Synthesis and characterization of gelatin nanoparticles using CDI/NHS as a non-toxic cross-linking system

Gelatin nanoparticles, cross-linked by a mixture of a water soluble carbodiimide (CDI) and N-hydroxysuccinimide (NHS) as a non-toxic cross-linking system, was prepared. The conventional two step desolvation method with acetone as the non-solvent was used. The mean size and size distribution as well as the morphology of the formed nanoparticles were evaluated and compared with those of nanoparticles cross-linked by glutaraldehyde (GA) as the most commonly used cross-linking agent. Furthermore, intrinsic viscosities of the nanoparticles cross-linked by CDI/NHS and GA were measured and compared under various conditions. The results showed the formation of smoother and more homogeneous nanoparticles with smaller size when CDI/NHS used as cross-linking agent under the same synthesis condition. Moreover, nanoparticles encapsulating paracetamol as a model drug were produced by the two different cross-linking agents and were characterized for drug entrapment and loading efficiencies and in vitro drug release. Both drug entrapment and loading efficiencies was higher in the CDI/NHS cross-linked nanoparticles; however, the release kinetics was comparable to that of nanoparticles cross-linked with GA. The differences in the characteristics of CDI/NHS and GA cross-linked nanoparticles were attributed to the different nature of network structures formed by the two cross-linking agents. On the whole, these results suggested that CDI/NHS cross-linked nanoparticles have high potential to be used for drug delivery application in preference to the nanoparticles synthesized by toxic cross-linking agents.     

磁性壳聚糖-5-氟尿嘧啶纳米粒的制备及体外释药性能

采用交联-聚合法在超声波的作用下,制备了磁性壳聚糖-5-氟尿嘧啶纳米粒(M CN-Fu)。透射电子显微镜(TEM)和红外光谱(IR)等分析结果表明,M CN-Fu粒子外形规整,分散性好,粒径主要在50 nm~60 nm之间。紫外-可见光谱分析结果表明,M CN-Fu的载药量为21.3%,在磷酸盐缓冲溶液(pH=7.2)中,30 h的累积释药率为67.6%,具有良好缓释性能,并具有良好磁响应性能。     

Gelatin nanoparticles produced by a simple W/O emulsion as delivery system for methotrexate

Biodegradable hydrophilic gelatin nanoparticles, containing different initial amounts of methotrexate (MTX), were prepared using a simple solvent evaporation technique based on a single water-in-oil emulsion and stabilized by the use of glutaraldehyde as cross-linking agent. The effects of several parameters on particle size, drug encapsulation efficiency and drug release were investigated. Size and shape of the nanoparticles were examined by scanning electron microscopy. The release of MTX was monitored in vitro and the mechanism of release was studied. Particles with a mean diameter of 100–200 nm were produced, which were able to release MTX following a diffusion-controlled mechanism of release. It was observed that the initial amount of MTX used for sample loading did not have any effect on the pattern of release, while it affected the amount of drug entrapped into the nanoparticles and also both the release rate and the total amount of drug released.     

Coating of polyurethane scaffolds with collagen: comparison of coating and cross-linking techniques

Collagen has been coated successfully onto numerous hydrophilic polymer scaffolds to improve cell adhesion. Due to the hydrophobic nature of thermoplastic polyurethane (TPU), coating with aqueous collagen solution is problematic for such scaffolds. This study facilitated the coating of TPU with collagen and compared cross-linking and coating techniques. Three different cross-linking methods were compared. Both thermal and glutaraldehyde methods showed proof of cross-linking; however glutaraldehyde seemed to be superior to the other methods. The use of human urine as a wetting agent and the chemical glutaraldehyde had no effect on a cytotoxicity test performed by means of a WST-1 assay with a fibroblastic cell line. Three different coating techniques for porous TPU scaffolds were also investigated: ultrasound, pressurized air and injection. Of these, injection performed best. This method facilitated a coating of 100% of the porous scaffolds examined, which was verified by staining, FTIR and SEM.     

Preparation and optimization of doxorubicin-loaded albumin nanoparticles using response surface methodology

Background: The objective of this work was to optimize the preparation of doxorubicin-loaded albumin nanoparticles (Dox-A-Nps) through desolvation procedures using response surface methodology (RSM). A central composite design (CCD) for four factors at five levels was used in this study.

Method: Albumin nanoparticles were prepared through a desolvation method and were optimized in the aid of CCD. Albumin concentration, amount of doxorubicin, pH values, and percentage of glutaraldehyde were selected as independent variables, particle size, zeta potential, drug loading, encapsulation efficiency, and nanoparticles yield were chosen as response variables. RSM and multiple response optimizations utilizing a quadratic polynomial equation were used to obtain an optimal formulation.

Results: The optimal formulation for Dox-A-Nps was composed of albumin concentration of 17?mg/ml, amount of doxorubicin of 2?mg/ml, pH value is 9 and percentage of glutaraldehyde of 125% of the theoretic amount, under which the optimized conditions gave rise to the actual average value of mean particle size (151?±?0.43?nm), zeta potential (?18.8?±?0.21 mV), drug loading efficiency (21.4?±?0.70%), drug entrapment efficiency (76.9?±?0.21%) and nanoparticles yield (82.0?±?0.34%). The storage stability experiments proved that Dox-A-Nps stable in 4°C over the period of 4 months. The in vitro experiments showed a burst release at the initial stage and followed by a prolonged release of Dox from albumin nanoparticles up to 60?h.

Conclusions: This study showed that the RSM-CCD method could efficiently be applied for the modeling of nanoparticles, which laid the foundation of the further research of immuno nanoparticles.     

Method of fabrication of submicron composite microparticles of hydroxyapatite and ferromagnetic nanoparticles for a protein drug carrier

Composite microparticles of hydroxyapatite (HAp) and ferromagnetic nanoparticles were prepared using algenic acid gel cross-linked by bivalent calcium ions. The resultant size of the composite microparticle could be reduced to the submicron scale using extremely vigorous stirring by a homogenizer at 16 000 r.p.m. The instantaneous gellation induced by the bivalent ion cross-linking was revealed to be advantageous for combining the constituent nanoparticles of HAp and ferromagnetic nanoparticles. Since the structural arrest occurs instantly at the moment of cross-linking by the bivalent calcium ion, the added constituent HAp/ferromagnetic nanoparticles do not have time to be segregated apart from the binding gel; consequently, the submicron-scale composite state of these constituents could be instantaneously immobilized as in the dispersed state in liquid by the applied high shear. Thus, the instantaneous gellation characteristic of the bivalent ion cross-linking is preferable for incorporating different nanoparticles. The retention ability of the test protein (cytochrome c) was examined using the bincinchoninic assay method. Comparison of the protein retention ability with the control sample without HAp nanoparticles revealed that the incorporated HAp predominantly contributes to the retention ability of cytochrome c.     

Microstructure and characteristic properties of gelatin/chitosan scaffold prepared by a combined freeze-drying/leaching method

A combined freeze-drying and particulate leaching method for scaffold synthesis showed an improvement in the horizontal microstructure of the gelatin/chitosan scaffolds. Type and concentration of the cross-linking agent, freezing temperature, concentration of the polymeric solution and gelatin/chitosan weight ratio were the variables affecting the scaffold properties. Assessment of the tensile properties of the scaffolds revealed that for a scaffold with 50% chitosan, glutaraldehyde, as a cross-linking agent, created much tighter polymeric network compared to N,N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDC). However, in the case of gelatin scaffolds, EDC was identified as the stronger cross-linker. Compressive behavior of the scaffolds satisfied formulations obtained from the theoretical modeling of the low-density, elastomeric foams. The investigation of the scaffold degradation indicated that the increase in the mechanical strength of the scaffolds would not always reduce their degradation rate.     

Design and evaluation of D-α tocopheryl polyethylene glycol 1000 succinate emulsified poly-ϵ-caprolactone nanoparticles for protein/peptide drug delivery

Background: Incorporation of proteins/peptide drugs into nanoparticulate drug delivery system is one of the effective approaches to increase the stability of protein/peptide drugs against enzymatic degradation, to release them in a controlled fashion and to achieve site-specific drug delivery.

Objective: Our goal was to design and evaluate poly-?-caprolactone (PCL) nanoparticles using bovine serum albumin (BSA) as a model protein. d-α-tocopheryl polyethylene glycol 1000 (vitamin E TPGS) was used as an emulsifier in the fabrication of these nanoparticles.

Methods: Double emulsion solvent evaporation method was employed to formulate BSA-loaded PCL nanoparticles and the nanoparticles thus prepared were further characterized.

Results: The size of BSA-loaded PCL nanoparticles were in the range of 400–500?nm with a polydispersity index (PDI) of 0.195 and zeta potential was about ?28.6 mV. Scanning electron microscopy (SEM) confirmed the presence of smooth and spherical surface of nanoparticles. Encapsulation efficiency was about 85% and a yield of 70–75% was attained. BSA was released in a biphasic pattern with an initial 20% release within 2?h followed by a slower release patter over 5 days. Flow cytometry and fluorescence microscopy was used to study the uptake of these nanoparticles. Circular dichroism (CD) results showed that there was no significant effect of formulation conditions on the secondary structure of BSA.

Conclusion: Based on the results obtained, these TPGS-emulsified PCL nanoparticles proved to be potential carriers for the delivery of protein/peptide drugs.     

戊二醛改性壳聚糖/ 纳米TiO2 复合膜的抗水性研究

采用戊二醛作为交联剂与壳聚糖进行交联,以提高壳聚糖/ 纳米TiO2 膜的抗水性。采用三元二次通用旋转组合设计实验方法,建立了力学性能、吸水率与戊二醛用量、交联时间、TiO2 溶胶添加量的二次回归数学模型,得出了制备戊二醛改性壳聚糖/ 纳米TiO2 膜的最佳工艺条件为交联剂用量0. 10 g/ (100 mL)乙酸溶液,交联时间4. 37 h,TiO2 溶胶添加量0. 08 mL/ (100 mL)的乙酸溶液。上述条件下得到的膜的拉伸强度为5. 69MPa,吸水率为133. 66%。与壳聚糖/ 纳米TiO2 膜相比,吸水率下降了72. 8%。     

Protein crystals as scanned probes for recognition atomic force microscopy

Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.     

Preparation of cross-linked sodium alginate microparticles using glutaraldehyde in methanol

Polymeric sodium alginate microparticles were prepared by precipitating sodium alginate in methanol, followed by cross-linking with glutaraldehyde. The extent of cross-linking was controlled by the time of exposure to glutaraldehyde. The topology of microparticles was characterized by scanning electron microscopy (SEM), which indicated smooth surfaces. The equilibrium swelling experiments were carried out in water to observe the effect of cross-linking and drug loading for better utility of microparticles. It was found that swelling decreased, but drug loading increased, with an increase in cross-linking of the matrix.     

Preparation of Cross-Linked Sodium Alginate Microparticles Using Glutaraldehyde in Methanol

Polymeric sodium alginate microparticles were prepared by precipitating sodium alginate in methanol, followed by cross-linking with glutaraldehyde. The extent of cross-linking was controlled by the time of exposure to glutaraldehyde. The topology of microparticles was characterized by scanning electron microscopy (SEM), which indicated smooth surfaces. The equilibrium swelling experiments were carried out in water to observe the effect of cross-linking and drug loading for better utility of microparticles. It was found that swelling decreased, but drug loading increased, with an increase in cross-linking of the matrix.     

Sustained release of methotrexate through liquid-crystalline folate nanoparticles

To make chemotherapy more effective, sustained release of the drug is desirable. By controlling the release rates, constant therapeutic levels can be achieved which can avoid re-administration of drug. This helps to combat tumors more effectively with minimal side effects. The present study reports the control release of methotrexate through liquid-crystalline folate nanoparticles. These nanoparticles are composed of highly ordered folate self-assembly which encapsulate methotrexate molecules. These drug molecules can be released in a controlled manner by disrupting this assembly in the environment of monovalent cations. The ordered structure of folate nanoparticles offers low drug losses of about 4–5 %, which is significant in itself. This study reports the size-control method of forming methotrexate encapsulated folate nanoparticles as well as the release of methotrexate through these nanoparticles. It has been demonstrated that methotrexate release rates can be controlled by controlling the size of the nanoparticles, cross-linking cation and cross-linking concentration. The effect of different factors like drug loading, release medium, and pH of the medium on methotrexate release rates was also studied.     

The Production and Evaluation of Orally Administered Insulin Nanoparticles

In an attempt to overcome the proteolytic degradation and poor absorption of orally administered insulin, nanoparticles were made from insulin. These 200 nm particles were made from a Neutral insulin Injection (Actrapid, Novo) by desolvation and crosslinking with glutaraldehyde. The purified nanoparticles were found to be absorbed from the intestinal tract of mice as well as normal and diabetic rats. The blood glucose concentrations in some animals could be reduced to about 15 to 20? of the starting level, 3 hours after administration of between 35 and 70 mg of nanoparticles per 100 g body weight. The nanoparticles appear to exert a slower but more pronounced response than similarly administered Actrapid although no dose response relationship was established. The doses of nanoparticles needed preclude the development of a commercially viable product.     

Insulin-loaded nanoparticles are prepared by alginate ionotropic pre-gelation followed by chitosan polyelectrolyte complexation

Alginate nanoparticles were prepared from dilute alginate sol by inducing a pre-gel with calcium counter ions, followed by polyelectrolyte complex coating with chitosan. Particles in the nanometer size range were obtained with 0.05% alginate and 0.9 mM Ca2+. The mean particle size was influenced by time and stirring speed of nanoparticle preparation, by alginate guluronic acid content and chitosan molecular weight and by the initial alginate:chitosan mass ratio. The association efficiency of insulin into alginate nanoparticles, as well as loading capacity were mainly influenced by the alginate:chitosan mass ratio. Under optimized size conditions, the association efficiency and loading capacities were as high as 92% and 14.3%, respectively. Approximately 50% of the protein was partially retained by the nanoparticles in gastric pH environment up to 24 hours while a more extensive release close to 75% was observed under intestinal pH conditions. Mild formulation conditions, optimum particle size range obtained, high insulin entrapment efficiency, and resistance to gastrointestinal release seem to be synergic and promising factors toward development of an oral insulin delivery form.     

Template-synthesized protein nanotubes

A layer-by-layer deposition strategy for preparing protein nanotubes within the pores of a nanopore alumina template membrane is described. This method entails alternately exposing the template membrane to a solution of the desired protein and then to a solution of glutaraldehyde, which acts as cross-linking agent to hold the protein layers together. The number of layers of protein that make up the nanotube walls can be controlled at will by varying the number of alternate protein/glutaraldehyde cycles. After the desired number of layers have been deposited on the pore walls, the alumina template can be dissolved to liberate the protein nanotubes. We show here that glucose oxidase nanotubes prepared in this way catalyze glucose oxidation and that hemoglobin nanotubes retain their heme electroactivity. Furthermore, for the glucose oxidase nanotubes, the enzymatic activity increases with the nanotube wall thickness.     

Sonochemical preparation of TiO2 nanoparticles

TiO₂ nanoparticles with mean diameter of about 20 nm, average crystallite size of 15 nm and BET specific surface area of 78.88 m²/g, were prepared through a simple sonochemical method and initial treatment in 10 M NaOH aqueous solution. XRD results exhibited that the obtained nanoparticles composed of pure rutile phase. During the initial treatment with NaOH aqueous solution, the Ti–O–Ti bonds in the TiO₆ octahedra structure of the raw material were broken, and new octahedras formed after ultrasonic irradiation. It is supposed that edge sharing was favored during ultrasonic treatment, leading to formation of the rutile nanoparticles.     

Superparamagnetism of Cu2Se nanoparticles

Cu2Se nanoparticles were synthesized using the standard Schlenk line and glove box techniques, with the hot-injection method. The X-ray diffraction (XRD) analysis showed that the initial nanoparticles were formed in a stoichiometric Cu2Se phase with a cubic structure. When the nanoparticles are exposed to air, the diffraction peaks shift to higher angles. This suggests that the nanoparticles are changed to a nonstoichiometric Cu2-deltaSe phase with copper vacancies. The mean size of the nanoparticles was about 7 nm. The magnetic results show that the initial nanoparticles were diamagnetic, and after 1-week air exposure, they became paramagnetic. This dramatic change from diamagnetic to paramagnetic can be explained by the oxidation of Cu+ into Cu2+ at the nanoparticle surface. In addition, the superparamagnetic properties were observed to have a blocking temperature of 150 K. The coercive field decreased as the temperature approached the blocking temperature, and eventually vanished above the blocking temperature.     

Key parameters affecting the initial leaky effect of hemoglobin-loaded nanoparticles as blood substitutes

In order to realize long-term carrying/delivering oxygen and minimize the adverse effects of free hemoglobin (Hb) in vivo, Hb is desired to be confined in Hb-loaded nanoparticles (HbP), a novel blood substitute with potential clinical applications, and thus functions as the native red blood cells (RBCs). However, the initial burst release of Hb (“leaky effect”) greatly underscores the significance of this work. The study described here wants to disclose the key preparative parameters, including polymer, excipients in the inner aqueous phase and solvent profile, affecting the Hb release behavior (the initial 24 h) from HbP fabricated by commonly used solvent diffusion/evaporation double emulsion technique. The results demonstrate that PEGlytated polymers, regardless of two- or tri-block copolymers show slower release compared with the corresponding non-PEGlytated ones. The higher polymer concentration yields lower initial release. PEG200, added as excipient facilitates Hb burst effect to about 38.4%, almost 17% increase compared to the control (∼21%), whereas, PVA and Poloxamer188, due to amphiphilic nature, can effectively attenuate this leakage to about 13.0 and 5.1%, respectively. The diffusion/extraction rate from oil phase and the subsequent evaporation rate from the aqueous continuous phase of solvents impose different influences on Hb release. To reduce the burst effect, the initial diffusion/extraction rate should be slow, whereas, the concomitant evaporation rate should be as fast as possible. The results obtained here will be guidance’s for the future tailored design of more desirable polymersome nanoparticle blood substitutes.     

Development of a novel vaccine delivery system based on Gantrez nanoparticles

The adjuvant capacity of a novel vaccine vector "Gantrez-nanoparticles" (NP) towards coated or encapsulated ovalbumin (OVA) was investigated. OVA nanoparticles were prepared by a solvent displacement method previously described. The protein was incorporated during the manufacturing process (OVA-encapsulated nanoparticles) or after the preparation (OVA-coated nanoparticles). The mean size of the different nanoparticle formulations was lower than 300 nm, and the OVA content ranged approximately from 67 microg/mg nanoparticles (for OVA-coated nanoparticles) to 30 microg/mg nanoparticles (for OVA-encapsulated nanoparticles). All the OVA-NP formulations were capable of amplifying the antibodies titres (IgG1 and IgG2a) in mice after a single subcutaneous inoculation with respect free OVA or OVA adsorbed to Alum. Furthermore, the elicited response was, for some formulations, predominantly Th1 subtype. Thus, the formulation that contained mainly the antigen inside, and with a low concentration of cross-linking agent, displayed the best potential to induce a Th1 response after 35 days post-immunisation. These results are highly suggestive for the use of Gantrez nanoparticles as an efficient antigen delivery system, especially when a long lasting Th1 cytokine response is required.