Respirable PLGA Microspheres Containing Rifampicin for the Treatment of Tuberculosis: Manufacture and Characterization

Purpose. Particles with aerodynamic diameters of 1–5m deposit in the periphery of the lungs and are phagocytized by alveolarmacrophages, the primary site of Mycobacterium tuberculosisinfection. Aerosols of biodegradable polymeric microspheres containingantitubercular agents may be delivered to the lungs to improve the treatmentof tuberculosis. Methods. Poly(lactide-co-glycolide) (PLGA) microspherescontaining rifampicin were prepared using solvent evaporation and spraydrying methods. The solvent evaporation process was optimized usingfactorial experimental design and surface response methodology. Themorphology, particle size, drug loading, and dissolution of microspheres wasevaluated. Results. The spray dried rifampicin loaded PLGAmicroparticles were shriveled, unlike the spherical particles produced bysolvent evaporation. Drug loadings of 20;pc and 30;pc were achieved forsolvent evaporation and spray dried products, respectively. The particlesprepared by solvent evaporation and spray drying had 3.45 m and 2.76m median diameters by volume, respectively. Conclusions. Respirable rifampicin loaded PLGAmicrospheres were produced by both solvent evaporation and spray dryingmethods. These particles are being evaluated in an animal model oftuberculosis.     

Prime-boost vaccination based on DNA and protein-loaded microspheres for tuberculosis prevention

We evaluated the use of a vaccine formulation based on a mixture of two different PLGA microspheres, composed by faster and slower release profiles, containing DNA encoding hsp65 and the recombinant hsp65 protein, respectively, aiming to DNA priming and protein boost after a single dose vaccination. The combination of PLGA50:50 microspheres containing DNA-hsp65 and trehalose dimycolate (TDM) with PLGA75:25 microspheres containing recombinant hsp65 (prime-boost Me) was able to induce high levels of anti-hsp65 specific antibodies. The serum levels of these specific antibodies remained high during 90 days after vaccination, whereas the DNA Me formulation based only in DNA-hsp65 plus TDM-loaded microspheres was not able to sustain the high antibody levels during the same period. Production of IFN-gamma was significant in animals vaccinated with both formulations, while the prime-boost Me vaccinated mice sustained higher levels of this cytokine during all the evaluation period. Thus, prime-boost strategy by using biodegradable microspheres seems to be a promising strategy to stimulate long-lasting immune response.     

Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA

Purpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 m. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.     

Attenuated Salmonella and Shigella as Carriers for DNA Vaccines

The discovery that genes can be functionally transferred from bacteria to mammalian cells has suggested the possible use of bacterial vectors as gene delivery vehicles for vaccines. Attenuated invasive human intestinal bacteria, such as Salmonella and Shigella, have been used as plasmid DNA vaccine carriers and their potency has been evaluated in several animal models. This delivery system allows the administration of DNA vaccines together with associated bacterial immunostimulators directly to professional antigen presenting cells via human mucosal surfaces. Various strategies have been taken to improve the use of this delivery system to achieve robust immune responses at both mucosal and systemic sites of the immunized animals.     

PLGA and PHBV Microsphere Formulations and Solid-State Characterization: Possible Implications for Local Delivery of Fusidic Acid for the Treatment and Prevention of Orthopaedic Infections

Purpose  To develop and characterize the solid-state properties of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) microspheres for the localized and controlled release of fusidic acid (FA). Methods  The effects of FA loading and polymer composition on the mean diameter, encapsulation efficiency and FA released from the microspheres were determined. The solid-state and phase separation properties of the microspheres were characterized using DSC, XRPD, Raman spectroscopy, SEM, laser confocal and real time recording of single microspheres formation. Results  Above a loading of 1% (w/w) FA phase separated from PLGA polymer and formed distinct spherical FA-rich amorphous microdomains throughout the PLGA microsphere. For FA-loaded PLGA microspheres, encapsulation efficiency and cumulative release increased with initial drug loading. Similarly, cumulative release from FA-loaded PHBV microspheres was increased by FA loading. After the initial burst release, FA was released from PLGA microspheres much slower compared to PHBV microspheres. Conclusions  A unique phase separation phenomenon of FA in PLGA but not in PHBV polymers was observed, driven by coalescence of liquid microdroplets of a DCM-FA-rich phase in the forming microsphere. Electronic supplementary material   The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

基于离子交换色谱法检测结核治疗性DNA疫苗纯度的方法建立

目的 建立并验证结核治疗性DNA疫苗纯度的高效液相色谱测定方法。方法 采用TSK gel DNA-NPR（4.6 mm ×75 mm, 2.5 µm）色谱柱,流动相：以20 mmol·L^-1 三羟甲基氨基甲烷（pH=9）为流动相A,以20 mmol·L^-1三羟甲基氨基甲烷-1 mol·L^-1 氯化钠（pH=9）为流动相B,梯度洗脱,柱温：25 ℃;流速：0.5 mL·min^-1;检测波长：260 nm。结果 质粒DNA在0.5～2.5 µg的范围内与峰面积呈良好线性关系,R²=0.999 8,该法专属性、精密度和耐用性良好,检测限为0.001 mg·mL^-1,供试品在 24 h内稳定。结论 该方法符合检测结核治疗性DNA疫苗纯度的要求,适用于该产品的质量控制。     

Identification of promising DNA GyrB inhibitors for Tuberculosis using pharmacophore‐based virtual screening,molecular docking and molecular dynamics studies

In this study, we searched for potential DNA GyrB inhibitors using pharmacophore‐based virtual screening followed by molecular docking and molecular dynamics simulation approaches. For this purpose, a set of 248 DNA GyrB inhibitors was collected from the literature and a well‐validated pharmacophore model was generated. The best pharmacophore model explained that two each of hydrogen bond acceptors and hydrophobicity regions were critical for inhibition of DNA GyrB. Good statistical results of the pharmacophore model indicated that the model was robust in nature. Virtual screening of molecular databases revealed three molecules as potential antimycobacterial agents. The final screened promising compounds were evaluated in molecular docking and molecular dynamics simulation studies. In the molecular dynamics studies, RMSD and RMSF values undoubtedly explained that the screened compounds formed stable complexes with DNA GyrB. Therefore, it can be concluded that the compounds identified may have potential for the treatment of TB.     

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle

Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an “expected percentage of protection” above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.     

Nanopharmaceutical approach using pelargonidin towards enhancement of efficacy for prevention of alloxan-induced DNA damage in L6 cells via activation of PARP and p53

Alloxan is an environmental food contaminant that causes DNA damage in living cells and induces hyperglycemia. Pelargonidin (PG), an active ingredient found in extract of various fruits and vegetables, has been nanoencapsulated (NPG) with poly-lactide-co-glycolide (PLGA) and tested for efficacy in prevention of alloxan (ALX)-induced DNA damage in L6 cells in vitro. Glucose uptake, reactive oxygen species (ROS) generation, glucose transporter 4, glucokinase levels and mechanism of activation of DNA repair proteins (PARP and p53) have been studied in ALX-induced L6 cells. Drug–DNA interaction has been analyzed using calf thymus DNA as target through circular dichroism and melting temperature profile. NPGs were physico-chemically characterized by standard protocols using dynamic light scattering and transmission electron microscopy. Pre-treatment with both PG and/or NPG was effective in reducing ALX-induced oxidative stress and showed favourable effects for protection against DNA damage by activating DNA repair cascades. Results suggested ∼10-fold increase in efficacy of NPG than PG in prevention of alloxan-induced oxidative stress and DNA damage.     

Clinical potential of electroporation for gene therapy and DNA vaccine delivery

ABSTRACT

Introduction: Electroporation allows efficient delivery of DNA into cells and tissues, thereby improving the expression of therapeutic or immunogenic proteins that are encoded by plasmid DNA. This simple and versatile method holds a great potential and could address unmet medical needs such as the prevention or treatment of many cancers or infectious diseases.

Areas covered: This review explores the electroporation mechanism and the parameters affecting its efficacy. An analysis of past and current clinical trials focused on DNA electroporation is presented. The pathologies addressed, the protocol used, the treatment outcome and the tolerability are highlighted. In addition, several of the possible optimization strategies for improving patient compliance and therapeutic efficacy are discussed such as plasmid design, use of genetic adjuvants for DNA vaccines, choice of appropriate delivery site and electrodes as well as pulse parameters.

Expert opinion: The growing number of clinical trials and the results already available underline the strong potential of DNA electroporation which combines both safety and efficiency. Nevertheless, it remains critical to further increase fundamental knowledge to refine future strategies, to develop concerted and common DNA electroporation protocols and to continue exploring new electroporation-based therapeutic options.     

The Effect of CTAB Concentration in Cationic PLG Microparticles on DNA Adsorption and in Vivo Performance

Purpose. Cationic PLG microparticles with adsorbed DNA have previously been shown to efficiently target antigen presenting cells in vivo for generating higher immune responses in comparison to naked DNA. In this study we tried to establish the role of surfactant (CTAB) concentration on the physical behavior of these formulations. Methods. Cationic PLG microparticle formulations with adsorbed DNA were prepared using a solvent evaporation technique. Formulations with varying CTAB concentrations and a fixed DNA load were prepared. The loading efficiency and 24 h DNA release was evaluated for each formulation. Select formulations were tested in vivo. Results. Higher CTAB concentration correlated with higher DNA binding efficiency on the microparticles and lower in vitro release rates. Surprisingly though, the in vivo performance of formulations with varying CTAB concentration was comparable to one another. Conclusions. Cationic PLG microparticles with adsorbed DNA, as described here, offer a robust way of enhancing in vivo responses to plasmid DNA.     

Current strategies for DNA therapy based on lipid nanocarriers

Introduction: Lipid based nanocarriers represent one of the most widely used strategies for the delivery of gene molecules. This review focuses on current strategies for the use of these nanocarriers that could open new horizons in DNA therapy and offer an opportunity to support the transition from resource-based approaches towards knowledge-based strategies.

Areas covered: The present review highlights the most promising approaches focusing on the development of safe, stable, and effective lipid-based carriers capable of delivering DNA to the proper target sites and cells. In addition, we intend to provide some insights in to future strategies that should be considered in order to break down barriers in the transformation of DNA basic-science breakthroughs into clinical applications.

Expert opinion: On the basis of the significant advances in the design of lipid nanocarriers our impression is that they are, with respect to other systems, in a ‘pole’ position in the DNA therapy development race.     

The Preparation,Characterization, and Evaluation of Cationic Microparticles for DNA Vaccine Delivery

Pharmaceutical Research -     

DNA条形码鉴定洋金花及其伪品

为建立有毒中药洋金花及其伪品DNA条形码鉴定方法,采用国际通用的条形码序列ITS2、psbA-trnH、matK和rbcL对洋金花原植物白花曼陀罗及其伪品毛曼陀罗、曼陀罗和木本曼陀罗4个种共计20份材料进行了比较研究。PCR及测序成功率分别为ITS2(100%)、matK(100%)、psbA-trnH(90%)、rbcL(85%)。采用CodonCode Aligner进行序列拼接,采用MEGA 4.1计算白花曼陀罗及其伪品的种内、种间的K2P距离,并基于K2P模型构建NJ树。结果显示ITS2序列共有30个单核苷酸多态性(SNPs)位点、psbA-trnH序列有33个单碱基的插入和缺失I。TS2和psbA-trnH序列种间遗传距离大于种内,matK和rbcL种内和种间没有明显Barcoding Gap。4个条形码序列及其组合获得的分子系统树(ITS2、psbA-trnH、matK、rbcL、matK+rbcL)均分成了两大支,木本曼陀罗单聚为一支,该分子证据支持将Brugmansia提升为属水平。实验结果表明ITS2及psbA-trnH序列可以作为洋金花及其伪品鉴定用的条形码序列。     

Biodegradable PLGA Microspheres Loaded with Ganciclovir for Intraocular Administration. Encapsulation Technique,in Vitro Release Profiles,and Sterilization Process

Purpose. The purpose of this work was to obtain a sterilized formulation consisting of biodegradable microspheres of poly (DL-lactide-co-glycolide) (PLGA) for intraocular sustained release of ganciclovir. Methods. Microspheres were prepared using a dispersion of ganciclovir in fluorosilicone oil (FSiO) that was further dispersed in an acetone solution of PLGA [50/50 and inherent viscosity 0.41 dl/g], and emulsified in silicone oil with a surfactant. Once prepared, the formulation was exposed with an effective radiation dose of 2.5 megarads. The release rate data of ganciclovir from the sterilized and nonsterilized batches were compared using the similarity factor (f₂). Results. The dispersion of the drug in FSiO contributed to achieving a drug payload of up to 95% of the theoretical in the 300-500 m microspheres. Ten mg released ganciclovir in vitroat 1.3 g/h for the first 21 days, but decreased to 0.2 g/h from day 25 until the end of the release study (42 days). No significant differences in the amounts of encapsulated drug (=0.05) were observed between the sterilized and nonsterilized microspheres. Furthermore, dissolution profiles of formulations behaved similarly before and after gamma radiation exposure. Conclusions. The technique of microsphere preparation described resulted in high ganciclovir loading (95%) and prolonged drug release. The ganciclovir formulation behaved similarly before and after the sterilization process.     

Synchronous conjugation of i-motif DNA and therapeutic siRNA on the vertexes of tetrahedral DNA nanocages for efficient gene silence

The functionality of DNA biomacromolecules has been widely excavated, as therapeutic drugs, carriers, and functionalized modification derivatives. In this study, we developed a series of DNA tetrahedron nanocages (Td), via synchronous conjugating different numbers of i-(X) and therapeutic siRNA on four vertexes of tetrahedral DNA nanocage (aX-Td@bsiRNA, a+b = 4). This i-motif-conjugated Td exhibited good endosomal escape behaviours in A549 tumor cells, and the escape efficiency was affected by the number of i-motif. Furthermore, the downregulating mRNA and protein expression level of epidermal growth factor receptor (EGFR) caused by this siRNA embedded Td were verified in A549 cells. The tumor growth inhibition efficiency of the 2X-Td@2siRNA treated group in tumor-bearing mice was significantly higher than that of non-i-motif-conjugated Td@2siRNA (3.14-fold) and free siRNA (3.63-fold). These results demonstrate a general strategy for endowing DNA nanostructures with endosomal escape behaviours to achieve effective in vivo gene delivery and therapy.     

Design and development of some thiazole‐based flavanoids as novel antibacterial against pathogens causing surgical site infection for possible benefit in bone trauma via inhibition of DNA gyrase

In this study, a novel class of hybrid thiazole‐based flavanoid derivatives were synthesized and characterized by FT ‐IR , ¹H‐NMR , ¹³C‐NMR , mass and elemental analysis. These derivatives were evaluated for antibacterial activity for possible benefit in bone trauma via inhibition of DNA gyrase enzyme. Results suggested that compounds 9n , 9o , and 9p showed considerable inhibition of DNA gyrase with considerable activity against tested forty strains of Staphylococcus aureus clinical isolates. Moreover, compound 9n showed hydrogen bonding with LYS 460 along with low binding free energy of ?4.36 kcal/mol against DNA gyrase enzyme. The hemolytic activity of the potent compounds showed mild to no activity together with excellent pharmacokinetics, suggesting to have a potential for the development of designed compounds as novel antibacterial agents.     

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1′-hydroxyestragole. The model was extended by linking the area under the curve for 1′-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1′-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.     

Defining in vivo dose-response curves for kidney DNA adduct formation of aristolochic acid I in rat,mouse and human by an in vitro and physiologically based kinetic modeling approach

Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.     

Establishing a public health analytical service based on chemical methods for detecting and quantifying Pacific ciguatoxin in fish samples

A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 μg/kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 μg/kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p < 0.001, Fishers Exact Test).