Physicochemical characterization of poly(L-lactic acid) and poly(D,L-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier

Polymer nanoparticles have been used as non-viral gene delivery systems and drug delivery systems. In this study, biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) and poly(D,L-lactide-co-glycolide) (PLGA)/PEI nanoparticles were prepared and characterized as gene delivery systems. The PLA/PEI and PLGA/PEI nanoparticles, which were prepared by a diafiltration method, had spherical shapes and smooth surface characteristics. The size of nanoparticles was controlled by the amount of PEI, which acted as a hydrophilic moiety, which effectively reduced the interfacial energy between the particle surface and the aqueous media. The nanoparticles showed an excellent dispersive stability under storage in a phosphate-buffered saline solution for 12 days. The positive zeta-potentials for the nanoparticles decreased and changed to negative values with increasing plasmid DNA (pDNA) content. Agarose gel electrophoresis showed that the complex formation between the nanoparticles and the pDNA coincided with the zeta-potential results. The results of in vitro transfection and cell viability on HEK 293 cells indicated that the nanoparticles could be used as gene delivery carriers.     

聚苹果酸在药物载体研究中的应用进展

聚苹果酸是一类具备生物可降解性,生物可吸收性及相容性的高分子,可作为生物医用材料广泛应用于药物释放系统和组织工程等领域。聚苹果酸作为药物载体可以控制药物释放,延长药物作用时间,降低药物的毒副作用,还可以加强制剂的靶向给药能力。此文对聚苹果酸的靶向给药机理,药物与载体的结合方式,载体药物的相容性以及释药分布等进行了综述,评价了该载体应用中存在的不足,展望了其应用前景。     

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.     

Characterization of rhodamine loaded PEG-g-PLA nanoparticles (NPs): effect of poly(ethylene glycol) grafting density

In our previous study, PEG-g-PLA nanoparticles were developed and characterized. The aim of the present work is to investigate the effect of PEG grafting density (% PEG inserted onto poly(d, l)-lactide, PLA backbone) on both physicochemical and biological properties (mainly plasma protein binding and in vitro macrophage uptake) of PEG-g-PLA NPs. Rhodamine B (RHO) loaded NPs were prepared from a 1:1 (wt/wt) blend of PLA and PEG-g-PLA copolymer of varying PEG grafting density (1, 7, or 20% mol/mol of lactic acid monomer) by an o/w emulsion solvent evaporation method. These NPs were characterized with regard to their morphology, size, surface charge, loading efficiency, and rhodamine release. The extent of protein adsorption to the surface of different NPs was qualitatively investigated by dynamic light scattering technique. Additionally, the in vitro macrophage uptake following incubation of RAW 264.7 cells with rhodamine loaded PEG-g-PLA and PLA particles was investigated by confocal laser scanning microscopy (CLSM). The amount of NPs phagocytosed following incubation of RAW 264.7 cells with different concentrations of rhodamine loaded PLA or pegylated NPs for 24h at 37 °C was also determined by fluorescence spectroscopy. ALL lyophilized NPs showed larger diameter in the range of 300-400 nm compared to freshly prepared NPs suspension indicating particle aggregation upon lyophilization. % EE of rhodamine was found to be between 10% and 68% wt/wt depending on PEG grafting density. The higher the grafting density of PEG over PLA backbone, the more the entrapment efficiency. All pegylated NPs showed low zeta potential (close to zero) values. In vitro release analysis revealed that rhodamine leaked from all nanoparticles at a very slow rate at physiological pH, thus making it suitable for both imaging and uptake studies with RAW 264.7 cells. All PEG-g-PLA NPs of different PEG grafting density were well tolerated and exhibited no toxicity to RAW 264.7 cells as seen by cell proliferation assays. Cellular uptake of NPs was mainly dependent on polymer type as well as PEG grafting density. Grafted copolymer NPs resulted in lower degree of macrophage uptake compared to PLA NPs in macrophages cell lines. The higher the PEG grafting density, the lower the uptake of NPs by macrophage cells. Minimum NPs uptake for all the investigated concentrations was achieved when the PEG grafting density was 7% mol/mol of lactic acid. When increasing the PEG grafting density in the nanoparticles above 7%, no significant reduction in NPs phagocytosis was achieved. Thus, this study shows that the optimal PEG density required for designing stealth PEG-g-PLA NPs suitable for drug delivery applications might vary from 4 to 7%.     

Core-shell type polymeric nanoparticles composed of poly(L-lactic acid) and poly(N-isopropylacrylamide)

Poly(L-lactic acid)/poly(N-isopropylacrylamide) (abbreviated as LN) block copolymers were synthesized and the LN nanoparticles were prepared by simple diafiltration method. The thermal transition of the LN nanoparticles was at 32.3 degrees C, the lower critical solution temperature (LCST) of the polymer. The fluorescence spectroscopy data showed that LN was self-assembled in water to form core-shell structure nanoparticles, and the critical association concentration (CAC) value was estimated as 1.3x10(-2) g/l. From the transmission electron microscope observations, the LN nanoparticles were spherically shaped and ranged in size between 30 and 50 nm below the LCST. The hydrated size was measured by photon correlation spectroscopy, and reversible size changes were investigated by the factor of temperature. The release of indomethacin from the LN nanoparticles was thermo-sensitive due to the unique characteristic of poly(N-isopropylacrylamide).     

Self-assembled polymeric nanoparticles of poly(ethylene glycol) grafted pullulan acetate as a novel drug carrier

Self-assembling nanospheres of hydrophobized pullulan have been developed. Pullulan acetate (PA), as hydrophobized pullulan, was synthesized by acetylation. Carboxymethylated poly(ethylene-glycol) (CMPEG) was introduced into pullulan acetate (PA) through a coupling reaction using N,N'-dicyclohexyl carbodiimide (DCC). A synthesized PA-PEG-PA (abbreviated as PEP) conjugate was confirmed by Fourier transform-infrared (FT-IR) spectroscopy. Since PEP conjugates have amphiphilic characteristics in aqueous solution, polymeric nanoparticles of PEP conjugates were prepared using a simple dialysis method in water. From the analysis of fluorescence excitation spectra primarily, the critical association concentration (CAC) of this conjugate was found to be 0.0063 g/L. Observations by scanning electron microscopy (SEM) showed the spherical morphologies of the PEP nanoparticles. The particle size distribution of the PEP conjugates was determined using photon correlation spectroscopy (PCS) and the intensity-average particle size was 193.3 +/- 13.53 nm with a unimodal distribution. Clonazepam (CNZ), as a model drug, was easy to entrap into polymeric nanoparticles of the PEP conjugates. The drug release behavior was mainly diffusion controlled from the core portion.     

Biodegradable thermo-sensitive nanoparticles from poly(L-lactic acid)/poly(ethylene glycol) alternating multi-block copolymer for potential anti-cancer drug carrier.

In order to produce biodegradable thermo-sensitive nanoparticles, alternating multi-block copolymers (MBC) were synthesized by coupling dicarboxylated poly(ethylene glycol) (PEG; M(w) 2000) with poly(L-lactic acid) (PLLA)/PEG/PLLA triblock copolymers. Three different multi-block copolymers were synthesized by varying PLLA molecular weight (800 (MBC1), 1600 (MBC2), and 2800 (MBC3)). The MBC formed self-assembled nanoparticles with a unimodal size distribution during a dialysis process. The nanoparticles (NP) had a spherical shape with a size range of 90-330 nm in diameter and critical aggregation concentrations in a range of 5.6-12.6 microg/mL, depending on PLLA length in MBC. The thermo-sensitivity of MBC NP was monitored by the changes in particle size and interior structure as a function of temperature. The particle size slightly decreased as increasing temperature from 37 to 42 degrees C. The interior structure of the NP responded to temperature by altering microviscosity. The microviscosity, measured by the anisotropy (r value) of a fluorescence probe, of MBC1 NP significantly changed with increasing temperature (r = 0.187 at 25 degrees C and 0.216 at 42 degrees C), while MBC2 and MBC3 showed negligible changes in the microviscosity. This indicates that the temperature-dependent interior structure of the NP relied on the portion of PLLA in MBC. The thermo-sensitivity affected to the drug release behavior and cell cytotoxicity. At 42 degrees C, doxorubicin (DOX) loaded MBC1 NP showed enhanced cytotoxicity (approximately 20 times) against Lewis Lung Carcinoma (LLC) cells when compared to that at 37 degrees C.     

pH-sensitive nanoparticles of poly(amino acid) dodecanoate complexes

Nanoparticles were formed by the complexation of poly( -arginine) (PLA), poly( -histidine) (PLH) and poly( -lysine) (PLL) with dodecanoic acid (C12). Dynamic light scattering, ζ potential measurements, atomic force microscopy, fluorescence, and circular dichroism spectroscopy were used for their characterization. It was found that the diameters of the poly( -arginine) dodecanoate (PLA-C12), poly( -histidine) dodecanoate (PLH-C12), and poly( -lysine) dodecanoate (PLL-C12) complex nanoparticles were in the range 120–200 nm. Furthermore, the pH-sensitive dissolution and the surface charges can be adjusted by choosing PLA, PLH and PLL. The particle stability against basic pH values increases with increasing pK_a value of the poly(amino acid) in the series PLH-C12, PLL-C12 and PLA-C12. The particles as such show a core-shell morphology. Their cores are formed by stoichiometric poly(amino acid) dodecanoate complexes while the shells stabilizing the particles are formed by cationic poly(amino acid) chains in an uncomplexed state. The particles were tested as containers for hydrophobic molecules such as pyrene, which served as a fluorescence probe for measuring the polarity within the particles, and Q₁₀ which functioned as a model drug. The maximum uptake of Q₁₀ into the nanoparticles is about 13% (w/w), thereby making the complexes attractive as simple drug carriers for controlled release purposes. Circular dichroism measurements revealed that the poly(amino acid) chains of PLA-C12 and PLL-C12 adopt predominantly an -helix and that of PLH-C12 a β-sheet.     

Evaluation of carrier capacity and release characteristics for poly( butyl 2-cyanoacrylate) nanoparticles

The characteristics of incorporation and adsorption of drugs onto poly(butyl 2-cyanoacrylate) nanoparticles has been studied using two model compounds, rose bengal and adriamycin. The maximum drug carrier capacity was found to be dependent on the method employed for loading of the compound, the nanoparticle composition, the compound itself and the pH of the solution. A linear correlation was found between the amount of compound incorporated into the nanoparticles and the time for 50% release. A novel bathocromic shift method (spectroscopy) has been used which enabled the concurrent determination of both free and nanoparticle bound compound without prior separation.     

Chitosan-g-PEG nanoparticles ionically crosslinked with poly(glutamic acid) and tripolyphosphate as protein delivery systems

In the present study chitosan grafted copolymers with poly(ethylene glycol) (CS-g-PEG) were prepared and studied using PEG with molecular weights 2000 and 5000g/mol. The materials were characterized using (1)H NMR, FTIR and WAXD techniques. These polyelectrolytes were ionically crosslinked with tripolyphosphate (TPP) and poly(glutamic acid) (PGA) at different polymer/crosslinking agent ratios (1:1, 2:1, 3:1 and 4:1, w/w) for the nanoencapsulation of bovine serum albumin (BSA). Prepared nanoparticles are spherical in shape with a mean diameter ranging from 150 to 600 nm. The size depends mainly to the molecular weight of the PEG and the crosslinking agent used. The PEG molecular weight also seems to affect the release rate of BSA especially the first burst effect which appears to be high in copolymers containing PEG5000, compared with copolymer prepared with PEG2000, and it is also higher when PGA was used as crosslinking agent, instead of TPP.     

Exploring the immunopotentiation of Chinese yam polysaccharide poly(lactic-co-glycolic acid) nanoparticles in an ovalbumin vaccine formulation in vivo

Biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) has been approved by the US Food and Drug Administration and has frequently been used to develop potential vaccine delivery systems. The immunoregulation and immunopotentiation of Chinese yam polysaccharide (CYP) have been widely demonstrated. In the current study, cell uptake mechanisms in dendritic cells (DCs) were monitored in vitro using confocal laser scanning microscopy, transmission electron microscopy, and flow cytometry. To study a CYP-PLGA nanoparticle-adjuvanted delivery system, CYP and ovalbumin (OVA) were encapsulated in PLGA nanoparticles (CYPPs) to act as a vaccine, and the formulation was tested in immunized mice. The CYPPs more easily underwent uptake by DCs in vitro, and CYPP/OVA could stimulate more effective antigen-specific immune responses than any of the single-component formulations in vivo. Mice immunized using CYPP/OVA exhibited more secretion of OVA-specific IgG antibodies, better proliferation, and higher cytokine secretion by splenocytes and significant activation of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. Overall, the CYPP/OVA formulation produced a stronger humoral and cellular immune response and a mixed Th1/Th2 immune response with a greater Th1 bias in comparison with the other formulations. In conclusion, the data demonstrate that the CYPP-adjuvanted delivery system has the potential to strengthen immune responses and lay the foundation for novel adjuvant design.     

姜黄素PLGA纳米粒的制备及制剂学性质分析

目的制备姜黄素(Curcumin,Cur)聚乳酸羟基乙酸共聚物(PLGA)纳米粒(Cur-PLGA-NPs)并对其理化性质进行考察。方法采用改良的自乳化溶剂挥发法制备纳米粒,通过正交设计,以粒径、包封率和载药量为评价指标优化处方工艺。结果制备Cur-PLGA-NPs的优化条件为PLGA 100 mg,泊洛沙姆188浓度1.0%,丙酮与乙醇体积比3∶1,有机相体积15 m L。按优化条件所制备的Cur-PLGA-NPs粒径为(120.33±2.44)nm,多分散系数为0.10±0.02,包封率为84.50%±1.13%,载药量为4.75%±0.22%。结论采用改良的自乳化溶剂挥发法成功制备了Cur-PLGA-NPs,为后续"纳米粒-脂质体系统"的研究奠定了基础,有望实现药物在肝脏的浓集。     

壳聚糖包衣对胰岛素聚酯纳米粒胃肠道吸收的促进作用

目的研究壳聚糖包衣胰岛素乳酸/羟基乙酸共聚物(PLGA)纳米粒对胰岛素胃肠道吸收的促进作用。方法以双乳化法制备了胰岛素PLGA复乳,壳聚糖用作稳定剂,制备了包衣纳米粒;观察了粒子大小、表面形态及Zeta电位;测定了包封率;考察了体外释药行为;以糖尿病大鼠评价降血糖水平。结果包衣纳米粒粒度分布均匀,隐约可见层状结构,壳聚糖可改变粒子表面Zeta电位,提高包封率,降低突释;灌服10 u·kg^-1包衣纳米粒,14～16 h降血糖水平显著高于未包衣纳米粒(P＜0.05),药理相对生物利用度提高到15.4%。结论壳聚糖包衣聚酯纳米粒可以促进胰岛素胃肠道吸收。     

利福喷丁/聚乳酸-羟基乙酸纳米粒的制备及处方工艺优化

目的： 研制负载利福喷丁的聚乳酸-羟基乙酸共聚物[poly（lactic-co-glycolic acid）,PLGA]纳米粒,并对其处方及制备工艺进行优化。方法： 采用快速膜乳化法制备利福喷丁/PLGA纳米粒。通过单因素实验考察了乳化剂浓度、PLGA浓度、油相/水相体积比、初乳制备转速、初乳制备时间、过膜压力、过膜次数对纳米粒制备的影响。在此基础上以粒径、载药率、包封率为评价指标,使用正交实验设计对纳米粒制备的处方工艺进行优化,以TOPSIS法进行多指标综合分析。然后对最优处方工艺进行验证,并对载药纳米粒的体外释药行为进行考察。结果： 经最优处方工艺制备的载药纳米粒,粒径（428±11.4）nm,粒径分布为（0.186±0.036）,包封率为（76.89±2.6）%,载药率为（10.89±1.2）%。用透视电镜观察呈均匀分布的球形。在体外药物释放实验中,药物在72 h内累计释放了78.81%。结论： 采用快速膜乳化可以简单快捷地制备均匀圆整、包封性好、具有良好缓释性能的利福喷丁/PLGA纳米粒,并为新型抗结核精准治疗的开发提供了基础。     

Development and characterization of poly(lactic-co-glycolic) acid nanoparticles loaded with copaiba oleoresin

Abstract

Copaiba oleoresin (CPO), obtained from Copaifera landgroffii, is described as active to a large number of diseases and more recently in the endometriosis treatment. In this work, poly(lactic-co-glycolic acid) (PLGA) nanoparticles containing CPO were obtained using the design of experiments (DOE) as a tool to optimize the production process. The nanoparticles optimized by means of DOE presented an activity in relation to the cellular viability of endometrial cells. The DOE showed that higher amounts of CPO combined with higher surfactant concentrations resulted in better encapsulation efficiency and size distribution along with good stability after freeze drying. The encapsulation efficiency was over 80% for all produced nanoparticles, which also presented sizes below 300?nm and spherical shape. A decrease in viability of endometrial stromal cells from ectopic endometrium of patients with endometriosis and from eutopic endometriotic lesions was demonstrated after 48?h of incubation with the CPO nanoparticles. The nanoparticles without CPO were not able to alter the cell viability of the same cells, indicating that this material was not cytotoxic to the tested cells and suggesting that the effect was specific to CPO. The results indicate that the use of CPO nanoparticles may represent a promising alternative for the treatment of endometriosis.     

表面修饰的灯盏花素聚乳酸纳米粒的制备和大鼠体内药动学*

目的:制备灯盏花素聚乳酸纳米粒并对其进行了表面修饰,同时考察了游离药物和纳米药物经大鼠尾静脉注射后在动物体内的药动学。方法:采用自乳化溶剂扩散法制备灯盏花素聚乳酸纳米粒,并用泊洛沙姆188对纳米粒进行表面修饰,采用反相高效液相色谱法(RP-HPLC)测定纳米粒的包封率、载药量和血浆样品中灯盏花素的含量,药时数据采用DASver 1.0药代计算程序处理。结果:载药纳米粒平均粒径为177.2和319.6 nm,多分散指数分别为(0.11±0.01)和(0.12±0.02),平均包封率及载药量分别为(86.9±0.9)%,(8.0±0.2)%和(93.1±0.6)%,(8.5±0.1)%,游离灯盏花素iv后呈二室模型,t1/2β为(0.81±0.14)m in,纳米组则呈一室模型,2种粒径的纳米粒的t1/2β分别为(8.90±0.16)m in(177.2 nm)和(13.90±0.07)m in(319.6 nm)。游离灯盏花素和2种粒径的灯盏花聚乳酸纳米粒的AUC0~t分别为(158.82±69.96),(1 476.25±51.22)和(704.95±25.39)mg.m in.L-1。经t检验,游离药物与纳米药物之间的t1/2β和AUC0~t均有统计学差异(P<0.01)。结论:灯盏花素制成纳米粒后明显增加了药物在动物体内的半衰期,延长了药物在体内的循环时间,且不同粒径的纳米粒对药动学有一定的影响。     

口服胰岛素聚乳酸纳米粒的研制

目的　研制一种新型的口服胰岛素 (INS)纳米粒制剂。方法　以溶剂 非溶剂法制备了INS PLA NP ,进行了形态、粒径、包封率、载药量等主要性质的考察。以糖尿病小鼠为动物模型 ,初步考察了口服INS PLA NP后的降血糖药效。结果　制得的INS PLA NP均匀圆整 ,平均粒径为 (84 .34± 14 .76 )nm ,平均包封率为 (6 5 .93± 3.4 5 ) % ,平均载药量为 (0 .6 2± 0 .0 3)u·mg-1。糖尿病小鼠口服 5 0 ,6 0 ,80u·kg-1剂量的INS PLA NP后均表现出显著的降血糖作用。而口服 4 0u·kg-1剂量的INS PLA NP无效。结论　使用PLA NP作INS口服给药的载体具有可行性。     

Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens

Injectable biodegradable polymeric particles (usually microspheres) represent an exciting approach to control the release of vaccine antigens to reduce the number of doses in the immunization schedule and optimize the desired immune response via selective targeting of antigen to antigen presenting cells. After the first couple of decades of their study, much progress has been made towards the clinical use of antigen-loaded microspheres. Poly(lactide-co-glycolic acids) (PLGAs) have been studied most commonly for this purpose because of their proven safety record and established use in marketed products for controlled delivery of several peptide drugs. PLGA microspheres have many desirable features relative to standard aluminum-based adjuvants, including the microspheres' ability to induce cell-mediated immunity, a necessary requirement for emergent vaccines against HIV and cancer. This review examines several impediments to PLGA microparticle development, such as PLGA-encapsulated antigen instability and deficiency of animal models in predicting human response, and describes new trends in overcoming these important issues. PLGA microparticles have displayed unprecedented versatility and safety to accomplish release of one or multiple antigens of varying physical-chemical characteristics and immunologic requirements, and have now met numerous critical benchmarks in development of long-lasting immunity after a single injected dose.