Synthesis and characterization of PLGA nanoparticles

Poly(lactide-co-glycolide) (PLGA) nanoparticles of different physical characteristics (size, size distribution, morphology, zeta potential) can be synthesized by controlling the parameters specific to the synthesis method employed. The aim of this review is to clearly, quantitatively and comprehensively describe the top-down synthesis techniques available for PLGA nanoparticle formation, as well as the techniques commonly used for nanoparticle characterization. Many examples are discussed in detail to provide the reader with an extensive knowledge base on the important parameters specific to the synthesis method described and ways in which these parameters can be manipulated to control the nanoparticle physical characteristics.     

Synthesis and characterization of PLGA nanoparticles

Poly(lactide-co-glycolide) (PLGA) nanoparticles of different physical characteristics (size, size distribution, morphology, zeta potential) can be synthesized by controlling the parameters specific to the synthesis method employed. The aim of this review is to clearly, quantitatively and comprehensively describe the top–down synthesis techniques available for PLGA nanoparticle formation, as well as the techniques commonly used for nanoparticle characterization. Many examples are discussed in detail to provide the reader with an extensive knowledge base on the important parameters specific to the synthesis method described and ways in which these parameters can be manipulated to control the nanoparticle physical characteristics.     

Incorporation of PLGA nanoparticles into porous chitosan-gelatin scaffolds: influence on the physical properties and cell behavior

Bone regeneration can be accelerated by localized delivery of appropriate growth factors/biomolecules. Localized delivery can be achieved by a 2-level system: (i) incorporation of biomolecules within biodegradable particulate carriers (nanoparticles), and (ii) inclusion of such particulate carriers (nanoparticles) into suitable porous scaffolds. In this study, freeze-dried porous chitosan–gelatin scaffolds (CH–G: 1:2 ratio by weight) were embedded with various amounts of poly(lactide-co-glycolide) (PLGA) nanoparticles, precisely 16.6%, 33.3% and 66.6% (respect to CH–G weight). Scaffolds loaded with PLGA nanoparticles were subjected to physico-mechanical and biological characterizations including morphological analysis, swelling and dissolution tests, mechanical compression tests and cell viability tests. Results showed that incorporation of PLGA nanoparticles into porous crosslinked CH–G scaffolds: (i) changed the micro-architecture of the scaffolds in terms of mean pore diameter and pore size distribution, (ii) reduced the dissolution degree of the scaffolds, and (iii) increased the compressive modulus. On the other hand, the water uptake behavior of CH–G scaffolds containing PLGA nanoparticles significantly decreased. The incorporation of PLGA nanoparticles did not affect the biocompatibility of CH–G scaffolds.     

Effects of surfactants on the properties of PLGA nanoparticles

The objective of this study was to investigate the physical characteristics of poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with two surfactants, Pluronic or the commonly used polyvinyl alcohol (PVA); and determine their in vitro efficiency as drug carriers for cancer therapy. Free surfactant cytotoxicity results indicated that Pluronic F127 (PF127) was most cytocompatible among the Pluronics tested and hence chosen for coating PLGA NPs for further studies. Release studies using doxorubicin (DOX) as a drug model showed sustained release of DOX from both PVA- and PF127-coated PLGA NPs (PLGA-PVA and PLGA-PF127, respectively) over 28 days. Further, there was no significant difference in human dermal fibroblasts and human aortic smooth muscle cell survival when exposed to both types of NPs. Cellular uptake studies demonstrated that uptake of both nanoparticle types was dose-dependent for both prostate and breast cancer cells. However, these cancer cells internalized more PLGA-PF127 NPs than PLGA-PVA NPs. Moreover, studies showed that drug-loaded PLGA-PF127 NPs not only killed more cancer cells than drug-loaded PLGA-PVA NPs, but also overcame drug resistance in LNCaP, MDA-MB-231, and MDA-MB-468 cancer cells on re-exposure. These results indicate that PLGA-PF127 NPs can form a promising system that not only delivers anti-cancer drugs, but also overcomes drug resistance, which is prevalent in most cancer cells.     

PEGylation strategies for active targeting of PLA/PLGA nanoparticles

This work evaluates various techniques for the incorporation of poly(ethylene glycol) (PEG) onto biodegradable nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA) with the purpose of providing a functional site for surface conjugation of targeting agents and for improving surface properties. The techniques compared were based on NP preparation with blends of PLGA and poloxamer or with block copolymers of PLGA/PLA with PEG. Blending of PLGA with poloxamer 407 resulted in the incorporation of the latter to up to a 43 wt % content. Direct conjugation of heterofunctional NH2-PEG-COOH to the surface of premade NPs was not highly effective. Preparation of copolymers of PLGA with PEG was determined to be more effective and versatile by polymerization of lactide and glycolide dimers onto the hydroxyl group of heterofunctional OH-PEG-COOH than by conjugation of the premade polymers with carbodiimide chemistry. NPs prepared with these copolymers confirmed the surface localization of PEG and proved to be useful for conjugation of mouse immumoglobulin as a model targeting agent.     

Effect of thiol functionalization on the hemo-compatibility of PLGA nanoparticles

In this study, an attempt was made to reduce the interaction of poly(D,L-lactic acid/glycolic acid) (PLGA) nanoparticles with the opsonins and phagocytic cells upon functionalization with thiol groups. Terminal carboxylic groups in PLGA were conjugated to the amino group of cysteine and nanoparticles were prepared by solvent evaporation technique. Detailed in vitro investigations were performed on PLGA and cysteine modified PLGA (Cys-PLGA) nanoparticles to asses their blood compatibility. The effect of these nanoparticles on the release of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) from human macrophage cells were evaluated. Thiolation was confirmed by fourier transform infrared spectroscopy and Ellman's assay; both PLGA and modified nanoparticles had average size in the range of 250 nm. Thiolation was an effective strategy in reducing the protein adsorption, complement activation, and platelet activation of PLGA nanoparticles. PLGA and modified PLGA nanoparticles were compatible with the blood cells and no hemolytic effect was detected. Particles were noncytotoxic on L929 cells and release of proinflammatory cytokines from macrophage cells was rather unaffected with the modification strategy. From these studies, it seems that thiolation of particulate delivery system is an interesting approach in manipulating the blood-particle interactions and appears to be an effective candidate for injectable drug delivery applications.     

Targeted delivery of antibiotics to intracellular chlamydial infections using PLGA nanoparticles

Chlamydia trachomatis and Chlamydia pneumoniae are intracellular bacterial pathogens that have been shown to cause, or are strongly associated with, diverse chronic diseases. Persistent infections by both organisms are refractory to antibiotic therapy. The lack of therapeutic efficacy results from the attenuated metabolic rate of persistently infecting chlamydiae in combination with the modest intracellular drug concentrations achievable by normal delivery of antibiotics to the inclusions within which chlamydiae reside in the host cell cytoplasm. In this research, we evaluated whether nanoparticles formulated using the biodegradable poly(d-L-lactide-co-glycolide) (PLGA) polymer can enhance the delivery of antibiotics to the chlamydial inclusion complexes. We initially studied the trafficking of PLGA nanoparticles in Chlamydia-infected cells. We then evaluated nanoparticles for the delivery of antibiotics to the inclusions. Intracellular trafficking studies show that PLGA nanoparticles efficiently concentrate in inclusions in both acutely and persistently infected cells. Further, encapsulation of rifampin and azithromycin antibiotics in PLGA nanoparticles enhanced the effectiveness of the antibiotics in reducing microbial burden. Combination of rifampin and azithromycin was more effective than the individual drugs. Overall, our studies show that PLGA nanoparticles can be effective carriers for targeted delivery of antibiotics to intracellular chlamydial infections.     

The uptake and intracellular fate of PLGA nanoparticles in epithelial cells

Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic-co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (～2 h), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4–24 h. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.     

荧光示踪PLGA纳米粒与HL60细胞间相互作用

目的 纳米粒作为药物载体在临床诊断和治疗中有着广泛的研究和应用,其跨细胞膜进入细胞内部的过程与其生物效应直接相关,研究纳米粒与细胞间相互作用可揭示其相关机制.方法 通过荧光示踪法研究荧光素标记的PLGA纳米粒与HL60细胞间相互作用,采用激光共聚焦显微镜定量分析了纳米粒的入胞进程.结果 PLGA纳米粒与HL60细胞间相互作用具有很强的温度依赖性,其中受体介导的细胞内吞机制在纳米粒的入胞过程中起到了重要作用.结论 PLGA纳米粒与HL60细胞间相互作用的研究为纳米药物的设计和应用提供了一定的理论基础.     

Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors

Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications.     

PLGA–lecithin–PEG core–shell nanoparticles for controlled drug delivery

Current approaches to encapsulate and deliver therapeutic compounds have focused on developing liposomal and biodegradable polymeric nanoparticles (NPs), resulting in clinically approved therapeutics such as Doxil/Caelyx and Genexol-PM, respectively. Our group recently reported the development of biodegradable core–shell NP systems that combined the beneficial properties of liposomal and polymeric NPs for controlled drug delivery. Herein we report the parameters that alter the biological and physicochemical characteristics, stability, drug release properties and cytotoxicity of these core–shell NPs. We further define scalable processes for the formulation of these NPs in a reproducible manner. These core–shell NPs consist of (i) a poly(d,l-lactide-co-glycolide) hydrophobic core, (ii) a soybean lecithin monolayer, and (iii) a poly(ethylene glycol) shell, and were synthesized by a modified nanoprecipitation method combined with self-assembly. Preparation of the NPs showed that various formulation parameters such as the lipid/polymer mass ratio and lipid/lipid–PEG molar ratio controlled NP physical stability and size. We encapsulated a model chemotherapy drug, docetaxel, in the NPs and showed that the amount of lipid coverage affected its drug release kinetics. Next, we demonstrated a potentially scalable process for the formulation, purification, and storage of NPs. Finally, we tested the cytotoxicity using MTT assays on two model human cell lines, HeLa and HepG2, and demonstrated the biocompatibility of these particles in vitro. Our data suggest that the PLGA–lecithin–PEG core–shell NPs may be a useful new controlled release drug delivery system.     

Delivery of rapamycin by PLGA nanoparticles enhances its suppressive activity on dendritic cells

The purpose of this study was to evaluate the effect of rapamycin delivery by poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles on the maturation of dendritic cells (DCs). DCs were generated from mouse bone marrow and exposed to particulate and soluble rapamycin without any additional treatment, or with pre- or posttreatment with lipopolysaccharide (LPS). Annexin V-FITC/PI staining was performed on DC cultures to assess the viability of DCs during study. Surface phenotype of DCs was characterized for the expression of maturation markers, that is, MHC class II, CD86, and CD40 by flow cytometry. Cell culture supernatants were analyzed for the production of TGF-beta, IL-12, and IL-10 cytokines using sandwich ELISA method. DCs from Balb/C mice were cocultured with T cells from C57BL/6 mice and allogenic mixed lymphocyte reaction was assessed by [3H]-Thymidine incorporation. Unlike free rapamycin that has shown little if any effect on the expression of maturation markers in immature DCs, PLGA encapsulated rapamycin decreased the expression of all maturation markers under the study, that is, MHC class II, CD86, and CD40, significantly. LPS pre- or posttreated DCs demonstrated decreased expression of MHC class II, CD86, and CD40 in the presence of soluble or encapsulated rapamycin. The cytokine secretion profiles revealed high levels of TGF-beta and very low levels of IL-10 and IL-12 production. Rapamycin in soluble or encapsulated form significantly inhibited mixed lymphocyte reaction in DCs. The inhibitory effect of rapamycin on the maturation of DCs with respect to DC phenotype, cytokine production, and functional effects on the proliferation of T cells was significantly increased by PLGA delivery.     

吉西他滨PLGA 纳米颗粒对小鼠Lewis 肺癌细胞增殖抑制效应的实验研究

目的:通过体外实验探讨GemcitabineC18-PLGA 纳米颗粒(GemC18-PLGA-NPs)对Lewis 肺癌细胞株(LLC)的增殖抑制效应。方法: 以LLC 细胞为研究对象分为4 个给药组: GemC18-PLGA-NPs 组、PLGA-NPs 组、GemC18 组和Gemcitabine HCl 组(GemHCl 组),MTT 法观察不同药物浓度对肿瘤细胞生长的抑制作用及细胞毒性作用,并检测给药24、48和72 h 后肿瘤细胞的存活率及IC50 值。采用流式细胞术检测不同给药组48 和72 h 的细胞凋亡率。结果:MTT 试验结果提示GemC18-PLGA-NPs、GemC18 和GemHCl 组均对细胞有显著抑制作用,且GemHCl 组的存活率最低,GemC18 组次之,GemC18-PLGA-NPs 组的存活率最高。72 h 后GemC18-PLGA-NPs 的细胞存活率在1 μmol/ L 浓度时明显超过了GemHCl(P<0.05),表明其有显著的药物缓释作用;PLGA-NPs 组对细胞抑制轻微,且不具有时间和浓度的依赖性。结论:GemC18-PLGA 纳米颗粒对LLC 细胞具有显著的体外细胞增殖抑制作用,且具有良好的生物相容性和缓释性。     

载紫杉醇PLGA/F68纳米粒逆转多药耐药用于乳腺癌的治疗

目的载紫杉醇聚乳酸聚羟基乙酸共聚物（PLGA）/F68纳米粒逆转耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞多药耐药的可行性研究。方法使用超声乳化溶剂挥发法分别制备载紫杉醇PLGA和载紫杉醇PLGA/F68纳米粒（10%）,并对载紫杉醇纳米粒进行表征。载紫杉醇纳米粒的体外释放研究使用高效液相色谱进行分析。最后研究载紫杉醇纳米粒在耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞的细胞摄取和细胞毒性（PLGA/F68组、PLGA组和泰素组）。结果纳米粒呈球形,表面粗糙多孔,平均粒径250 nm左右,粒径分布比较窄,体外药物释放呈双相释放模型。载紫杉醇PLGA/F68纳米粒能够被耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞摄取。载紫杉醇PLGA/F68纳米粒比载紫杉醇PLGA纳米粒（P〈0.05）和泰素（TaxolR）（P〈0.05）有更高的细胞毒性。结论载紫杉醇PLGA/F68纳米粒能够逆转耐紫杉醇人乳腺癌细胞MCF-7/Taxol细胞的多药耐药,药用辅料Pluronic F68在乳腺癌治疗中具有潜在的应用前景。     

地高辛-PLGA抗肿瘤纳米粒子的制备及其抗肿瘤活性的初步研究

目的 制备载地高辛的聚乳酸-羟基乙酸共聚物(PLGA)纳米粒子,提高地高辛的生物利用度,降低其毒副作用.方法 建立测定地高辛-PLGA纳米粒子载药量和包封率的高效液相色谱法;采用乳化溶剂挥发法制备地高辛-PLGA纳米粒子,并通过单因素实验优化制备条件;采用噻唑蓝法评价地高辛和地高辛-PLGA纳米粒子的抗肿瘤能力.结果 以粒径为筛选条件的单因素实验结果表明,制备地高辛-PLGA纳米粒子的最佳条件为PLGA 30 mg,地高辛2 mg,二氯甲烷3 ml,聚乙烯醇质量分数0.5％,超声功率200 W.此制备条件下得到的地高辛-PLGA纳米粒子的粒径约231 nm,包封率为74.61％,载药量为5.37％,且其抗肿瘤活性优于地高辛,差异具有统计学意义(P＜0.05).结论 以PLGA为载体材料制备地高辛-PLGA纳米粒子可增强地高辛的抗肿瘤作用.     

Transferrin-conjugated magnetic silica PLGA nanoparticles loaded with doxorubicin and paclitaxel for brain glioma treatment

The effective treatment of malignant brain glioma is hindered by the poor transport across the blood–brain barrier (BBB) and the low penetration across the blood-tumor barrier (BTB). In this study, transferrin-conjugated magnetic silica PLGA nanoparticles (MNP-MSN-PLGA-Tf NPs) were formulated to overcome these barriers. These NPs were loaded with doxorubicin (DOX) and paclitaxel (PTX), and their anti-proliferative effect was evaluated in vitro and in vivo. The in vitro cytotoxicity of drug-loaded NPs was evaluated in U-87 cells. The delivery and the subsequent cellular uptake of drug-loaded NPs could be enhanced by the presence of magnetic field and the usage of Tf as targeting ligand, respectively. In particular, cells treated with DOX-PTX-NPs-Tf with magnetic field showed the highest cytotoxicity as compared to those treated with DOX-PTX-NPs-Tf, DOX-PTX-NPs, DOX-PTX-NPs-Tf with free Tf. The in vivo therapeutic efficacy of drug-loaded NPs was evaluated in intracranial U-87 MG-luc2 xenograft of BALB/c nude mice. In particular, the DOX-PTX-NPs-Tf treatment exhibited the strongest anti-glioma activity as compared to the PTX-NPs-Tf, DOX-NPs-Tf or DOX-PTX-NPs treatment. Mice did not show acute toxicity after administrating with blank MNP-MSN-PLGA-Tf NPs. Overall, MNP-MSN-PLGA-Tf NPs are promising carriers for the delivery of dual drugs for effective treatment of brain glioma.     

Sustained release of dexamethasone from hydrophilic matrices using PLGA nanoparticles for neural drug delivery

The release of the anti-inflammatory agent dexamethasone (DEX) from nanoparticles of poly(lactic-co-glycolic acid) (PLGA) embedded in alginate hydrogel (HG) matrices was investigated. DEX-loaded PLGA nanoparticles were prepared using a solvent evaporation technique and were characterized for size, drug loading, and in-vitro release. The crosslinking density of the HG was studied and correlated with drug release kinetics. The amount of DEX loaded in the nanoparticles was estimated as approximately 13 wt%. The typical particle size ranged from 400 to 600 nm. The in-vitro release of DEX from NPs entrapped in the HG showed that 90% of the drug was released over 2 weeks. The impedance of the NP-loaded HG coatings on microfabricated neural probes was measured and found to be similar to the unmodified and uncoated probes. The in-vivo impedance of chronically implanted electrodes loaded with DEX was maintained at its initial level, while that of the control electrode increased by 3 times after about 2 weeks after implantation until it stabilized at approximately 3 MOmega. This improvement in performance is presumably due to the reduced amount of glial inflammation in the immediate vicinity of the DEX-modified neural probe.     

Molecular weight distribution changes during degradation and release of PLGA nanoparticles containing epirubicin HCl

The molecular dynamics of the degradation process of poly(lactic-co-glycolic acid) nanospheres were investigated during the degradation process usually observed when these polymers are used as controlled release carriers. The molecular weight distribution of PLGA samples was determined over a period of 32 days by accurately analyzing the molecular weight distribution of the polymer as a function of time as degradation progressed. The molecular weight distribution shifted gradually to lower average molecular weights over 32 days, with significantly smaller molecular weight components appearing at 8-12 days. In addition, the degradation of nanospheres containing epirubicin HCI was analyzed and increasing the amount of epirubicin from 1.7 to 3.4 to 6.7 wt% was found to hasten the degradation of the nanoparticles and subsequently affect the release behavior from these particles. This is believed to be the first time that such molecular dynamics have been presented for the degradation of PLGA nanoparticle formulations containing a drug for controlled delivery.     

In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A

Although oral vaccination has numerous advantages over the commonly used parenteral route, degradation of vaccine and its low uptake in the lymphoid tissue of the gastrointestinal (GI) tract still impede their development. In this study, the model antigen ovalbumin (OVA) and the immunostimulant monophosphoryl lipid A (MPLA) were incorporated in polymeric nanoparticles based on poly(D,L-lactide-co-glycolide) (PLGA). These polymeric carriers were orally administered to BALB/c mice (Bagg albino, inbred strain of mouse) and the resulting time-dependent systemic and mucosal immune responses towards OVA were assessed by measuring the OVA-specific IgG and IgA titers using an enzyme-linked immunosorbent assay (ELISA). PLGA nanoparticles were spherical in shape, around 320 nm in size, negatively charged (around -20 mV) and had an OVA and MPLA payload of 9.6% and 0.86%, respectively. A single immunization with formulation containing (OVA + MPLA) incorporated in PLGA nanoparticles induced a stronger IgG immune response than that induced by OVA in PBS solution or OVA incorporated into PLGA nanoparticles. Moreover, significantly higher IgA titers were generated by administration of (OVA + MPLA)/PLGA nanoparticles compared to IgA stimulated by control formulations, proving the capability of inducing a mucosal immunity. These findings demonstrate that co-delivery of OVA and MPLA in PLGA nanoparticles promotes both systemic and mucosal immune responses and represents therefore a suitable strategy for oral vaccination.