbP with suitable size, surface charge, and surface hydrophilicity, has a promising potential as long-circulating oxygen carriers with desirable biocompatibility and biofunctionality.     

Nicotine–magnesium aluminum silicate microparticle surface modified with chitosan for mucosal delivery

Magnesium aluminum silicate (MAS), a negatively charged clay, and nicotine (NCT), a basic drug, can interact electrostatically to form microparticles. Chitosan (CS) was used for the surface modification of the microparticles, and a lyophilization method was used to preserve the original particle morphology. The microparticles were characterized in terms of their physicochemical properties, NCT content, mucoadhesive properties, and release and permeation across porcine esophageal mucosa. The results showed that the microparticles formed via electrostatic interaction between MAS and protonated NCT had an irregular shape and that their NCT content increased with increasing NCT ratios in the microparticle preparation solution. High molecular weight CS (800 kDa) adsorbed to the microparticle surface and induced a positive surface charge. CS molecules intercalated into the MAS silicate layers and decreased the crystallinity of the microparticles, leading to an increase in the release rate and diffusion coefficient of NCT from the microparticles. Moreover, the microparticle surface modified with CS was found to have higher NCT permeation fluxes and mucoadhesive properties, which indicated the significant role of CS for NCT mucosal delivery. However, the enhancement of NCT permeation and of mucoadhesive properties depended on the molecular weight and concentration of CS. These findings suggest that NCT-MAS microparticle surface modified with CS represents a promising mucosal delivery system for NCT.     

Rhodamine-loaded poly(lactic-co-glycolic acid) nanoparticles for investigation of in vitro interactions with breast cancer cells

Nanoparticle-based drug delivery systems are considered promising for the delivery of imaging agents and drugs for the detection and treatment of illnesses, including cancer. Investigation of nanoparticle interactions with the diseased cells can lead to better designs. In this work, poly(lactic-co-glycolic acid) nanoparticles loaded with rhodamine 6G were prepared by nanoprecipitation with high encapsulation efficiency. In vitro release studies demonstrated that rhodamine escaped from the nanoparticles at a very slow rate at physiological pH, thus making it ideal for imaging studies. At acidic pH this agent was released quickly, suggesting charge interactions between the polymer and rhodamine. Microscopy and flow cytometry studies show higher uptake in MDA-MB-231 breast cancer cells when exposed to rhodamine-loaded nanoparticles than to rhodamine in solution.     

Preparation and evaluation of chitosan–itraconazole co-precipitated nanosuspension for ocular delivery

The purpose of the present study was to prepare itraconazole-loaded chitosan nanosuspension and evaluate it for ocular delivery. Itraconazole-loaded chitosan nanosuspension was prepared by controlled co-precipitation of chitosan and itraconazole from aqueous acetate solution using a combination of pH change and non-solvent addition. The co-precipitated suspension was evaluated for particle size, zeta-potential, entrapment efficiency and solubility study. It was observed that co-precipitation of itraconazole and chitosan from chitosan–lysine system in the presence of Poloxamer-188 as stabiliser provided nanosuspension of the smallest particle size with a 12-fold increase in aqueous saturation solubility of itraconazole and the fastest in vitro release. Transmission electron micrographs of the nanosuspension showed ovoid-shaped particles. A comparative evaluation of the itraconazole (1%, w/v) nanosuspension with commercial itraconazole suspension (1%, w/v) revealed a significantly higher percentage cumulative permeation of itraconazole across the isolated goat cornea from the nanosuspension dosage form as compared to the commercial suspension (P < 0.01).     

Development of new ionic gelation strategy: Towards the preparation of new monodisperse and stable hyaluronic acid/β-cyclodextrin-grafted chitosan nanoparticles as drug delivery carriers for doxorubicin

In the present study, β-cyclodextrin-grafted chitosan nanoparticles (β-CD-g-CS NPs) were prepared using a new ionic gelation strategy involving a synergistic effect of NaCl (150 mmol/L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mmol/L), and water bath sonication. This new strategy afforded smaller and more monodisperse β-CD-g-CS NPs vs. the classical ionic gelation method. New HA/β-CD-g-CS NPs were also prepared using the above-mentioned strategy by adding hyaluronic acid (HA) to the β-CD-g-CS copolymer at different weight ratios until the ZP values conversion. The best result was obtained with the weight ratio of w(HA):w(β-CD-g-CS) = 2:1 and furnished new spherical and smooth HA/β-CD-g-CS NPs. Furthermore, the stability of β- CD-g-CS NPs and HA/β-CD-g-CS NPs at 4°C in physiological medium (pH 7.4) was compared for 3 weeks period and showed that HA/β-CD-g-CS NPs were more stable all maintaining their monodispersity and high negative ZP values compared to β-CD-g-CS NPs. Finally, preliminary study of HA/β-CD-g-CS NPs as carrier for the controlled release of the anticancer drug doxorubicin was investigated. These new HA/β-CD-g-CS NPs can potentially be used as drug delivery and targeting systems for cancer treatment.     

Electrospinning of curcumin loaded chitosan/poly （lactic acid） nanofilm and evaluation of its medicinal characteristics

The curcumin loaded chitosanlpoly （lactic acid） （PLA） nanoflbers were produced using electrospinning. Box-Behnken experimental design was used for the optimization of variables （-1, 0, ＋ 1 coded level） like chitosan/PLA strength （% w/v）, curcumin strength （% w/v） and applied voltage （kV） to obtain uniform fiber diameter. The morphology of nanofibers was shown by SEM. Molecular interactions and the presence of each chemical compound of curcumin loaded chitosanlPLA fibers were characterized by FTIR and EDX analysis. Antioxidant, drug release and in vitro cytotoxicity tests were performed to evaluate the suitability of nanofibers that would be used for wound healing. In vivo wound healing studies on excision and incision wounds created on rat model showed significant reduction of wound area when compared to untreated. The better healing efficiency can be attributed to the presence of curcumin and chitosan.     

In vitro bioactivity of sol–gel-derived hydroxyapatite particulate nanofiber modified titanium

A chemically-etched titanium surface was modified by electrospinning a sol–gel-derived hydroxyapatite (HAp) that was subjected to calcination within the temperature range of 200–1400°C in the normative atmospheric condition. After heat treatment, crystal structures of the filmed titanium oxide and HAp on the titanium’s surface were identified using wide-angle X-ray diffraction. A highly porous layer of HAp was found to have formed on the oxidized titanium surfaces. The surfaces of three different samples; (1) electrospun HAp, (2) HAp calcined at 600°C, and (3) HAp calcined at 800°C, were investigated for their ability to foster promotion, proliferation, and differentiation of human osteoblasts (HOB) (in the 9th passage) in vitro up to 6 days. Among the three samples, cells cultured on the HAp calcined at 800°C titanium surfaces displayed the best results with regard to adhesion, growth, and proliferation of HOB. This novel method for fabrication of titanium substrates would provide a promising improvement for titanium-based medical devices over the current standards, which lack such substrates. These titanium substrates explicitly provide enhanced HOB proliferation in terms of both desired surface properties and their produced bulk quantity.     

In vitro release dynamics of model drugs from psyllium and acrylic acid based hydrogels for the use in colon specific drug delivery

Psyllium is medicinally important gel forming polysaccharides. Keeping in view, the pharmacological importance of psyllium and drug delivery devices based on hydrogels, psyllium, if suitably tailored to prepare the hydrogels, can act as the double potential candidates for the novel drug delivery systems. Therefore, it is an attempt to prepared psyllium and acrylic acid based pH sensitive novel hydrogels by using N,N'-methylenebisacrylamide (N,N-MBAAm) as crosslinker and ammonium persulfate (APS) as initiator for the use in colon specific drug delivery. The present paper discusses the swelling kinetics of the hydrogels and release dynamics of model drugs (tetracycline hydrochloride, insulin and tyrosine) from drug-loaded hydrogels, for the evaluation of the swelling mechanism and drug release mechanism from the polymeric networks .The effect of pH on the swelling kinetics and release pattern of drugs have been studied by varying the pH of the release medium. It has been observed that swelling and release of drugs from the hydrogels occurred through non-Fickian or anomalous diffusion mechanism in distilled water and pH 7.4 buffer. It shows that the rate of polymer chain relaxation and the rate of drug diffusion from these hydrogels are comparable.     

Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo

Background: Although piperine can inhibit cells of tumors, the poor water solubility restricted its clinical application. This paper aimed to develop mixed micelles based on Soluplus^® and D-α-tocopherol polyethylene glycol succinate (TPGS) to improve the aqueous solubility and anti-cancer effect.

Methods: Piperine-loaded mixed micelles were prepared using a thin-film hydration method, and their physicochemical properties were characterized. The cellular uptake of the micelles was confirmed by confocal laser scanning microscopy in A549 lung cancer cells and HepG₂ liver cancer cells. In addition, cytotoxicity of the piperine mixed micelles was studied in A549 lung cancer cells and HepG₂ liver cancer cells. Free piperine or piperine-loaded Soluplus^®/TPGS mixed micelles were administered at an equivalent dose of piperine at 3.2?mg/kg via a single intravenous injection in the tail vain for the pharmacokinetic study in vivo.

Results: The diameter of piperine-loaded Soluplus^®/TPGS (4:1) mixed micelles was about 61.9?nm and the zeta potential –1.16?±?1.06?mV with 90.9% of drug encapsulation efficiency and 4.67% of drug-loading efficiency. Differential scanning calorimetry (DSC) studies confirmed that piperine is encapsulated by the Soluplus^®/TPGS. The release results in vitro showed that the piperine-loaded Soluplus^®/TPGS mixed micelles presented sustained release behavior compared to the free piperine. The mixed micelles exhibited better antitumor efficacy compared to free piperine and physical mixture against in A549 and HepG₂ cells by MTT assay. The pharmacokinetic study revealed that the AUC of piperine-loaded mixed micelles was 2.56 times higher than that of piperine and the MRT for piperine-loaded mixed micelles was 1.2-fold higher than piperine (p?Conclusion: The results of the study suggested that the piperine-loaded mixed micelles developed might be a potential nano-drug delivery system for cancer chemotherapy. These results demonstrated that piperine-loaded Soluplus^®/TPGS mixed micelles are an effective strategy to deliver piperine for cancer therapy.     

In vitro evaluation of freeze-dried bone allografts combined with platelet rich plasma and human bone marrow stromal cells for tissue engineering

Freeze-dried bone allograft (FDBA) might be more effective in combination with platelet rich plasma (PRP) and bone marrow stromal cells (BMSC) in accelerating bone healing. The isolation of BMSC through density gradient (pBMSC) is not extensively applicable in clinical practice, because it increases the risk of infection. Alternatively, BMSC can be concentrated by simple centrifugation (wBMSC) directly in the operating room. However, we do not know if wBMSC act in the same way as pBMSC. BMSC from 10 donors were tested whether, in the presence of a combination of FDBA and autologous PRP, the osteogenic differentiation of the cells concentrated by simple centrifugation (wBMSC + FDBA + PRP) was similar to that of pBMSC. Cell-associated alkaline phosphatase, osterix and fibroblast growth factor-2 were higher in wBMSC + FDBA + PRP. In conclusion, the combination of FDBA and PRP had a favouring effect on the differentiation towards osteoblasts and allowed BMSC concentrated by simple centrifugation to differentiate as fast as BMSC purified by density gradient.     

In vitro release of cisplatin from sol–gel processed organically modified silica xerogels

SiO₂, SiO₂/PEG and SiO₂/PDMS xerogels were examined as polymeric carriers for the controlled release of cisplatin—an antineoplasmic medicine. Drug/carrier systems were prepared by the sol–gel method. The effect of organic substitution of the silica xerogel matrix and drying conditions on the release of cisplatin was evaluated. Based on the presented results of the study it may be stated that sol–gel method is useful for entrapping a cisplatin in the pores of organically modified silica gels and for releasing cisplatin mainly in the way of diffusion from the pores of the lattice under the in vitro conditions. The use of organic impurities in silica gel increased the release of cisplatin from xerogel (from 62% to 97% within 7 days), and thermal treatment of all xerogels with cisplatin at the temperature of 80 °C resulted in the acceleration of the drug release (2 days) and increase of the released drug (89–98%).     

Lipid nanoparticles of zaleplon for improved oral delivery by Box–Behnken design: optimization,in vitro and in vivo evaluation

Purpose: Zaleplon (ZL) is a hypnotic drug prescribed for the management of insomnia and convulsions. The oral bioavailability of ZL was low (～30%) owing to poor water solubility and hepatic first-pass metabolism. The cornerstone of this investigation is to develop and optimize solid lipid nanoparticles (SLNs) of ZL with the aid of Box–Behnken design (BBD) to improve the oral bioavailability.

Methods: A design space with three formulation variables at three levels were evaluated in BBD. Amount of lipid (A₁), amount of surfactant (A₂) and concentration of co-surfactant (%) (A₃) were selected as independent variables, whereas, particle size (B₁), entrapment efficiency (B₂) and zeta potential (ZP, B₃) as responses. ZL-SLNs were prepared by hot homogenization with ultrasonication method and evaluated for responses to obtain optimized formulation. Morphology of nanoparticles was observed under SEM. DSC and XRD studies were examined to understand the native crystalline behavior of drug in SLN formulations. Further, in vivo studies were performed in Wistar rats.

Results: The optimized formulation with 132.89?mg of lipid, 106.7?mg of surfactant and 0.2% w/v of co-surfactant ensued in the nanoparticles with 219.9?±?3.7?nm of size, ?25.66?±?2.83?mV surface charge and 86.83?±?2.65% of entrapment efficiency. SEM studies confirmed the spherical shape of SLN formulations. The DSC and XRD studies revealed the transformation of crystalline drug to amorphous form in SLN formulation. In conclusion, in vivo studies in male Wistar rats demonstrated an improvement in the oral bioavailability of ZL from SLN over control ZL suspension.

Conclusions: The enhancement in the oral bioavailability of ZL from SLNs, developed with the aid of BBD, explicated the potential of lipid-based nanoparticles as a potential carrier in improving the oral delivery of this poorly soluble drug.     

In vitro evaluation of compression-coated glycyl-l-histidyl-l-lysine–Cu(II) (GHK–Cu2+)-loaded microparticles for colonic drug delivery

Glycyl-l-histidyl-l-lysine–Cu(II) (GHK–Cu²⁺)-loaded Zn-pectinate microparticles in the form of hydroxypropyl cellulose (HPC) compression-coated tablets were prepared and their in vitro behavior tested. GHK–Cu²⁺ delivery to colon can be useful for the inhibition of matrix metalloproteinase, with the increasing secretion of tissue inhibitors of metalloproteinases (TIMPS),which are the major factors contributing in mucosal ulceration and inflammation in inflammatory bowel disease. The concentration of peptide was determined spectrophotometrically. The results obtained implied that surfactant ratio had a significant effect on percent production yield (1.25 to 1.75 w/w; 72.22% to 80.84%), but cross-linking agent concentration had not. The entrapment efficiency (EE) was found to be in the range of 58.25–78.37%. The drug-loading factor significantly increased the EE; however, enhancement of cross-linking agent concentration decreased it. The release of GHK–Cu²⁺ from Zn-pectinate microparticles (F1–F8) in simulated intestinal fluid was strongly affected by cross-linking agent concentration and drug amount (50?mg for F1–F6; 250?mg for F7–F8), but not particularly affected by surfactant amount. Release profiles represented that the microparticles released 50–80% their drug load within 4?h. Therefore, the optimum microparticle formulation (F8) coated with a relatively hydrophobic polymer HPC to get a suitable colonic delivery system. The optimum colonic delivery tablets prepared with 700?mg HPC-SL provided the expected delayed release with a lag time of 6?h. The effects of polymer viscosity and coat weight on GHK–Cu²⁺ release were found to be crucial for the optimum delay of lag time. The invention was found to be promising for colonic delivery.     

A simple and efficient electrochemical sensor for folic acid determination in human blood plasma based on gold nanoparticles–modified carbon paste electrode

Folic acid (FA) is a water soluble vitamin that exists in many natural species. The lack of FA causes some deficiencies in human body, so finding a simple and sensitive method for determining the FA is important. A new chemically modified electrode was fabricated for determination of FA in human blood plasma using gold nanoparticles (AuNPs) and carbon paste electrode (CPE). Gold nanoparticles–modified carbon paste electrode (AuNPs/CPE) was characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The experimental parameters such as pH, scan rate (ν) and amount of modifier were studied by cyclic voltammetry and the optimized values were chosen. The electrochemical parameters such as diffusion coefficient of FA (D_FA), electrode surface area (A) and electron transfer coefficient (α) were calculated. Square wave voltammetry as an accurate technique was used for quantitative calculations. A good linear relation was observed between anodic peak current (i_pa) and FA concentration (C_FA) in the range of 6 × 10^? 8 to 8 × 10^? 5 mol L^? 1, and the detection limit (LOD) achieved 2.7 × 10^? 8 mol L^? 1, that is comparable with recently studies. This paper demonstrated a novel, simple, selective and rapid sensor for determining the FA in the biological samples.     

In vivo evaluation of titanium implants coated with bioactive glass by pulsed laser deposition

During the past years, different techniques, like chemical treatment, plasma spraying, sputtering, enamelling or sol–gel; and materials, like metals, hydroxylapatite, calcium phosphates, among others, have been applied in different combinations to improve the performance of prostheses. Among the techniques, Pulsed Laser Deposition (PLD) is very promising to produce coatings of bioactive glass on any metal alloy used as implant. In this work the biocompatibility of PLD coatings deposited on titanium substrates was examined by implantation in vivo. Different coating compositions were checked to find the most bioactive that was then applied on titanium and implanted into paravertebral muscle of rabbit.     

Development and in vitro–in vivo evaluation of a water-in-oil microemulsion formulation for the oral delivery of troxerutin

Objective: The main objective of this study was to develop and evaluate a W/O microemulsion formulation of troxerutin to improve its oral bioavailability.

Methods: The W/O microemulsion was optimized using a pseudo-ternary phase diagram and evaluated for physical properties. In vitro MDCK cell permeability studies were carried out to evaluate the permeability enhancement effect of microemulsion, and in vivo absorption of troxerutin microemulsion in the intestine was compared with that of solution after single-dose administration (56.7?mg/kg) in male Wistar rats.

Results: The optimal formulation consisted of lecithin, ethanol, isopropyl myristate and water (23.30/11.67/52.45/12.59 w/w) was physicochemical stable and the mean droplet size was about 50.20?nm. In vitro study, the troxerutin-loaded microemulsion showed higher intestinal membrane permeability across MDCK monolayer when compared with the control solution. The W/O microemulsion can significantly promote the intestinal absorption of troxerutin in rats in vivo, and the relative bioavailability of the microemulsion was about 205.55% compared to control solution.

Conclusion: These results suggest that novel W/O microemulsion could be used as an effective formulation for improving the oral bioavailability of troxerutin.     

Functionalised gold nanoparticles for selective induction of in vitro apoptosis among human cancer cell lines

The interaction of citrate- and polyethylene imine (PEI)-functionalised gold nanoparticles (GNP) with cancer cell lines with respect to the cellular response was studied. It was found that GNP/citrate nanoparticles were able to induce apoptosis in human carcinoma lung cell lines A549, but GNP/PEI did not show any reduction in the viability of the cells in human breast cancer cell line MCF-7 and A549 cell lines. FACS data confirmed that the number of apoptotic cells increased with increase in the concentration of GNP/citrate nanoparticles. Decline in cellular expansion and changes in the nuclear morphology were noted after the treatment of GNP/citrate nanoparticles on A549 cell lines, which itself is a direct response for stress induction. The induction of cellular apoptosis was further confirmed by DNA fragmentation assay. These data confirm the potential of GNP/citrate nanoparticle to evoke cell-specific death response in the A549 cell lines.