Cell targeting peptide conjugation to siRNA polyplexes for effective gene silencing in cardiomyocytes

To deliver siRNA specifically to cardiomyocytes with a high transfection efficiency, primary cardiomyocyte-targeting (PCM) and/or cell-penetrating (Tat) peptides were incorporated into the siRNA. With the addition of plasmid DNA, these peptide-conjugated siRNAs were able to form compact and stable nanosized polyplex particles with bioreducible poly(CBA-DAH). The peptide-modified siRNA polyplexes enhanced the cellular uptake and the gene-silencing capacity of the siRNA in cardiomyocytes without significant immunogenicity or cytotoxicity. These findings demonstrate that the cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important strategy for cell-specific gene therapy in gene-mediated disease states.     

Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to enhance siRNA activity

Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a approximately 10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine 'apparent' siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.     

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.     

Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to enhance siRNA activity

Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a ～10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine ‘apparent’ siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.     

Triamcinolone acetonide nanoparticles incorporated in thermoreversible gels for age-related macular degeneration

Age-related macular degeneration (AMD) is one of the leading causes of blindness in the US affecting millions yearly. It is characterized by intraocular neovascularization, inflammation and retinal damage which can be ameliorated through intraocular injections of glucocorticoids. However, the complications that arise from repetitive injections as well as the difficulty posed by targeting the posterior segment of the eye make this interesting territory for the development of novel drug delivery systems (DDS). In the present study, we described the development of a DDS composed of triamcinolone acetonide-encapsulated PEGylated PLGA nanoparticles (NP) incorporated into PLGA–PEG–PLGA thermoreversible gel and its use against VEGF expression characteristic of AMD. We found that the NP with mean size of 208?±?1.0?nm showed uniform size distribution and exhibited sustained release of the drug. We also demonstrated that the polymer can be injected as a solution and transition to a gel phase based on the biological temperature of the eye. Additionally, the proposed DDS was non-cytotoxic to ARPE-19 cells and significantly reduced VEGF expression by 43.5?±?3.9% as compared to a 1.53?±?11.1% reduction with triamcinolone. These results suggest the proposed DDS will contribute to the development of novel therapeutic strategies for AMD.     

The impact of AT1002 on the delivery of ritonavir in the presence of bioadhesive polymer, carrageenan

New insights into the modification of the tight junctions theoretically offer the opportunity to regulate the diffusion barrier and then make it possible to investigate a permeation enhancer of low-bioavailability therapeutic agents. AT1002, a minimum biologically active fragment of zonula occludens toxin which reversibly opens intercellular tight junctions after binding to the Zonulin receptor, increased the transport of various molecular weight markers or low-bioavailability agents. The objective of this study was continuously to evaluate the permeation-enhancing ability of AT1002 in the presence of the bioadhesive agent, carrageenan after intranasal administration of the antiretroviral drug, ritonavir, and the permeation enhancement ratio compared with the previous results. The permeation-enhancing effect of AT1002 was significantly promoted by the bioadhesive agent, carrageenan. The administration of ritonavir with AT1002 and carrageenan resulted in a 2.55-fold increase in AUC(0-240min) and a 2.48-fold increase in C(max) compared with the control group. However, AT1002 in the absence of carrageenan did not produce a statistic enhancement in the absorption of ritonavir. Hence, AT1002 together with the addition of carrageenan may open a new approach of research in the tight junction modulated permeation enhancer, and allow the development of the mucosal drug delivery of therapeutic agents.     

Layer-by-layer assembly of poly(l-glutamic acid)/chitosan microcapsules for high loading and sustained release of 5-fluorouracil

Hollow polyelectrolyte microcapsules based on poly(l-glutamic acid) (PLGA) and chitosan (CS) with opposite charges were fabricated by layer-by-layer (LbL) assembly technique using melamine formaldehyde (MF) microparticles as sacrificial templates. The LbL assembly of polyelectrolytes and the resultant PLGA/CS microcapsules were characterized. A hydrophilic anticancer drug, 5-fluorouracil (5-FU), was chosen to investigate the loading and release properties of the microcapsules. The PLGA/CS microcapsules show high loading capacity of 5-FU under conditions of high drug concentration and salt adding. The high loading can be ascribed to spontaneous deposition of 5-FU induced by hydrogen bonding between 5-FU and PLGA/CS microcapsules. The PLGA/CS microcapsules show sustained release behavior. The release rate of 5-FU drastically slows down after loading in PLGA/CS microcapsules. The 5-FU release from PLGA/CS microcapsules can be best described using Ritger-Peppas or Baker-Londale models, indicating the diffusion mechanism of 5-FU release from the PLGA/CS microcapsules. In vitro cytotoxicity evaluation by the MTT assay shows good cell viability over the entire concentration range of PLGA/CS microcapsules. Therefore, the novel PLGA/CS microcapsules are expected to find application in drug delivery systems because of the properties of biodegradability, high loading, sustained release and cell compatibility.     

Nanoporous multilayer poly(L-glutamic acid)/chitosan microcapsules for drug delivery

Nanoporous poly(L-glutamic acid)/chitosan (PLGA/CS) multilayer microcapsules were fabricated by layer-by-layer (LbL) assembly using the porous silica particles as sacrificial templates. The LbL assembled nanoporous PLGA/CS microcapsules were characterized by Zeta-potential analyzer, FTIR, TGA, SEM, TEM and CLSM. 5-Fluorouracil (5-FU) was chosen as model drug. The drug loading content of PLGA/CS microcapsules depends on loading time, loading temperature, pH value and NaCl concentration. High loading capacity of microcapsules can be achieved by simply adjusting pH value and salt concentration. Moreover, 5-Fu loaded microcapsules take on a sustained release behavior, especially in an acid solution, in contrast to burst release of bare 5-Fu. The kinetics of 5-Fu release from PLGA/CS microcapsules conforms to Korsmeyer-Peppas and Baker-Lonsdale models, the mechanism of which can be ascribed to priority of drug diffusion and subordination of polymer degradation. The MTT cytotoxicity assay in vitro reveals the satisfactory anticancer activity of the drug-loaded PLGA/CS microcapsules. Therefore, the novel nanoporous PLGA/CS microcapsules is expected to find application in drug delivery systems.     

The influence of stabilizer and bioadhesive polymer on the permeation-enhancing effect of AT1002 in the nasal delivery of a paracellular marker

Permeation enhancers are of major interest to improve the low bioavailability of therapeutic agents due to poor membrane permeation. AT1002, a six-amino acid fragment of Zonula occludens toxin, was reported to possess permeation-enhancing effects. However, further studies were suggested to focus on the peptide nature of AT1002 like stability and membrane clearance to accurately reflect its permeation-enhancing potential. Thus, this paper focused on the susceptibility of AT1002 for identifying additives to minimize the instability of AT1002, and the permeation-enhancing effect of AT1002 when co-administered with a bioadhesive polymer. The stability study showed that AT1002 were unstable in neutral to basic pH conditions and with increasing incubation time, and 5% dextrose and the 1% mixture of amino acids (arginine, cysteine, glycine) significantly minimized the instability of AT1002 at pH 7.4 for at least 6 hours, respectively. In the intranasal study of a paracellular marker, the administration of mannitol with AT1002 in 5% dextrose solution led to statistically significant 3.14- and 2.17-fold increases in C_max and AUC_0-360min in the presence of carrageenan over the control. Thus, the addition of carrageenan as a bioadhesive polymer and dextrose as a stabilizer together with AT1002 may allow the development of the mucosal drug delivery of low-bioavailability therapeutic agents.     

The influence of AT1002 on the nasal absorption of molecular weight markers and therapeutic agents when co-administered with bioadhesive polymers and an AT1002 antagonist, AT1001

Objectives The purpose of this study was to demonstrate the effects of the tight junction permeation enhancer, AT1002, on the nasal absorption of molecular weight markers and low bioavailable therapeutic agents co‐administered with bioadhesive polymers or zonulin antagonist. Methods The bioadhesive polymers, carrageenan and Na‐CMC, were prepared with AT1002 to examine the permeation‐enhancing effect of AT1002 on the nasal absorption of inulin, calcitonin and saquinavir after nasal administration to Sprague–Dawley rats. Blood samples were collected over a 6‐hour period from a jugular cannula. In addition, we determined whether AT1002 exerts a permeation‐enhancing effect via activation of PAR‐2 specific binding to a putative receptor of zonulin. To examine this zonulin antagonist, AT1001, was administered 30 min prior to dosing with an AT1002/inulin solution and blood samples were collected over a 6‐hour period. Key findings The bioadhesive polymers did not directly increase the absorption of inulin, calcitonin and saquinavir, but promoted the permeation‐enhancing effect of AT1002 when delivered nasally, thereby significantly increasing the absorption of each drug. Pre‐treatment with AT1001 antagonized the zonulin receptor and significantly minimized the permeation‐enhancing effect of AT1002. Conclusion These findings will assist in understanding the permeation‐enhancing capability of and the receptor binding of AT1002. Further, combining AT1002 with carrageenan supports the development of the mucosal delivery of therapeutic agents that have low bioavailability even with bioadhesive agents.     

A biodegradable drug delivery system for the treatment of postoperative inflammation

Cataract surgery is often performed in patients suffering from associated pathologies. Our goal is to develop a biodegradable drug delivery system (DDS) combined with the artificial intraocular lens (IOL). DDS were manufactured using poly(D,L-lactide-co-glycolide), or PLGA, and were loaded with triamcinolone acetonide (TA). The loading capacity was approximately 1050 microg of TA per DDS. The higher the molecular weight of PLGA (34,000, 48,000 and 80,000Da), the slower was the release of TA in vitro. Cataract surgery was performed on the right eye of rabbits. IOL was inserted with (i) no DDS, (ii) unloaded DDS PLGA48000, (iii) one loaded DDS PLGA48000, (iv) two loaded DDS. The number of inflammatory cells and the protein concentration were measured in the aqueous humor (AH). Unloaded DDS showed good ocular biocompatibility. One DDS PLGA48000 loaded with TA significantly reduced postoperative ocular inflammation. Two loaded DDS PLGA48000 was even more effective in inhibiting such inflammation. On long-term observation (days 63 and 84), reduction of inflammation could be obtained by insertion of one DDS PLGA48000 and a second DDS PLGA80000. Therefore, our "all in one" system is very promising since it could replace oral treatment and reduce the number of intraocular injections.     

Preparation and in vitro characterization of amifostine biodegradable microcapsules.

The purpose of this project was to develop sustained release microcapsules of amifostine. The microcapsules were prepared using solvent evaporation technique. The effect of several formulation variables on the characteristics of the microcapsules was studied. The formulation variables studied were drug loading, polymer (polylactide-co-glycolide) (PLGA) concentration, and the amount of gelatin in the initial aqueous phase. The drug loading was studied at three different levels (5, 10, and 25 mg); the PLGA concentration was studied at two levels (500 and 1000 mg); and the amount of gelatin used ranged from 2 to 14 mg. In general, the microcapsules were less than 155 microm in diameter with median size between 50 and 80 microm. While the use of higher amounts of PLGA significantly increased the median size of the microcapsules, using higher amounts of amifostine had no significant effect, irrespective of the amount of PLGA. The use of gelatin, within the range 2-14 mg, did not show any significant effect on the particle size distribution. Scanning electron microscopy (SEM) of the microcapsules revealed that all nine formulations yielded spherical particles. The use of 500 mg PLGA with 10 or 25 mg amifostine yielded microcapsules with porous surfaces. The surface pores, however, were not present in microcapsules prepared using 1000 mg PLGA. The efficiency of encapsulation decreased significantly from 63 to 24% when the amount of amifostine increased from 5 to 25 mg in the formulations using 500 mg PLGA. Similarly, the efficiency of encapsulation decreased from 87 to 23% when the amount of PLGA was doubled to 1000 mg. An increase in the amount of amifostine in the formulation using 500 mg PLGA also resulted in a significant increase in initial drug release (from 20 to 62%) within the first hour. These results were consistent with the porous morphology of these microcapsules. In general, all batches of microcapsules showed 24-96 h sustained drug release.     

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.     

Application of atelocollagen-mediated siRNA delivery for RNAi therapies

RNAi has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Novel treatments and drug discovery in pre-clinical studies based on RNAi are currently targeting a wide range of diseases, including viral infections and cancers by the local administration of synthetic small interfering RNA (siRNA) that target local lesions. Recently, specific methods for the systemic administration of siRNAs have been reported to treat non-human primates or a cancer metastasis model. In vivo siRNA-delivery technology is a key hurdle to the successful therapeutic application of RNAi. This article reviews the non-viral delivery system of atelocollagen for siRNA, which could be useful for functional screening of the genes in vitro and in vivo, and will provide a foundation for further development of RNAi therapeutics.     

Therapeutic potential of chemically modified siRNA: Recent trends

Small interfering RNAs (siRNAs) are one of the valuable tools to investigate the functions of genes and are also used for gene silencing. It has a wide scope in drug discovery through in vivo target validation. siRNA therapeutics are not optimal drug‐like molecules due to poor bioavailability and immunogenic and off‐target effects. To overcome the challenges associated with siRNA therapeutics, identification of appropriate chemical modifications that improves the stability, specificity and potency of siRNA is essential. This review focuses on the various chemical modifications and their implications in siRNA therapy.     

Recent advances in PLGA particulate systems for drug delivery

PLGA is a FDA-approved biocompatible and biodegradable polymer that is widely used in biomedical fields including drug delivery. Micro and nanoparticles based on PLGA have been extensively studied as drug delivery systems. Numerous studies proved that PLGA particulate systems are highly promising drug carriers for tumor targeting as well as pulmonary, oral, ophthalmic and vaginal delivery. PLGA particles can load a variety of classes of drugs including peptides, proteins and siRNA, protect unstable drugs in the body and have an ability to adapt versatile surface functionalities. PLGA particle systems have evolved with advancement of nano and biotechnology in the past decade. This review focuses on novel and innovative PLGA-based particulate drug delivery carriers in recent years.     

The economics of topical immunomodulators for the treatment of atopic dermatitis

Atopic dermatitis is a common, chronic, relapsing inflammatory skin disease frequently affecting infants and children. The worldwide prevalence of atopic dermatitis is estimated to be 5--20% of the paediatric population. First-line therapy has generally consisted of dry skin care, avoidance of triggers, application of topical corticosteroids, and administration of antihistamines and oral antibacterials. Topical corticosteroids improve the lesions of atopic dermatitis; however, concern on the part of physicians and patients regarding adverse effects has led to reluctance to utilise topical corticosteroids early and especially for prolonged periods. Topical immunomodulators (TIMs), including tacrolimus ointment and pimecrolimus cream, were recently introduced for the treatment of atopic dermatitis.Clinical data show that TIMs are effective in atopic dermatitis, yet do not cause the significant adverse effects associated with topical corticosteroids. Questions remain regarding the place of TIMs as a treatment for atopic dermatitis and how to use them most effectively, from both therapeutic and pharmacoeconomic standpoints. Specifically, two major issues remain unresolved: (i) how TIMs measure up to other therapies, especially topical corticosteroids; and (ii) how members of the TIM drug class compare against each other.Previous research has established that atopic dermatitis has a significant impact on quality of life (QOL) and carries a substantial economic burden. Some studies have also measured the utility of various atopic dermatitis disease states. While there is a need for further research, early economic studies provide evidence that TIMs positively affect the QOL of patients and families. In certain patients, TIMs may be cost effective and have an acceptable incremental cost utility compared with topical corticosteroids.Making cost-effectiveness comparisons between tacrolimus and pimecrolimus is challenging because there are limited head-to-head comparative data. Given currently available efficacy data, the results of one study suggest that tacrolimus may be more cost effective than pimecrolimus in paediatric patients with moderate atopic dermatitis.The full economic and QOL benefits of both agents are yet to be completely understood. The studies reviewed herein are the first to delineate the pharmacoeconomic benefits of TIMs in atopic dermatitis, and lay the foundation for future analyses. TIMs represent an exciting advance in the treatment of atopic dermatitis. Additional research will help determine the proper place of TIMs among the current array of therapeutic options for atopic dermatitis.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.