Acoustic monitoring of a fluidized bed coating process

Bioeliminable co-polymers based on poly(methacryloylglycylglycine-OH(x)-co-hydroxypropylmethacrylamide(y)) were successfully converted into nanoparticles by using the co-precipitation technique. Human serum albumin (HSA) and a modified (beta-cyclodextrin were used, respectively, as model protein drug and stabilizer. Nanoparticles were characterized from a dimensional and morphological point of view by means of laser diffraction granulometry and scanning electron microscopy (SEM). The prepared nanoparticles displayed a monomodal diameter distribution in the range of 130 nm, confirmed by SEM micrographs. Protein loading efficiency and drug release kinetics investigations, carried out on bioeliminable nanoparticles loaded with fluoresceinated HSA (HSA-FITC), showed that protein loading is in the range of 60% with a typical time controlled release profile. In vitro cytotoxicity investigations of the polymer matrices and resulting nanoparticles were carried out by using different assays aimed at the evaluation of the interactions of the materials with cell metabolism and the cell membrane. On the whole, bioeliminable polymers and nanoparticles resulted in high cytocompatibility thus suggesting their suitability for biomedical applications.     

介孔二氧化硅阿霉素纳米粒的制备及体外释放考察

目的制备载阿霉素的介孔二氧化硅纳米粒(MSN),并对其体外释放进行初步研究。方法通过聚合法制备MSN,应用透射电镜表征纳米粒的形态,动态光散射粒径测定仪测定粒子的平均粒径及分布。紫外可见分光光度法评价载药量、包封率及体外释放。结果纳米粒分布均一,平均粒径约70 nm(PDI<0.1)。药物的载药量和包封率分别为(20.38±3.58)%和(55.29±5.17)%。纳米粒经24 h恒温振荡释放达平衡,在pH 5.5磷酸盐缓冲液中累积释放分数达到95%。结论 MSN具有较高的药物载药量,有望成为一种新型的化疗药物载体。     

Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel

Background

Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier in tumor targeting but suffer from the high level of opsonization by reticuloendothelial system due to their hydrophobic structure. As a result surface modification of these nanoparticles has been widely studied as an essential step in their development. Among various surface modifications, human serum albumin (HSA) possesses advantages including small size, hydrophilic surface and accumulation in leaky vasculature of tumors through passive targeting and a probable active transport into tumor tissues.

Methods

PLGA nanoparticles of docetaxel were prepared by emulsification evaporation method and were surface conjugated with human serum albumin. Fourier transform infrared spectrum was used to confirm the conjugation reaction where nuclear magnetic resonance was utilized for conjugation ratio determination. In addition, transmission electron microscopy showed two different contrast media in conjugated nanoparticles. Furthermore, cytotoxicity of free docetaxel, unconjugated and conjugated PLGA nanoparticles was studied in HepG2 cells.

Results

Size, zeta potential and drug loading of PLGA nanoparticles were about 199 nm, −11.07 mV, and 4%, respectively where size, zeta potential and drug loading of conjugated nanoparticles were found to be 204 nm, −5.6 mV and 3.6% respectively. Conjugated nanoparticles represented a three-phasic release pattern with a 20% burst effect for docetaxel on the first day. Cytotoxicity experiment showed that the IC50 of HSA conjugated PLGA nanoparticles (5.4 μg) was significantly lower than both free docetaxel (20.2 μg) and unconjugated PLGA nanoparticles (6.2 μg).

Conclusion

In conclusion surface modification of PLGA nanoparticles through HSA conjugation results in more cytotoxicity against tumor cell lines compared with free docetaxel and unconjugated PLGA nanoparticles. Albumin conjugated PLGA nanoparticles may represent a promising drug delivery system in cancer therapy.     

Doxorubicin-loaded galactose-conjugated poly(d,l-lactide-co-glycolide) nanoparticles as hepatocyte-targeting drug carrier

The objective of this work is to produce doxorubicin-loaded galactose-conjugated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to be specifically recognised by human hepatoma cellular carcinoma (Hep G2) cells and assess NPs cytotoxicity. Doxorubicin-unloaded and doxorubicin-loaded galactose-conjugated PLGA NPs were prepared using an emulsion method and characterised for morphology, size, drug release behaviour, Hep G2 recognition and cell cytotoxicity. The produced doxorubicin-loaded PLGA-galactose-conjugate nanoparticles (PLGA-GAL NPs) are spherical in shape with a size of 365?±?74?nm, a drug encapsulation efficiency of 69% and released in a biphasic pattern with higher release rates at pH 5. In vitro cell studies confirmed the specific interaction between the receptors of Hep G2 and the PLGA-GAL NPs. Cell cytotoxicity tests showed that unloaded NPs are non-toxic and that doxorubicin-loaded NPs caused a cellular viability decrease of around 80%, therefore representing a promising approach to improve liver-specific drug delivery.     

泊洛沙姆188-PLGA纳米给药系统对抗耐药肿瘤的研究

目的 利用泊洛沙姆188对PLGA进行化学修饰,制备包载阿霉素的纳米粒,并评价纳米粒在人耐药乳腺癌细胞中的摄取能力及毒性。方法 通过EDC/NHS法合成泊洛沙姆188-PLGA,通过核磁共振对其结构进行表征并测定临界胶束浓度;通过纳米沉淀法制备包载阿霉素的纳米粒,通过粒度仪对纳米粒的粒径及分布进行分析,通过细胞摄取实验及细胞毒性实验对纳米粒的摄取效果及毒性进行评价。结果 成功合成了泊洛沙姆188-PLGA,并制备了粒径在140 nm左右的纳米粒,该纳米粒在人耐药乳腺癌细胞中有较好的摄取效果及较强的毒性。结论 泊洛沙姆188能够逆转耐药,增强耐药细胞对化疗药物的敏感程度。     

载阿霉素介孔二氧化硅纳米粒的细胞毒性及细胞摄取

目的制备载阿霉素的介孔二氧化硅纳米粒(mesoporous silica nanoparticles,MSN),对其理化性质及细胞摄取行为进行初步研究。方法通过聚合法制备MSN,透射电镜表征纳米粒的形态,动态光散射粒径测定仪测定粒子的平均粒径及分布。紫外分光光度计测定阿霉素的负载行为,MTT比色分析法研究粒子的细胞毒性,激光共聚焦显微镜观察其人乳腺癌MCF-7细胞对载药粒子的摄取。结果纳米粒分布均一,平均粒径约70 nm(PDI<0.1),载药量质量分数达到20%。MCF-7细胞对载药粒子的摄取较快,空白纳米粒具有较低的细胞毒性。结论介孔二氧化硅纳米粒具有较高的药物载药量和良好的生物相容性,能较快地被对人乳腺癌MCF-7细胞摄取,有望成为一种新型的药物化疗载体。     

Influence of surfactants, polymer and doxorubicin loading on the anti-tumour effect of poly(butyl cyanoacrylate) nanoparticles in a rat glioma model

Poly(n-butyl cyanoacrylate) nanoparticles coated with polysorbate-80 can enable the transport of bound drugs across the blood-brain barrier (BBB) after i.v. injection. In the present study the influence of different formulation parameters on the anti-tumoural effects of doxorubicin nanoparticles against glioblastoma 101/8 was investigated. The manufacturing parameters of poly(alkyl cyanoacrylate) doxorubicin-loaded nanoparticles were optimized concerning drug loading. The nanoparticles were coated with different surfactants and injected intravenously on days 2, 5 and 8 after intra-cranial implantation of glioblastoma 101/8 to rats. The survival times of all doxorubicin containing preparations, including a doxorubicin solution, increased the survival times significantly compared to untreated tumour-bearing rats. The most pronounced increase in survival was obtained with the poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80 and 35% of these animals survived for over 180 days (termination of the experiments). The other nanoparticle preparations yielded lower survival times. Poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80-coated proved to be very efficient against glioblastoma 101/8. The data suggest that the interaction of nanoparticles with the blood after injection as well as the enhanced permeability and retention effect (EPR effect) contributed differently to the anti-tumoural efficacy depending on nanoparticle formulation and surface properties.     

Influence of surfactants,polymer and doxorubicin loading on the anti-tumour effect of poly(butyl cyanoacrylate) nanoparticles in a rat glioma model

Poly(n-butyl cyanoacrylate) nanoparticles coated with polysorbate-80 can enable the transport of bound drugs across the blood–brain barrier (BBB) after i.v. injection. In the present study the influence of different formulation parameters on the anti-tumoural effects of doxorubicin nanoparticles against glioblastoma 101/8 was investigated. The manufacturing parameters of poly(alkyl cyanoacrylate) doxorubicin-loaded nanoparticles were optimized concerning drug loading. The nanoparticles were coated with different surfactants and injected intravenously on days 2, 5 and 8 after intra-cranial implantation of glioblastoma 101/8 to rats. The survival times of all doxorubicin containing preparations, including a doxorubicin solution, increased the survival times significantly compared to untreated tumour-bearing rats. The most pronounced increase in survival was obtained with the poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80 and 35% of these animals survived for over 180 days (termination of the experiments). The other nanoparticle preparations yielded lower survival times. Poly(n-butyl cyanoacrylate) doxorubicin-loaded nanoparticles coated with polysorbate 80-coated proved to be very efficient against glioblastoma 101/8. The data suggest that the interaction of nanoparticles with the blood after injection as well as the enhanced permeability and retention effect (EPR effect) contributed differently to the anti-tumoural efficacy depending on nanoparticle formulation and surface properties.     

采用星点设计-效应面法优化及制备阿霉素白蛋白纳米粒

目的采用星点设计-效应面法优化阿霉素白蛋白纳米粒的制备工艺。方法采用去溶剂化-固化交联法制备阿霉素白蛋白纳米粒。以白蛋白质量浓度(X1:1ρ,g.L-1)、阿霉素的质量浓度(X2:2ρ,g.L-1)、pH值(X3)及白蛋白理论交联度(X4,%)为考察对象,以纳米粒平均粒径(Y1:d,nm)、zeta电位(Y2:V,mV)、载药量(Y3:w1,%)和包封率(Y4:w2,%)为评价指标,以四因素五水平的星点设计-效应面法筛选出最佳制备工艺;并采用透射电镜观察制得纳米粒的形态。结果优化后的处方工艺为:白蛋白质量浓度为17 g.L-1、阿霉素质量浓度为2 g.L-1、pH值为9、白蛋白理论交联度为125%。以此条件制得的纳米粒平均粒径为(151±0.43)nm,zeta电位为-(18.8±0.21)mV,载药量为(21.4±0.70)%,包封率为(76.9±0.21)%,均与预测值偏差较小。结论阿霉素白蛋白纳米粒的制备采用星点设计-效应面法设计并优化是可行的。     

Treatment of glioblastoma with poly(isohexyl cyanoacrylate) nanoparticles

Glioblastomas belong to the most devastating cancer diseases. For this reason, polysorbate 80 (Tween 80)-coated poly(isohexyl cyanoacrylate) (PIHCA) (Monorex) nanoparticles loaded with doxorubicin were developed and tested for their use for the treatment of glioblastomas. The preparation of the nanoparticles resulted in spherical particles with high doxorubicin loading. The physico-chemical properties and the release of doxorubicin from the PIHCA-nanoparticles were analysed, and the influence on cell viability of the rat glioblastoma 101/8-cell line was investigated. In vitro, the empty nanoparticles did not show any toxicity, and the anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution, represented by IC(50) values. The in vivo efficacy was then tested in intracranially glioblastoma 101/8-bearing rats. Rats were treated with 3 × 1.5mg/kg doxorubicin and were sacrificed 18 days after tumour transplantation. Histological and immunohistochemical analyses were carried out to assess the efficacy of the nanoparticles. Tumour size, proliferation activity, vessel density, necrotic areas, and expression of glial fibrillary acidic protein demonstrated that doxorubicin-loaded PIHCA-nanoparticles were much more efficient than the free drug. The results suggest that poly(isohexyl cyanoacrylate) nanoparticles hold great promise for the non-invasive therapy of human glioblastomas.     

贝伐单抗介导的阿霉素白蛋白纳米粒的制备与优化

目的采用Box-Behnken效应面法优化贝伐单抗介导的阿霉素白蛋白纳米粒制备工艺。方法在采用去溶剂化-固化交联法制备出的阿霉素白蛋白纳米粒的基础上,用异型双功能交联剂NHS-PEG3500-MAL作为偶联剂,将贝伐单抗偶联到载药白蛋白纳米粒表面。以2-亚氨基硫烷盐酸盐用量、载药白蛋白纳米粒与贝伐单抗质量比、NHS-PEG3500-MAL用量为影响因素,以纳米粒粒径、载药量和包封率作为评价指标,用三因素三水平的Box-Be-hnken效应面法设计试验,并对免疫纳米粒的制备进行优化;考察制备出的免疫纳米粒的抗体活性保存率及稳定性。结果优化后的处方是2-亚氨基硫烷盐酸盐50μg、DOX-A-NPs与Bevacizumab质量比为27.5 mg.mg-1、NHS-PEG3500-MAL用量为8.8 mg,以此处方制得的免疫纳米粒粒径为(216.1±2.31)nm、载药量为(28.93±0.94)%、包封率为(80.39±2.83)%,测得值与预测值相差较小。结论采用Box-Behnken效应面法优化并制备阿霉素白蛋白免疫纳米粒是可行的。     

Interaction of folate-conjugated human serum albumin (HSA) nanoparticles with tumour cells

Folic acid has been previously demonstrated to mediate intracellular nanoparticle uptake. Here, we investigated cellular uptake of folic acid-conjugated human serum albumin nanoparticles (HSA NPs). HSA NPs were prepared by desolvation and stabilised by chemical cross-linking with glutaraldehyde. Folic acid was covalently coupled to amino groups on the surface of HSA NPs by carbodiimide reaction. Preparation resulted in spherical HSA NPs with diameters of 239 ± 26 nm. As shown by size exclusion chromatography, 7.40 ± 0.90 μg folate was bound per mg HSA NPs. Cellular NP binding and uptake were studied in primary normal human foreskin fibroblasts (HFFs), the human neuroblastoma cell line UKF-NB-3, and the rat glioblastoma cell line 101/8 by fluorescence spectrophotometry, flow cytometry, and confocal laser scanning microscopy. Covalent conjugation of folic acid to HSA NPs increased NP uptake into cancer cells but not into HFFs. Free folic acid interfered with cancer cell uptake of folic acid-conjugated HSA NPs but not with uptake of folic acid-conjugated HSA NPs into HFFs. These data suggest that covalent linkage of folic acid can specifically increase cancer cell HSA NP uptake.     

Preparation and in vitro characterization of HSA-mPEG nanoparticles.

Surface modified human serum albumin (HSA) nanoparticles with a size of approximately 150 nm in diameter were prepared from a PEG-HSA conjugate, methoxy-polyethylene glycol modified human serum albumin (HSA-mPEG) using a coacervation method and crosslinked with glutaraldehyde. The zeta-potential of the surface modified nanoparticles was significantly lower than that of unmodified HSA nanoparticles. The existence of a hydrated steric barrier surrounding the nanoparticles was confirmed by electrolyte and pH induced flocculation tests. The surface modified nanoparticles showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.     

Adsorption of obidoxime onto human serum albumin nanoparticles: Drug loading,particle size and drug release

The standard treatment of poisoning by organophosphorous compounds such as paraoxon includes the administration of oximes. Due to their inability to rapidly cross the blood–brain barrier (BBB) in therapeutically relevant concentrations, these drugs possess insufficient activity in the central nervous system. Since human serum albumin (HSA) nanoparticles enable the delivery of a variety of drugs across the BBB into the brain, in the present study the antidote obidoxime was bound to these particles by adsorption. The resulting sorption isotherms showed a best fit to Langmuir isotherms indicating that obidoxime adsorbs to HSA nanoparticles forming a monolayer. A maximum drug loading of 93.5 µg obidoxime/mg of nanoparticles at pH 8.3 was calculated. At higher concentrations the particle diameter increased significantly with obidoxime concentration leading to instable particle systems. The in vitro release of obidoxime from HSA nanoparticles showed a rapid release of the drug from the nanoparticles within 3 h.     

Desolvation process and surface characterisation of protein nanoparticles

The objective of the present study was to characterise and optimise the desolvation process of human serum albumin (HSA) for the preparation of nanoparticles and to characterise the resulting colloidal system. Following the desolvation of the protein, the resulting nanoparticles were stabilised by the addition of varying amounts of glutaraldehyde or by heat denaturation. The particle size, zeta potential, and the number of available amino groups on the surface of the nanoparticles were determined. The amino groups were quantified by a spectrophotometric method using 2,4, 6-trinitrobenzenesulfonic acid (TNBS). The results indicated that the particle size depended mainly on the amount of desolvating agent added, but not on the amount of cross-linker or the kind of cross-linking procedure. Increasing amounts of glutaraldehyde reduced the number of amino groups on the surface of HSA nanoparticles and also decreased the zeta potential of the carrier system. The temperature and heat denaturation time only had an influence on the stability of the nanoparticles but not on the amount of amino groups or the particle size. It was shown that heat denatured HSA nanoparticles possessed the greatest number of amino groups on their surface. Additional experiments for the characterisation of gelatin A and B nanoparticles were performed.     

Albumin nanoparticles with predictable size by desolvation procedure

Nanoparticles prepared of human serum albumin (HSA) represent promising carriers for drug delivery. Desolvating procedure is a common method to obtain protein-based particles. However process parameters have a great influence on size, size distribution and particle yield of the resulting formulation. Therefore, it is crucial to investigate their effects on particle preparation. This study is focused on the influence of different desolvating agents on the resulting particle characteristics and cytotoxicity in cell culture. Another aspect of the study was the influence of different stirring rates, pre-stirring of the HSA solution and the continuous addition of desolvating agent during the preparation process. The results indicate that it is possible to predict the particle size depending on dielectric constant of the desolvation medium. Remarkably, combination of methanol and ethanol used as desolvating agent was able to produce very small spherical HSA nanoparticles in a size range between 50 and 80?nm.     

Optimization of the preparation process for human serum albumin (HSA) nanoparticles

Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.     

Albumin nanoparticles with predictable size by desolvation procedure

Nanoparticles prepared of human serum albumin (HSA) represent promising carriers for drug delivery. Desolvating procedure is a common method to obtain protein-based particles. However process parameters have a great influence on size, size distribution and particle yield of the resulting formulation. Therefore, it is crucial to investigate their effects on particle preparation. This study is focused on the influence of different desolvating agents on the resulting particle characteristics and cytotoxicity in cell culture. Another aspect of the study was the influence of different stirring rates, pre-stirring of the HSA solution and the continuous addition of desolvating agent during the preparation process. The results indicate that it is possible to predict the particle size depending on dielectric constant of the desolvation medium. Remarkably, combination of methanol and ethanol used as desolvating agent was able to produce very small spherical HSA nanoparticles in a size range between 50 and 80?nm.     

Human serum albumin (HSA) nanoparticles: reproducibility of preparation process and kinetics of enzymatic degradation

Nanoparticles prepared from human serum albumin (HSA) are versatile carrier systems for drug delivery and can be prepared by an established desolvation process. A reproducible process with a low batch-to-batch variability is required for transfer from the lab to an industrial production. In the present study the batch-to-batch variability of the starting material HSA on the preparation of nanoparticles was investigated. HSA can build dimers and higher aggregates because of a free thiol group present in the molecule. Therefore, the quality of different HSA batches was analysed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). The amount of dimerised HSA detected by SEC did not affect particle preparation. Higher aggregates of the protein detected in two batches by AUC disturbed nanoparticle formation at pH values below 8.0. At pH 8.0 and above monodisperse particles between 200 and 300 nm could be prepared with all batches, with higher pH values leading to smaller particles. Besides human derived albumin a particle preparation was also feasible based on recombinant human serum albumin (rHSA). Under comparable preparation conditions monodisperse nanoparticles could be achieved and the same effects of protein aggregates on particle formation were observed. For nanoparticulate drug delivery systems the enzymatic degradation is a crucial parameter for the release of an embedded drug. For this reason, besides the particle preparation process, particle degradation in the presence of different enzymes was studied. Under acidic conditions HSA as well as rHSA nanoparticles could be digested by pepsin and cathepsin B. At neutral pH trypsin, proteinase K, and protease were suitable for particle degradation. It could be shown that the kinetics of particle degradation was dependent on the degree of particle stabilisation. Therefore, the degree of particle stabilisation will influence drug release after cellular accumulation of HSA nanoparticles.     

A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation

Nanoparticles consisting of human serum albumin (HSA) play an emerging role in the development of new drug delivery systems. Many of these protein-based colloidal carriers are prepared by the well-known desolvation technique, which has shown to be a robust and reproducible method for the laboratory-scale production of HSA nanoparticles. The aim of the present study was to upscale the ethanolic desolvation process utilizing the paddle stirring systems Nanopaddle I and II in combination with a HPLC pump in order to find the optimal conditions for the controlled desolvation of up to 2000 mg of the protein. For characterization of the HSA nanoparticles particle size, zeta potential as a function of the pH, polydispersity index and particle content were investigated. The particle content was determined by microgravimetry and by a turbidimetry to allow optimized in-process control for the novel desolvation technique. Furthermore the sedimentation coefficient was measured by analytical ultracentrifugation (AUC) to gain deeper insight into the size distribution of the nanoparticles. The formed nanocarriers were freeze dryed to achieve a solid preparation for long-term storage and further processing. Particles ranging in size between 251.2 ± 27.0 and 234.1 ± 1.5 nm and with a polydispersity index below 0.2 were achieved.