Chitosan-modified poly(D,L-lactide-co-glycolide) nanospheres for plasmid DNA delivery and HBV gene-silencing

Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles (NS) formulated using the biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA) for plasmid DNA (pDNA) delivery. A cationic polymer, Chitosan (CHS), was incorporated in the PLGA matrix to improve pDNA loading efficiency and cellular uptake ability. PLGA-CHS NS were prepared by a spontaneous emulsion diffusion (SED) method, and various formulation factors were investigated. Spherical nanoparticles with particle size of around 60 nm were obtained under optimum formulation condition. The effectiveness of pDNA-loaded PLGA-CHS nanoparticles in expressing the indicative enhanced Green Fluorescent Protein (eGFP) and in slicing Hepatitis B virus (HBV) gene were examined in HepG2.2.15 cells. CHS-modified PLGA NS exhibited much higher loading efficiency than unmodified PLGA NS. CHS-PLGA NS showed a positive zeta potential, while plain-PLGA NS were negatively charged. EGFP expression studies by observation with confocal leaser scanning microscopy (CLSM) indicated that pDNA-loaded CHS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. The corresponding results showed that the HBV gene-silencing efficiency of CHS-PLGA NS was higher than those of plain-PLGA NS and naked pDNA. Thus, CHS-PLGA NS containing pDNA could provide an effective pDNA delivery system in vitro, showing that such an approach could be useful in the treatment of viral diseases in vivo.     

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.     

Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods

Controlled release of plasmid DNA (pDNA) from biodegradable poly lactic-co-glycolic acid (PLGA) microparticles has the potential to enhance transgene expression. However, barriers to this approach include limited encapsulation efficiency, pDNA damage during fabrication and confinement of the microparticles inside phagolysosomal compartments. Combining PLGA with poly ethyleneimine (PEI) can improve protection of pDNA during fabrication, increase encapsulation efficiencies and impart the PLGA microparticles with the capacity to escape the phagolysosomal compartments. This study compares three promising formulation methods for preparing PLGA PEI pDNA microparticles and evaluates for buffering capacity, cellular uptake, transfection efficiency and toxicity. In the first method, PLGA PEI pDNA microparticles are prepared by entrapping pDNA in blended PLGA/PEI using the double emulsion water-in-oil-in-water solvent evaporation technique (PA). In a second approach, PEI-pDNA polyplexes are prepared and then entrapped in PLGA microparticles using a double emulsion solvent evaporation method (PB). Microparticles prepared using formulation methods PA and PB are then compared against PLGA microparticles with PEI conjugated to the surface using carbodiimide chemistry (PC); 0.5% PVA is identified as the optimum concentration of surfactant for generating the strongest transfection efficiencies. N:P ratios of 5 and 10 are selected for preparation of each group. Gel electrophoresis demonstrates that all PLGA microparticle formulations have strong pDNA binding capacity. An MTT assay shows that in vitro cytotoxicity of PLGA PEI microparticles is significantly lower than PEI alone. PLGA PEI pDNA microparticles mediate higher cellular uptake efficiency and consequently higher transgene expression than unmodified PLGA microparticles in COS7 and HEK293 cells. Preparing PEI-pDNA polyplexes prior to entrapment in PLGA microparticles (PB) results in the highest pDNA loading. This is 2.5-fold higher than pDNA loading in unmodified PLGA microparticles. PLGA PEI pDNA microparticles prepared using method PB generates the strongest transfection efficiencies, which are 500-fold higher than unmodified PLGA pDNA microparticles in HEK293 cells and 1800-fold higher in COS-7 cells. The highest transfection efficiencies generated from microparticles prepared using method PB is achieved using an N:P ratio of 5.     

Versatility of biodegradable poly(D,L-lactic-co-glycolic acid) microspheres for plasmid DNA delivery.

In this study, we have optimized different formulations of DNA encapsulated into PLGA microspheres by correlating the protocol of preparation and the molecular weight and composition of the polymer, with the main characteristics of these systems in order to design an efficient non-viral gene delivery vector. For that, we prepared poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles with an optimized water-oil-water double emulsion process, by using several types of polymers (RG502, RG503, RG504, RG502H and RG752), and characterized in terms of size, zeta potential, encapsulation efficiency (EE%), morphology, DNA conformation, release kinetics, plasmid integrity and erosion. The size of the particles ranged between 0.7 and 5.7 microm depending on the protocol of formulation and the molecular mass of the polymer used. The microspheres prepared by using in their formulation polymers of high molecular weight (RG503 and RG504) were bigger in size than in the case of using a lower molecular weight polymer (RG502). The EE (%) of plasmid DNA increased with increasing the molecular mass of the polymer and by using the most hydrophilic polymer RG502H, which contains terminal acidic groups in its structure. The plasmid could be encapsulated without compromising its structural and functional integrity. Also a protective effect of PLGA on endonuclease digestion is observed. Plasmid DNA release from microspheres composed of low molecular weight or hydrophilic polymers, like RG502H, was faster than from particles containing high molecular weight or hydrophobic polymers. These PLGA microspheres could be an alternative to the viral vectors used in gene therapy, given that may be used to deliver genes and other bioactive molecules, either very rapidly or in a controlled manner.     

Poly(L-lactic acid)/polyethylenimine nanoparticles as plasmid DNA carriers

Non-viral vectors such as liposomes, polycations, and nanoparticles have been used as gene delivery systems. In this study, we prepared and characterized biodegradable poly(L-lactic acid) (PLA)/polyethylenimine (PEI) nanoparticles as gene carriers. pCMV/β-gal and pEGFP-C1 were utilized as model plasmid DNAs (pDNA). Nanoparticles were prepared using a double emulsion-solvent evaporation technique, and their pDNA binding capacity was assessed by agarose gel electrophoresis. Transfection was studied in HEK 293 and HeLa cell lines, and the transfection efficiencies were determined by β-galactosidase assay or flow cytometry. Three kinds of PLA/PEI systems were studied by varying the molecular weight of PEI. The PLA/PEI 25K system had a higher transfection efficiency than the PLA/PEI 0.8K or PLA/PEI 750K systems. The transfection efficiency was found to be dependent on the ratio of PLA/PEI nanoparticles to pDNA with an optimum ratio of 60:1 (w/w). The cytotoxicity was dependent on the quantity of PLA/PEI nanoparticles used, but it was comparable to that of commercial Lipofectin™. These results demonstrate the potential of PLA/PEI nanoparticles as gene carriers.     

阳离子PLGA载基因纳米粒的初步研究

目的制备并评价阳离子PLGA载基因纳米粒(pDNA-CTAB-NPs)。方法纳米沉淀法制备空白阳离子PLGA纳米粒(CTAB-NPs),与报告基因pEGFP复合,考察其理化性质、抗核酸酶降解能力、体外释药特性、细胞毒性及其在A549细胞中的转染情况。结果pDNA-CTAB-NPs呈球形或类球形,平均粒径175.5 nm,Zeta电位+12.54mV,pDNA结合充分(>95%),抗核酸酶降解能力强,具有缓释效果,符合Higuchi释放动力学方程(r=0.997 7),细胞毒性较低,能在A549细胞中表达。结论pDNA-CTAB-NPs是一种制备工艺简单,性能良好,极富潜力的非病毒纳米基因载体。     

Critical Determinants in PLGA/PLA Nanoparticle-Mediated Gene Expression

PURPOSE: The aim of the study was to determine the critical determinants in nanoparticle-mediated gene transfection. It was hypothesized that different formulation parameters could affect the nanoparticle characteristics and hence its gene transfection. METHODS: Nanoparticles encapsulating plasmid DNA encoding for firefly luciferase were formulated using polylactide (PLA) and poly (D,L-lactide-co-glycolide) (PLGA) polymers of different compositions and molecular weights. A multiple-emulsion solvent-evaporation method with polyvinyl alcohol (PVA) as an emulsifier was used to formulate DNA-loaded nanoparticles. Gene expression of nanoparticles was determined in breast cancer (MCF-7) and prostate cancer (PC-3) cell lines. RESULTS: Nanoparticles formulated using PLGA polymer demonstrated greater gene transfection than those formulated using PLA polymer, and this was attributed to the higher DNA release from PLGA nanoparticles. Higher-molecular-weight PLGA resulted in the formation of nanoparticles with higher DNA loading, which demonstrated higher gene expression than those formulated with lower-molecular-weight PLGA. In addition, the nanoparticles with lower amount of surface-associated PVA demonstrated higher gene transfection in both the cell lines. Higher gene transfection with these nanoparticles was attributed to their higher intracellular uptake and cytoplasmic levels. Further study demonstrated that the molecular weight and the degree of hydrolyzation of PVA used as an emulsifier also affect the gene expression of nanoparticles. CONCLUSIONS: Results thus demonstrate that the DNA loading in nanoparticles and its release, and the surface-associated PVA influencing the intracellular uptake and endolysosomal escape of nanoparticles, are some of the critical determinants in nanoparticle-mediated gene transfection.     

Low-molecular-weight polyethylenimine enhanced gene transfer by cationic cholesterol-based nanoparticle vector

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.     

Development of nanosomes using high‐pressure homogenization for gene therapy

Objectives The aim of this project was to develop a novel lipid‐based formulation suitable for gene therapy. Methods Novel nanosize liposome (nanosome) formulations containing pDNA (plasmid DNA) were developed using high‐pressure homogenization (HPH). The effect of lipid concentration was studied at two levels: 3 mm and 20 mm . The preformed nanosomes were incubated for 18–20 h with pDNA or pDNA/protamine sulfate (PS) complex. The physical properties of the pDNA nanosomes were compared by particle size distribution and zeta‐potential measurements. Their biological properties were also compared by pDNA efficiency of encapsulation/complexation, integrity, nuclease digestion, transfection efficiency and cell cytotoxicity. Key findings pDNA nanosomes prepared with 20 mm lipid (nanosomes : pDNA : PS at a ratio of 8.6 : 1 : 2) had particle sizes of 170–422 nm (90% confidence). The zeta‐potential of the formulation was 49.2 ± 1.5 mV, and the pDNA encapsulation/complexation efficiency was ～98%. pDNA nanosomes prepared with 3 mm lipid (nanosomes : pDNA : PS at a ratio of 2.09 : 1 : 2) had particle sizes of 140–263 nm (90% confidence). The zeta‐potential of this formulation was 36.4 ± 1.2 mV, and the pDNA encapsulation/complexation efficiency was ～100%. However, a comparison of the efficiency of transfection indicated that pDNA nanosomes prepared with low‐concentration lipids (3 mm ) showed significantly higher transfection efficiency compared with the pDNA nanosomes prepared with high‐concentration lipids (20 mm ), as well as those prepared with Fugene‐6 (a commercially available transfection reagent). This particular formulation (pDNA nanosomes, 3 mm lipids) also showed significantly less cytotoxicity compared with the other pDNA nanosome formulations. Conclusions To conclude, these results indicate that condensing pDNA with PS followed by subsequent complexation with low‐concentration nanosomes generated from HPH can produce a pDNA nanosome formulation that will boost transfection efficiency, while minimizing cytotoxicity. This new technology appears to be an efficient tool for future commercial or large‐scale manufacture of DNA delivery systems for gene therapy.     

Poly(L-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 approximately 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Stability and release characteristics of poly(D,L-lactide-co-glycolide) encapsulated CaPi-DNA coprecipitation

The aims of this work were to determine the stability of DNA-calcium-phosphate coprecipitation (CaPi-DNA) against various conditions during double emulsification microencapsulation and assess the release and physicochemical characteristics of poly(D,L-lactide-co-glycolide) (PLGA) microparticles loading CaPi-DNA. CaPi-DNA prepared at pH 6.5 showed a good stability with over 60% CaPi-DNA remained after emulsification, but no more than 40% at pH 8.0. Polyvinyl alcohol (PVA, 1-5%) could make over 80% CaPi-DNA (pH 7.0) preserved after homogenization. The dichloromethane (DCM), mixture of DCM and ethyl acetate, ether and n-hexane (1:1) exhibited neglectable influence on CaPi-DNA under homogenization. PLGA had influenced on CaPi-DNA without any additional stabilizer, in particular, PLGA (75:25, 4%, w/v) demonstrated a profound damage with only about 10% of the original CaPi-DNA remained. PLGA microparticles loading CaPi-DNA were spherical in shape with size range of 2.0-5.0microm, and entrapment efficiency 30-50%. CaPi-DNA was found to increase the stability of pDNA in PLGA microparticles without losing its structure integrity. The release of CaPi-DNA from microparticles showed a low burst release (<7.5%) within 24h and following sustained release process. The amount of cumulated CaPi-DNA release over 30 days was: 17.6% for PLGA (lactide:glycolide=50:50), 27.3% for PLGA (65:35) and 44.8% for PLGA (75:25) microparticles, respectively. The encapsulation of CaPi-DNA in microparticles could significantly protect CaPi-DNA from degradation of nuclease with average over 80% of total DNA recovery. These results suggested that the encapsulation of CaPi-DNA in PLGA microparticles could improve stability of pDNA.     

Calcium phosphate embedded PLGA nanoparticles: a promising gene delivery vector with high gene loading and transfection efficiency

In the purpose of increasing incorporation efficiency and improving the release kinetics of plasmid DNA (pDNA) from poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles, a facile method for the fabrication of calcium phosphate (CaPi) embedded PLGA nanoparticles (CaPi-pDNA-PLGA-NPs) was developed. The effect of several preparation factors on the particle size, incorporation efficiency, pDNA release and transfection efficiency in vitro was studied by Single Factor Screening Method. These preparation factors included the molecular weight (MW), hydrolysis degree (HD) of polyvinyl alcohol (PVA), sonication power and time, composition of organic phase, initial concentration of calcium phosphate and calcium (Ca) to phosphate ion (P) ratio (Ca/P ratio), etc. The CaPi-pDNA-PLGA-NPs made according to the optimal formulation were spherical in shape observed by transmission electron microscopy (TEM) with a mean particle size of 207±5 nm and an entrapment efficiency of 95.7±0.8%. Differential scanning calorimetry (DSC) suggested that there existed interaction between the DNA-calcium-phosphate (CaPi-pDNA) complexes and the polymeric matrices of PLGA. X-ray diffractometry (XRD) further proved the conclusion and indicated that the CaPi-pDNA was in weak crystallization form inside the nanoparticles. The Brunauer-Emmett-Teller (BET) surface area measurement demonstrated that the CaPi-pDNA-PLGA-NPs are mesoporous with specific surface area of 57.5m(2)/g and an average pore size of 96.5 ?. The transfection efficiency of the CaPi-pDNA-PLGA-NPs on human embryonic kidney 293 (HEK 293) cells in vitro was 22.4±1.2%, which was much higher than those of both the pDNA loaded PLGA nanoparticles (pDNA-PLGA-NPs) and the CaPi-pDNA embedded PLGA microparticles (CaPi-pDNA-PLGA-MPs). The CaPi-pDNA-PLGA-NPs are promising vectors for gene delivery.     

Skin photoprotection improvement: synergistic interaction between lipid nanoparticles and organic UV filters

The main objective of this study was to prepare two types of nanoparticles with poly(d,l-lactide-co-glycolide) (PLGA) and polyethylenimine (PEI) polymers. Plasmid DNA (pDNA) was adsorbed either on PLGA/PEI nanoparticles, or as PEI/DNA complex onto the surface of PLGA nanoparticles. Both types of nanoparticles were prepared by the double emulsion method. The nanoparticles were characterized by their size, zeta potential and pDNA or PEI/DNA complex adsorption. The PEI/DNA complex adsorption was confirmed with ethidium bromide assay. pDNA adsorption onto PLGA/PEI nanoparticles (PLGA/PEI-DNA) was studied by electrophoresis on agarose gel. Cytotoxicity and transfection efficiency of both types of nanoparticle and PEI/DNA complexes formulations were studied in head and neck squamous carcinoma cell line (FaDu). To improve endosomal release, photochemical internalization (PCI) was used. The zeta potential increased when the PEI/DNA complex adsorbed onto PLGA nanoparticles (PLGA-PEI/DNA). Optimal pDNA adsorption efficiency was achieved for nitrogen/phosphorous ratio≥20/1. In vitro transfection and cells viability on FaDu cells with or without PCI were found to be variable depending on the type and concentration of nanoparticles. The results showed that transfection efficiency for PLGA/PEI-DNA or PLGA-PEI/DNA nanoparticles ranged between 2 and 80%, respectively. PCI was found to slightly improve the transfection efficiency for all formulations.     

Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Vancomycin release from poly(D,L-lactide) and poly(lactide-co-glycolide) disks

A biodegradable and biocompatible polymeric system was developed for the controlled release of vancomycin for the treatment of brain abscesses. Poly(D,L-lactic acid) (PLA) and its copolymers poly(lactide-co-glycolide) PLGA 90:10 and PLGA 70:30, were prepared. Polymer disks containing vancomycin (VN) were prepared by solvent casting from methylene chloride solutions. Degradation of the polymer disk was studied by scanning electron microscopy, NMR and GPC. SEM revealed an increasing degree of degradation with time with both PLGAs, the effect being more distinct in the PLGA with the higher glycolide content (PLGA 70:30), which was confirmed with GPC, which showed both a decrease in the molecular weights of PLGA and a decrease in the heterogeneity index (chain length distribution) upon incubation in isotonic phosphate buffer at 37#176;C for up to 5 weeks. NMR showed a decrease in the CH 2 contents of the copolymers, implying that the glycolide component of the copolymers is being preferentially degraded. In situ, vancomycin release behaviour of the disks in pH 7.4 phosphate buffer saline (PBS) was followed for ~2 months in a static system. It was observed that release was according to Higuchi kinetics (Q vs. t 1/2) , and introduction of low molecular weight PLA or hydrophilic compounds like PEG increased the release rate.     

Chitosan nanoparticles for plasmid DNA delivery: effect of chitosan molecular structure on formulation and release characteristics

Chitosan can be useful as a nonviral vector for gene delivery. Although there are several reports to form chitosan-pDNA particles, the optimization and effect on transfection remain insufficient. The chitosan-pDNA nanoparticles were formulated using complex coacervation and solvent evaporation techniques. The important parameters for the encapsulation efficiency were investigated, including molecular weight and deacetylation degree of chitosan. We found that encapsulation efficiency of pDNA is directly proportional with deacetylation degree, but there is an inverse proportion with molecular weight of chitosan. DNA-nanoparticles in the size range of 450-820 nm depend on the formulation process. The surface charge of the nanoparticles prepared with complex coacervation method was slightly positive with a zeta potential of +9 to +18 mV; nevertheless, nanoparticles prepared with solvent evaporation method had a zeta potential approximately +30 mV. The pDNA-chitosan nanoparticles prepared by using high deacetylation degree chitosan having 92.7%, 98.0%, and 90.4% encapsulation efficiency protect the encapsulated pDNA from nuclease degradation as shown by electrophoretic mobility analysis. The release of pDNA from the formulation prepared by complex coacervation was completed in 24 hr whereas the formulation prepared by evaporation technique released pDNA in 96 hr, but these release profiles are not statistically significant compared with formulations with similar structure (p > .05). According to the results, we suggest nanoparticles have the potential to be used as a transfer vector in further studies.     

Vancomycin release from poly(D,L-lactide) and poly(lactide-co-glycolide) disks.

A biodegradable and biocompatible polymeric system was developed for the controlled release of vancomycin for the treatment of brain abscesses. Poly(D,L-lactic acid) (PLA) and its copolymers poly(lactide-co-glycolide) PLGA 90:10 and PLGA 70:30, were prepared. Polymer disks containing vancomycin (VN) were prepared by solvent casting from methylene chloride solutions. Degradation of the polymer disk was studied by scanning electron microscopy, NMR and GPC. SEM revealed an increasing degree of degradation with time with both PLGAs, the effect being more distinct in the PLGA with the higher glycolide content (PLGA 70:30), which was confirmed with GPC, which showed both a decrease in the molecular weights of PLGA and a decrease in the heterogeneity index (chain length distribution) upon incubation in isotonic phosphate buffer at 37 degrees C for up to 5 weeks. NMR showed a decrease in the CH2 contents of the copolymers, implying that the glycolide component of the copolymers is being preferentially degraded. In situ, vancomycin release behaviour of the disks in pH 7.4 phosphate buffer saline (PBS) was followed for approximately 2 months in a static system. It was observed that release was according to Higuchi kinetics (Q vs. t(1/2)), and introduction of low molecular weight PLA or hydrophilic compounds like PEG increased the release rate.     

Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles prepared by high pressure homogenization for paclitaxel chemotherapy

High pressure homogenization was employed in the current work to prepare poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) for controlled release of paclitaxel. The prepared drug-loaded PLGA NPs were found of spherical shape with a size of 200-300 nm. The drug encapsulation efficiency ranged from 34.8+/-1.6 to 62.6+/-7.9% depending on the homogenization pressure and cycles. Paclitaxel was released from the nanoparticles in a biphasic profile with a fast release rate in the first 3 days followed by a slow first-order release. A higher or comparable cytotoxicity against glioma C6 cells was found for the drug formulated in the PLGA NPs in comparison with the free drug Taxol. Confocal laser scanning microscopy (CLSM) evidenced internalization of the fluorescent coumarin 6-loaded PLGA NPs by the C6 cells. The freeze-dried nanoparticles were found to possess excellent water redispersability. The high pressure homogenization could be applied for large industrial scale production of nanoparticles for drug delivery.     

5-Fluorouracil-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(lactide-co-glycolide) polymers: characterization and drug release

5-Fluorouracil (5-FU), a hydrosoluble anti-neoplastic drug, was encapsulated in microspheres of poly(D,L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) polymers using the spray-drying technique, in order to obtain small size microspheres with a significant drug entrapment efficiency. Drug-loaded microspheres included between 47 +/- 11 and 67 +/- 12 microg 5-FU mg(-1) microspheres and the percentage of entrapment efficiency was between 52 +/- 12 and 74 +/- 13. Microspheres were of small size (average diameter: 0.9 +/- 0.4-1.4 +/- 0.8 microm microspheres without drug; 1.1 +/- 0.5-1.7 +/- 0.9 microm 5-FU-loaded microspheres) and their surface was smooth and slightly porous, some hollows or deformations were observed in microspheres prepared from polymers with larger Tg. A fractionation process of the raw polymer during the formation of microspheres was observed as an increase of the average molecular weight and also of Tg of the polymer of the microspheres. The presence of 5-FU did not modify the Tg values of the microspheres. Significant interactions between the drug and each one of the polymers did not take place and total release of the included drug was observed in all cases. The time needed for the total drug release (28-129 h) was in the order PLA > PLGA 75/25 > PLGA 50/50. A burst effect (17-20%) was observed during the first hour and then a period of constant release rate (3.52 +/- 0.82-1.46 +/- 0.26 microg 5-FU h(-1) per milligram of microspheres) up to 8 or 13 h, depending on the polymer, was obtained.     

Chitosan Nanoparticles for Plasmid DNA Delivery: Effect of Chitosan Molecular Structure on Formulation and Release Characteristics

Chitosan can be useful as a nonviral vector for gene delivery. Although there are several reports to form chitosan-pDNA particles, the optimization and effect on transfection remain insufficient. The chitosan-pDNA nanoparticles were formulated using complex coacervation and solvent evaporation techniques. The important parameters for the encapsulation efficiency were investigated, including molecular weight and deacetylation degree of chitosan. We found that encapsulation efficiency of pDNA is directly proportional with deacetylation degree, but there is an inverse proportion with molecular weight of chitosan. DNA-nanoparticles in the size range of 450–820 nm depend on the formulation process. The surface charge of the nanoparticles prepared with complex coacervation method was slightly positive with a zeta potential of +9 to +18 mV; nevertheless, nanoparticles prepared with solvent evaporation method had a zeta potential ～ +30 mV. The pDNA-chitosan nanoparticles prepared by using high deacetylation degree chitosan having 92.7%, 98.0%, and 90.4% encapsulation efficiency protect the encapsulated pDNA from nuclease degradation as shown by electrophoretic mobility analysis. The release of pDNA from the formulation prepared by complex coacervation was completed in 24 hr whereas the formulation prepared by evaporation tecnique released pDNA in 96 hr, but these release profiles are not statistically significant compared with formulations with similar structure p >. 05). According to the results, we suggest nanoparticles have the potential to be used as a transfer vector in further studies.