Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum

Small interfering RNAs (siRNA), RNA duplexes of approximately 21 nucleotides, offer a promising approach to specifically degrade RNAs in target cells by a process termed RNA interference. Insufficient in vivo-stability is a major problem of a systemic application of siRNAs in humans. The present study demonstrated that RNAse A-like RNAses degraded siRNAs in serum. The susceptibility of siRNAs towards degradation in serum was strongly enhanced by local clustering of A/Us within the siRNA sequence, i.e. regions showing low thermal stability, most notably at the ends of the molecule, and by 3'-overhanging bases. Importantly, inhibition of RNAse A family enzymes prevented the degradation and loss of silencing activity of siRNAs in serum. Furthermore, the degradation of siRNAs was considerably faster in human than in mouse serum, suggesting that the degradation of siRNAs by RNAse A family enzymes might be a more challenging problem in a future therapeutic application of siRNAs in humans than in mouse models. Together, the present study indicates that siRNAs are degraded by RNAse A family enzymes in serum and that the kinetics of their degradation in serum depends on their sequence. These findings might be of great importance for a possible future human therapeutic application of siRNAs.     

Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel

Purpose. Currently, most pDNA delivery systems based on synthetic polymers are either nonbiodegradable or not sensitive to the release environment. The primary objective of this study was to develop and evaluate an aqueous-based, thermosensitive, biodegradable and biocompatible triblock copolymer to control pDNA delivery in vitro and in vivo. Methods. The triblock copolymers, poly[ethylene glycol-b-(D, L-lactic acid-co-glycol acid)-b-ethylene glycol] (PEG-PLGA-PEG), were synthesized as previously described. The molecular weight and polydispersity of PEG-PLGA-PEG were monitored by gel permeation chromatography (GPC). The cytotoxicity of PEG-PLGA-PEG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide assay. The release of ³²P-labeled pDNA entrapped in aqueous dispersion of PEG-PLGA-PEG in 0.1 mol/L sodium phosphate buffer solution (pH 7.4) was studied at 37°C under agitation. Gene transfection efficiency was evaluated in a skin wound model in CD-1 mice. Results. The aqueous dispersion of PEG-PLGA-PEG flows freely at room temperature but form a gel at 37°C body temperature. The in vitro degradation of PEG-PLGA-PEG lasted for more than 30 days. The cytotoxicity of PEG-PLGA-PEG evaluated in HEK 293 cells was significantly lower than that of poly-L-lysine hydrochloride. The release profile of supercoiled pDNA from the polymer followed the zero-order kinetics up to 12 days. Maximal gene expression of luciferase was at 24 h in the skin wound of CD-1 mice and by 72 h, the expression dropped by nearly 94%. Conclusions. These results suggest hydrogel formed by PEG-PLGA-PEG could be a promising platform for delivery of pDNA, which represents a novel strategy that may serve as a non-viral vector for gene therapy in wound healing.     

Prospects for Intranasal Delivery of Neuropeptides to the Brain

Pharmaceutical Chemistry Journal - Intranasal administration (INA) of medicines has the advantages of being noninvasive, painless, simple, and convenient. The basic approaches to solving problems...     

Optimisation of Polyethylenimine-Based Gene Delivery to Mouse Brain

Abstract

Polyethylenimine (PEI) is proving to be an efficient and versatile vector for gene delivery in vivo. However, a limiting factor is the relatively short duration of gene expression in some sites. Given the particularly high levels of expression seen in the short term we postulated that loss of expression could result from overloading the nucleus with foreign DNA. To address this problem we first followed DNA delivery and localisation with digoxin-labelled plasmid DNA complexed with 22 kD linear PEI and used these complexes for intraventricular injection into brains of anaesthetised newborn mice. At 24 h post-injection, labelled DNA was found exclusively in the nuclear and perinuclear regions. We next carried out a dose response curve using decreasing amounts of DNA, either in a constant volume (2 μl) or at a constant concentration (500 ng/μl). In both conditions, transgene expression yield was maximum at 100 ng DNA per injection. Using this optimal amount of DNA increased yield of transgene expression significantly at 24 h and one week post-injection as compared to 1 μg DNA. A final point addressed was whether co-expressing an anti-apoptotic gene could enhance gene expression in the longer term. Co-expressing bcl-X_L with luciferase or LacZ significantly increased expression of both these genes at one week post-injection.     

Recent Advances in Brain Tumor Therapy: Local Intracerebral Drug Delivery by Polymers

New approaches to malignant glioma are being actively investigated. Local drug delivery directly to the site of the tumor is one novel approach that has been approved by the US FDA and other regulatory agencies worldwide. This agent, Gliadel, delivers the chemotherapeutic drug carmustine (BCNU) from a biodegradable polymer placed in the resection cavity after brain tumor surgery. Gliadel represents the first clinical application of polymer delivery for brain tumors, but the potential for this new methodology is far greater. In this review, we will briefly summarize the development of Gliadel from a laboratory idea to its current role as an approved treatment for gliomas. Then we will present the most recent work being done to expand the potential benefits of polymeric delivery for brain tumors. This work includes trials for its use as the initial therapy for gliomas, as well as its use against metastasis. Further clinical trials exploring the maximum-tolerated dose and the combination of Gliadel with systemic chemotherapeutic treatments such as temozolamide and O(6)-benzylguanine will be reviewed. Finally, we will present preclinical work on the efficacy of polymeric methods for delivering other chemotherapeutic agents, and a variety of novel compounds that modify brain tumor biology. This latter work represents potential future clinical applications of local polymeric drug delivery to the brain and other sites where cancers can occur.     

Delivery of 125I-NGF to the Brain via the Olfactory Route

The blood-brain barrier presents a major problem in the administration and testing of neurotropins as it prevents a sufficient concentration of these potential therapeutic agents from reaching the target areas of the human brain. The olfactory neuroepithelium is the only area of the body in which an extension of the central nervous system comes into direct contact with the environment. Following intranasal administration of ¹²⁵I-labeled nerve growth factor (¹²⁵I-NGF), radiolabel appeared rapidly in the olfactory bulb and other brain regions. Radiolabel accumulation in the olfactory bulb of the brain was a linear function of the intranasal dose and of the radiolabel concentration in the olfactory epithelium. Concentration of radiolabel in the olfactory bulb and brain with intranasal administration, but not with intravenous administration, suggests direct transport of label into the brain along the olfactory route following intranasal administration. The rapid appearance of label in the olfactory bulbs, cerebrum, and brain stem is more consistent with entry of label through intercellular clefts in the olfactory epithelium and extracellular transport along the olfactory neural pathway to reach the cerebrospinal fluid and brain than with uptake by olfactory neurons and subsequent intracellular axonal transport. At least 80% of the radiolabel found in the brain following intranasal delivery of ¹²⁵I-NGF precipitates in cold 5% trichloroacetic acid, suggesting that a significant amount of intact NGF reaches the brain. Preliminary studies using a sandwich enzyme-linked immunosorbent assay have confirmed the uptake of NGF into the brain following intranasal but not intravenous administration to rats. This is the first evidence for noninvasive delivery of unconjugated NGF to the brain.     

Glow Discharge Plasma Deposition (GDPD) Technique for the Local Controlled Delivery of Hirudin from Biomaterials

Purpose. Biomaterials which release locally high concentrations of antithrombotic agents should lessen the thrombogenicity of the materials. To evaluate this approach, we prepared novel polyurethane matrices loaded with hirudin and coated them with 2-hydroxyethyl methacrylate (HEMA) by glow discharge plasma deposition (GDPD) to reduce the release rate. Methods. Polyurethane (BioSpan^®) matrices containing hirudin and pore former (d-mannitol or BSA) were prepared by the solvent casting method. HEMA plasma deposition was then applied using GDPD technique to create a diffusional barrier film on the surface of the matrices. The effect of pore former and HEMA plasma coating on the release of hirudin was systematically investigated. Surface properties of matrices was also studied using Scanning Electron Microscopy (SEM) and Electron Spectroscopy for Chemical Analysis (ESCA). Results. The release of hirudin from BioSpan^® matrix could be controlled by changing the weight fraction and particle size of pore former. HEMA plasma treatment of matrices produced a thin, highly cross-linked film on the surface. The initial burst and subsequent release of hirudin was significantly reduced after HEMA plasma coating, which suggested that the plasma disposition acted as a diffusional barrier and limited the release of hirudin incorporated in the polyurethane matrix. Conclusions. The plasma coating served as a diffusional barrier, and could work to control the release kinetics of hirudin by changing the various plasma coating conditions. Local delivery of hirudin using these biomaterials at the site of cardiovascular diseases can have the advantage of regional high levels of hirudin, as well as lowering systemic hirudin exposure, thereby minimizing the possibility of side effects.     

Hydrogel Matrix Entrapping PLGA-Paclitaxel Microspheres: Drug Delivery with Near Zero-Order Release and Implantability Advantages for Malignant Brain Tumour Chemotherapy

Purpose

To develop paclitaxel-delivering PLGA microspheres entrapped in a gel matrix with sustained drug release properties and implantability advantages for local glioma chemotherapy.

Methods

Paclitaxel-loaded PLGA microspheres were fabricated using electrohydrodynamic atomization and entrapped by electrospray and gelation. The physicochemical characterizations were performed using scanning electron microscopy and differential scanning calorimetry. The influence of various parameters on the disintegration time was investigated. In vitro release of paclitaxel was quantified using high performance liquid chromatography. Cytotoxicity of the formulations was assessed by the quantification of IC₅₀ and caspase-3 activity against C6 glioma cells in vitro. The formulations were tested against a subcutaneous C6 glioma tumour in mice.

Results

Highly monodisperse gel beads containing a uniform microsphere distribution were obtained. Gelation using Ca²⁺ ions ensured entrapment of microspheres with high loading efficiency. With an increase in the gelation time, gelling bath concentration and decrease in microsphere loading, it was more difficult to disintegrate the beads and release the microspheres. The formulations demonstrated sustained drug release for more than 60 days at a near-constant rate and a low initial burst. Cell culture studies proved the cytotoxicity against C6 glioma and improved performance in comparison to Taxol®. The formulations could reduce subcutaneous tumour volume to a greater extent compared to Taxol® and the control.

Conclusions

Paclitaxel-loaded PLGA microspheres entrapped in an alginate gel matrix could be potential local chemotherapy implants to treat malignant glioma with critical advantages of implantability and sustained drug release with low initial burst.

     

Sustained Local Delivery of Structurally Diverse HIV-1 Microbicides Released from Sublimation Enthalpy Controlled Matrices

Purpose

Use of coital-dependent products to prevent HIV-1 transmission has resulted in mixed success. We hypothesize that incorporation of antiviral drug candidates into a novel controlled delivery system will prolong their activity, making their use coital independent, thus increasing their chance of prophylactic success.

Methods

Tenofovir, emtricitabine, and C5A peptide HIV microbicides were mechanically incorporated into matrices comprising a series of subliming solids. Matrix sublimation rates and drug release rates were measured in three in vitro and one in vivo environments intended to model human vaginal interior. Antiviral activity studies evaluating matrix incorporated microbicides were performed using in vitro cell cultures and human ectocervical explants.

Results

Drug release rates were identical to matrix sublimation rates, and were zero order. Differences in matrix material sublimation enthalpies determined drug release and matrix erosion rates in a thermodynamically definable manner, in vitro and in vivo. Durations of release ranging from several days to several months were readily achieved. Prolonged duration of anti HIV-1 activity was shown for matrix incorporated microbicides, using ectocervical explant and cell culture models of HIV-1 infection.

Conclusion

Subliming solid matrices show promise as a delivery system providing multi month intravaginal release of a wide range of HIV-1 microbicides.     

On The Rate and Extent of Drug Delivery to the Brain

To define and differentiate relevant aspects of blood-brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: Kp,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and Vu,brain (intra-brain distribution). The permeability of the blood-brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. Kp,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (Kp), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters Kp,uu, CLin, and Vu,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes.     

Poly Lactic Acid Based Injectable Delivery Systems for Controlled Release of a Model Protein,Lysozyme

The objective of this study was to evaluate the critical formulation parameters (i.e., polymer concentration, polymer molecular weight, and solvent nature) affecting the controlled delivery of a model protein, lysozyme, from injectable polymeric implants. The conformational stability and biological activity of the released lysozyme were also investigated. Three formulations containing 10%, 20%, and 30% (w/v) poly lactic acid (PLA) in triacetin were investigated. It was found that increasing polymer concentration in the formulations led to a lower burst effect and a slower release rate. Formulation with a high molecular weight polymer showed a greater burst effect as compared to those containing low molecular weight. Conformational stability and biological activity of released samples were studied by differential scanning calorimeter and enzyme activity assay, respectively. The released samples had significantly (P < 0.05) greater conformational stability and biological activity in comparison to the control (lysozyme in buffer solution kept at same conditions). Increasing polymer concentration increased both the conformational stability and the biological activity of released lysozyme. In conclusion, phase sensitive polymer-based delivery systems were able to deliver a model protein, lysozyme, in a conformationally stable and biologically active form at a controlled rate over an extended period.     

Delivery of Nerve Growth Factor to Brain Via Intranasal Administration and Enhancement of Brain Uptake

The main objective of the study was to investigate the efficacy of chitosan to facilitate brain bioavailability of intranasally administered nerve growth factor (NGF). In vitro permeability studies and electrical resistance studies were carried out across the bovine olfactory epithelium using Franz diffusion cells. The bioavailability of intranasally administered NGF in rat hippocampus was determined by carrying out brain microdialysis in Sprague–Dawley rats. The in vitro permeation flux across the olfactory epithelium of NGF solution without chitosan (control) was found to be 0.37 ± 0.06 ng/cm²/h. In presence of increasing concentration of chitosan (0.1%, 0.25%, and 0.5%, w/v) the permeation flux of NGF was found to be 2.01 ± 0.12, 3.88 ± 0.19, and 4.12 ± 0.21 ng/cm²/h respectively. Trans-olfactory epithelial electrical resistance decreased ～34.50 ± 4.06% in presence of 0.25% (w/v) chitosan. The C_max in rats administered with 0.25% (w/v) chitosan and NGF was 1008.62 ± 130.02 pg/mL, which was significantly higher than that for rats administered with NGF only 97.38 ± 10.66 pg/mL. There was ～14-fold increase in the bioavailability of intranasally administered NGF with chitosan than without chitosan. Chitosan can enhance the brain bioavailability of intranasally administered NGF. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3640–3646, 2009     

多层片给药系统在定位和控制释药中的应用

多层片给药系统由含药片层及屏障层组成，通过屏障层控制药物的释放。综述近年来该给药系统在缓控释方面的研究进展，介绍其在定位释药（颊部黏膜给药、胃内定位释药和结肠定位给药）和控制释药形式（双相型释药、零级释药、双峰型释药和延时型释药）方面的应用。     

Dose-Dependent Brain Delivery of Zidovudine Through the Use of a Zidovudine Chemical Delivery System

Pharmaceutical Research -     

Imaging of Cells and Nanoparticles: Implications for Drug Delivery to the Brain

A major challenge in the development of central nervous system drugs is to obtain therapeutic effective drug concentrations inside the brain. Many potentially effective drugs have never reached clinical application because of poor brain penetration. Currently, devices are being developed that may improve drug delivery into the brain. One approach involves the encapsulation of drugs into nanocarriers that are targeted to the brain, where the drug is released. Alternatively, living cells have been engineered to produce the pharmaceutical of interest at the target site. It is important to follow the fate of these drug delivery devices inside the body to verify their efficiency in reaching the brain. To this end, both ex-vivo approaches and in-vivo imaging techniques are used, including ex-vivo biodistribution, autoradiography, MRI, optical imaging, PET and SPECT. All these methods have their specific advantages and limitations. Consequently, selection of the tracking method should be based on the specific aims of the experiment. Here, we will discuss the methods that are currently applied for tracking brain drug delivery devices, including the most commonly used labels and labeling procedures for living cells and nanocarriers. Subsequently, we will discuss specific applications in tracking drug delivery devices.     

人脑恶性胶质瘤生物治疗的临床研究

报告２７例人脑胶质瘤生物治疗的临床结果，ＬＡＫ细胞／ＩＬ－２治疗组１２例，其中复发性星形细胞瘤１０例，丘脑胶质瘤２例，有效率为３３％。ＴＩＬ／ＩＬ－２治疗组１５例，星形细胞瘤及少枝胶质瘤均为术后肿瘤残留者，分别为１２例和３例，有效率为４７％。二组总有效率为４０％。临床研究结果表明ＬＡＫ细胞及ＴＩＬ／ＩＬ－２是治疗人脑胶质瘤安全、有一定疗效的新途径。     

Electrospun Micro- and Nanofibers for Sustained Delivery of Paclitaxel to Treat C6 Glioma in Vitro

Purpose The present study aims to develop electrospun PLGA-based micro- and nanofibers as implants for the sustained delivery of anticancer drug to treat C6 glioma in vitro.Methods PLGA and an anticancer drug—paclitaxel-loaded PLGA micro- and nanofibers were fabricated by electrospinning and the key processing parameters were investigated. The physical and chemical properties of the micro- and nanofibers were characterized by various state-of-the-art techniques, such as scanning electron microscope and field emission scanning electron microscope for morphology, X-ray photoelectron spectroscopy for surface chemistry, gel permeation chromatogram for molecular weight measurements and differential scanning calorimeter for drug physical status. The encapsulation efficiency and in vitro release profile were measured by high performance liquid chromatography. In addition, the cytotoxicity of paclitaxel-loaded PLGA nanofibers was evaluated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide MTT) assay on C6 glioma cell lines.Results PLGA fibers with diameters of around several tens nanometers to 10 μm were successfully obtained by electrospinning. Ultrafine fibers of around 30 nm were achieved after addition of organic salts to dilute polymer solution. The encapsulation efficiency for paclitaxel-loaded PLGA micro- and nanofibers was more than 90%. DSC results suggest that the drug was in the solid solution state in the polymeric micro- and nanofibers. In vitro release profiles suggest that paclitaxel sustained release was achieved for more than 60 days. Cytotoxicity test results suggest that IC50 value of paclitaxel-loaded PLGA nanofibers (36 μg/ml, calculated based on the amount of paclitaxel) is comparable to the commercial paclitaxel formulation-Taxol?.Conclusions Electrospun paclitaxel-loaded biodegradable micro- and nanofibers may be promising for the treatment of brain tumour as alternative drug delivery devices.     

Pharmacoproteomics of Brain Barrier Transporters and Substrate Design for the Brain Targeted Drug Delivery

Pharmaceutical Research - One of the major reasons why central nervous system (CNS)-drug development has been challenging in the past, is the barriers that prevent substances entering from the...