Chitosan nanoparticle as protein delivery carrier--systematic examination of fabrication conditions for efficient loading and release

Chitosan nanoparticles fabricated via different preparation protocols have been in recent years widely studied as carriers for therapeutic proteins and genes with varying degree of effectiveness and drawbacks. This work seeks to further explore the polyionic coacervation fabrication process, and associated processing conditions under which protein encapsulation and subsequent release can be systematically and predictably manipulated so as to obtain desired effectiveness. BSA was used as a model protein which was encapsulated by either incorporation or incubation method, using the polyanion tripolyphosphate (TPP) as the coacervation crosslink agent to form chitosan-BSA-TPP nanoparticles. The BSA-loaded chitosan-TPP nanoparticles were characterized for particle size, morphology, zeta potential, BSA encapsulation efficiency, and subsequent release kinetics, which were found predominantly dependent on the factors of chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The BSA loaded nanoparticles prepared under varying conditions were in the size range of 200-580nm, and exhibit a high positive zeta potential. Detailed sequential time frame TEM imaging of morphological change of the BSA loaded particles showed a swelling and particle degradation process. Initial burst released due to surface protein desorption and diffusion from sublayers did not relate directly to change of particle size and shape, which was eminently apparent only after 6h. It is also notable that later stage particle degradation and disintegration did not yield a substantial follow-on release, as the remaining protein molecules, with adaptable 3-D conformation, could be tightly bound and entangled with the cationic chitosan chains. In general, this study demonstrated that the polyionic coacervation process for fabricating protein loaded chitosan nanoparticles offers simple preparation conditions and a clear processing window for manipulation of physiochemical properties of the nanoparticles (e.g., size and surface charge), which can be conditioned to exert control over protein encapsulation efficiency and subsequent release profile. The weakness of the chitosan nanoparticle system lies typically with difficulties in controlling initial burst effect in releasing large quantities of protein molecules.     

Interaction Between Liposomes and Neutral Polymers: Effect of Adsorption on Drug Release

The processes of adsorption of two neutral polymers (poly(vinyl pyrrolidone), PVP and poly(vinyl alcohol), PVA) were investigated on liposomes composed of soy lecithin/dicetyl phosphate/cholesterol = 25:2:3 (molar ratio). The liposomes were prepared in buffered solution at pH = 7.4 and mixed with the solution of the measured polymers in the desired polymer/lipid (w/w) ratios. Adsorption was measured by determination of the equilibrium bulk concentration of the polymer. In the case of PVA quantitative adsorption measurements with a specific reagent were possible. Adsorption isotherms were recorded at 25 ± 1°C. It was concluded that adsorbed and unadsorbed PVA molecules are in equilibrium even at low polymer/ lipid ratios. The results were confirmed by dynamic laser light scattering (DLS), and thermal activity monitoring (TAM) experiments. Another group of the liposomes was prepared in 60 mM ammonium sulphate (pH = 5.0) and we filled the vesicles with a test dye, acridine orange (AO) using the pH-gradient (remote loading) method. The AO release property of liposomes was tested with a special vertical diffusion cell after we had made PVA adsorb on their surface in different PVA/lipid (w/w) ratios.     

Liposomes‐Camouflaged Redox‐Responsive Nanogels to Resolve the Dilemma between Extracellular Stability and Intracellular Drug Release

Liposomes have shown great promises for pharmaceutical applications, but still suffer from the poor storage stability, undesirable drug leakage, and uncontrolled drug release. Herein, liposomes‐camouflaged redox‐responsive nanogels platform (denoted as “R‐lipogels”) is prepared to integrate the desirable features of sensitive nanogels into liposomes to circumvent their intrinsic issues. The results indicate that drug‐loaded R‐lipogels with controlled size and high stability not only can achieve a very high doxorubicin (DOX)‐loading capacity (12.9%) and encapsulation efficiency (97.3%) by ammonium sulfate gradient method and very low premature leakage at physiological condition, but also can quickly release DOX in the reducing microenvironment of tumor cells, resulting in effective growth inhibition of tumor cells. In summary, the strategy given here provides a facile approach to develop liposomes–nanogels hybrid system with combined beneficial features of stealthy liposomes and responsive nanogels, which potentially resolves the dilemma between systemic stability and intracellular rapid drug release.     

Elucidation of the Mechanism of Enhanced Insulin Uptake and Release from pH Responsive Hydrogels

Summary: Environmentally responsive hydrogels composed of poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) have shown promise for oral insulin delivery due to their pH responsive complexation behavior. A series of hydrogel formulations were polymerized with varying amounts of crosslinker and varying monomer volume fraction. The mesh size of the network depended primarily on pH, varying from 8.0 to 27.2 nm. Insulin loading efficiency varied directly with crosslink density, ranging from 42.7 to 84.9% of available insulin loaded into the hydrogels. The release of insulin was performed with each polymer formulation at 5 pH levels ranging from 2.7 to 6.8. Insulin release was less than 20% for all formulations tested with insulin for the duration of the 3 hour release study for all pH levels considered except when the pH was 6.8, at which point the release occurred as a burst. Loading studies performed with insulin glargine, an insulin analog with an increased pI, showed the same trends as native insulin. However, the release of insulin glargine only occurred at a pH level above that of the pI of the protein. These results indicate that hydrogen bonds and ionic interactions between the protein and P(MAA-g-EG) may strongly influence its loading and release behavior in vitro.     

Controlled loading of building blocks into temporary self-assembled scaffolds for directed assembly of organic nanostructures

Using temporary self-assembled scaffolds to preorganize building blocks is a potentially powerful method for the synthesis of organic nanostructures with programmed shapes. We examined the underlying phenomena governing the loading of hydrophobic monomers into lipid bilayer interior and demonstrated successful control of the amount and ratio of loaded monomers. When excess styrene derivatives or acrylates were added to the aqueous solution of unilamellar liposomes made from saturated phospholipids, most loading occurs within the first few hours. Dynamic light scattering and transmission electron microscopy revealed no evidence of aggregation caused by monomers. Bilayers appeared to have a certain capacity for accommodating monomers. The total volume of loaded monomers is independent of monomer structure. X-ray scattering showed the increase in bilayer thickness consistent with loading monomers into bilayer interior. Loading kinetics is inversely proportional to the hydrophobicity and size of monomers. Loading and extraction kinetic data suggest that crossing the polar heads region is the rate limiting step. Consideration of loading kinetics and multiple equilibria are important for achieving reproducible monomer loading. The total amount of monomers loaded into the bilayer can be controlled by the loading time or length of hydrophobic lipid tails. The ratio of loaded monomers can be varied by changing the ratio of monomers used for loading or by the time-controlled replacement of a preloaded monomer. Understanding and controlling the loading of monomers into bilayers contributes to the directed assembly of organic nanostructures.     

Thermodynamic Studies of Insulin Loading into a Glucose Responsive Hydrogel Based on Chitosan‐polyacrylamide‐polyethylene Glycol

The insulin therapy constitutes the preferred treatment for Diabetes Mellitus (DM). The traditional insulin therapy, which consists of daily subcutaneous insulin injections to control blood glucose level, is not able to regulate the blood glucose level precisely. In this research, to facilitate the diabetic patient life, an intelligent drug delivery system based on a biodegrable biopolymer to control the insulin release, was designed. In this system, chitosan‐polyethylene glycol hydrogel and glucose oxidize play the role of drug carrier and glucose biosensor, respectively. To increase the hydrogel drug loading capacity, hydrogels with different PEG content were synthesized and insulin was loaded by swelling‐diffusion method into them. The loaded hydrogels were characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), High performance liquid chromatography (HPLC), and Thermogravimetric analysis (TGA). Finally, the thermodynamic study for insulin loading process was performed to investigate the stability of the drug in the system.     

Calcium Phosphate Nanocarriers Dual‐Loaded with Bovine Serum Albumin and Ibuprofen: Facile Synthesis,Sequential Drug Loading and Sustained Drug Release

A simple and green strategy is reported for the preparation, drug loading, and release properties of a drug delivery system consisting of calcium phosphate (CP) nanocarriers dual‐loaded with bovine serum albumin (BSA) and hydrophobic drug ibuprofen (IBU). The sequential loading of BSA and IBU in calcium phosphate nanocarriers and in vitro simultaneous release of BSA and IBU are realized and investigated. In this method, BSA, which is used as a model protein drug, is encapsulated in situ in calcium phosphate nanocarriers. Subsequently, the typical hydrophobic drug IBU is loaded in the BSA/CP drug delivery system, forming the IBU/BSA/CP dual drug delivery system. The experiments reveal that the preloaded BSA not only reduces the cytotoxicity of calcium phosphate nanocarriers but also significantly improves the IBU drug loading capacity in calcium phosphate nanocarriers and greatly extends the duration of drug release. Thus, the as‐prepared IBU/BSA/CP dual drug delivery system is promising for drug delivery applications.     

Permeability of insulin entrapped in liposome through the nasal mucosa of rabbits.

The permeability of liposome entrapping insulin through the nasal mucosa of rabbit has been studied and compared with the permeability of insulin solution with or without pretreatment by sodium glycocholate (GC). Insulin entrapped in liposome was not detected in the receiver cell using the diffusion cells with the nasal mucosa. On the other hand, permeability of insulin entrapped in liposome increased after the pretreatment of GC. The phospholipids which result from liposomes, were not observed in the receiver. Also, the GC remaining in the nasal mucosa was measured. Considering the mechanism of permeation of insulin entrapped in liposome through the nasal mucosa, the GC remaining in the nasal mucosa may cause the lysis of liposomes.     

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity.

An open-column layer-by-layer photo-immobilised pH gradient was introduced into two-dimensional chip electrophoresis with simplicity, reusability, improved separation performance and separation capacity.

     

Effect of formulation variables on entrapment efficiency and release characteristics of bovine serum albumin from carboxymethyl xanthan microparticles

Xanthan gum was derivatized to sodium carboxymethyl xanthan (SCMX) gum with a view to prepare bovine serum albumin (BSA)‐loaded carboxymethyl xanthan (CMX) microparticles through interaction with metal ion in a completely aqueous environment. The effect of various formulation variables, such as a pH of SCMX gum solution, concentration of BSA and SCMX gum, and gelation time on BSA entrapment efficiency and release of the protein in different media were studied. While BSA entrapment efficiency was found to decrease with increase in gelation time and initial BSA loading, the same was found to increase with increase in concentration of SCMX gum. Although the release of BSA in acidic medium was almost equal to that in alkaline medium, as compared up to 2 hr, the release in alkaline medium was found to be prolonged to a different extent depending upon the formulation variables. Copyright © 2008 John Wiley & Sons, Ltd.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.     

PLGA/TiO2纳米药物缓释载体的研究

首先利用硅烷偶联剂(KH550)对纳米二氧化钛表面进行预处理,得到氨基改性的二氧化钛,然后与带有高活性端基的丙交酯-乙交酯共聚物(PLGA)反应,制备纳米药物缓释载体PLGA/TiO2有机-无机杂化材料.通过核磁(1H-NMR)、傅里叶变换红外光谱仪(FTIR)、热重分析(TGA)、扫描电子显微镜(SEM)、透射电子显...     

Interaction of Liposomal Formulations of Meta-tetra(hydroxyphenyl)chlorin (Temoporfin) with Serum Proteins: Protein Binding and Liposome Destruction

mTHPC is a non polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy; however, neither was reported before. We studied drug binding to human serum proteins using size‐exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip^®) and PEGylated (Fospeg^®) liposomes does not affect equilibrium serum protein binding compared with solvent‐based mTHPC. At short incubation times the redistribution of mTHPC from Foslip^® and Fospeg^® proceeds by both drug release and liposomes destruction. At longer incubation times, the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared with conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long‐circulation times the pharmacokinetic behavior of Fospeg^® could be influenced by a combination of protein‐ and liposome‐bound drug. The study highlights the modes of interaction of photosensitizer‐loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in preclinical models, as well as the novel application of NTA to assess the destruction of liposomes.     

Ultrasound-Triggered Delivery of Iproplatin from Microbubble-Conjugated Liposomes

The Pt^IV prodrug iproplatin has been actively loaded into liposomes using a calcium acetate gradient, achieving a 3-fold enhancement in drug concentration compared to passive loading strategies. A strain-promoted cycloaddition reaction (azide- dibenzocyclooctyne) was used to attach iproplatin-loaded liposomes L(Pt) to gas-filled microbubbles (M), forming an ultrasound-responsive drug delivery vehicle [M−L(Pt)]. Ultrasound-triggered release of iproplatin from the microbubble-liposome construct was evaluated in cellulo. Breast cancer (MCF-7) cells treated with both free iproplatin and iproplatin-loaded liposome−microbubbles [M−L(Pt)] demonstrated an increase in platinum concentration when exposed to ultrasound. No appreciable platinum uptake was observed in MCF-7 cells following treatment with L(Pt) only or L(Pt)+ultrasound, suggesting that microbubble-mediated ultrasonic release of platinum-based drugs from liposomal carriers enables greater control over drug delivery.     

胰岛素反相制备色谱的方法开发

以胰岛素反相制备色谱方法的开发和优化为目标,通过考察色谱保留参数、峰展宽及样品流出曲线的浓度分布等色谱参数,对流动相梯度、色谱填料、载样量等色谱条件进行了优化,并建立了胰岛素制备色谱峰参数的描述方法。结果表明,所建立的方法可快速筛选出最适于胰岛素分离的色谱条件(包括流动相梯度及分离填料),即流动相中的强洗脱溶剂(有机相)需采取缓梯度窄区间的变化条件,筛选出的分离填料需具备峰向两侧展宽且展宽程度较小、样品最高浓度居中分布的特点。将方法用于实际胰岛素粗品的纯化制备,获得了杂质去除效果好、胰岛素纯度高的产品。该法为胰岛素反相色谱纯化制备方法的快速建立提供了指导,具有较强的实用价值,同时为发展大分子化合物的制备色谱方法提供了参考。     

Loading of gold nanoparticles inside the DPPC bilayers of liposome and their effects on membrane fluidities

Gold nanoparticles were loaded in the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposomes, named as gold-loaded liposomes. Above the gel to liquid-crystalline phase transition temperature, membrane fluidities of DPPC liposomes were changed by loading the gold nanoparticles. Compared with liposomes without loading the gold nanoparticles, gold-loaded liposomes showed the lower fluorescence anisotropy values. That is, the membrane fluidities of DPPC bilayer were increased by loading the gold nanoparticles. The membrane fluidities were increased as the amount of gold nanoparticles increased. The existence of gold nanoparticles in the DPPC bilayer was observed by transmission electron microscopy. Through the energy dispersive X-ray spectrometer, the particles in DPPC bilayer were confirmed to be gold nanoparticles.     

Effects of lysozyme and bovine serum albumin on membrane characteristics of dipalmitoylphosphatidylglycerol liposomes

The effects of adsorption of two kinds of proteins on the membrane characteristics of liposomes were examined at pH 7.4 in terms of adsorption amounts of proteins on liposomes, penetrations of proteins into liposomal bilayer membranes, phase transition temperature, microviscosity and permeability of liposomal bilayer membranes, using positively charged lysozyme (LSZ) and negatively charged bovine serum albumin (BSA) as proteins and negatively charged L-alpha-dipalmitoylphosphatidylglycerol (DPPG) liposomes. The saturated adsorption amount of LSZ was 720 g per mol of liposomal DPPG, while that of BSA was 44 g per mol of liposomal DPPG. The penetration of LSZ into DPPG lipid membranes was greater than that of BSA. The microviscosity in the hydrophobic region of liposomal bilayer membranes increased due to adsorption (penetration) of LSZ or BSA, while the permeability of liposomal bilayer membranes increased. The gel-liquid crystalline phase transition temperature of liposomal bilayer membranes was not affected by adsorption of LSZ or BSA, while the DSC peak area (heat of phase transition) decreased with increasing adsorption amount of LSZ or BSA. It is suggested that boundary DPPG makes no contribution to the phase transition and that boundary DPPG and bulk DPPG are in the phase-separated state, thereby increasing the permeability of liposomal bilayer membranes through adsorption of LSZ or BSA. A possible schematic model for the adsorption of LSZ or BSA on DPPG liposomes was proposed.     

Characterisation of electroosmotic flow in capillary electrochromatography columns

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.     

An easy sonochemical route for the encapsulation of tetracycline in bovine serum albumin microspheres

A one-step sonochemical process starting with the native BSA and tetracycline was employed to encapsulate the antibiotic drug in microspheres of BSA. The tetracycline loading studies showed that the maximum tetracycline loading capacity was found to be 65%. The antimicrobial activity of the tetracycline loaded in BSA microspheres was demonstrated on two bacterial strains that are sensitive to tetracycline.