Protamine-carboxymethyl cellulose magnetic nanocapsules for enhanced delivery of anticancer drugs against drug resistant cancers

Multidrug resistance is a major therapeutic challenge faced in the conventional chemotherapy. Nanocarriers are beneficial in the transport of chemotherapeutics by their ability to bypass the P-gp efflux in cancers. Most of the P-gp inhibitors under phase II clinical trial are facing failures and hence there is a need to develop a suitable carrier to address P-gp efflux in cancer therapy. Herein, we prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin against highly drug resistant HeLa cells. The experimental results revealed that improved cellular uptake, enhanced drug intensity profile with greater percentage of apoptotic cells was attained when doxorubicin loaded magnetic nanocapsules were used in the presence of external magnetic field. Hence, we conclude that this magnetic field assisted nanocapsule system can be used for delivery of chemotherapeutics for potential therapeutic efficacy at minimal dose in multidrug resistant cancers. From the Clinical Editor: Many cancer drugs fail when cancer cells become drug resistant. Indeed, multidrug resistance (MDR) is a major therapeutic challenge. One way that tumor cells attain MDR is by over expression of molecular pumps comprising of P-glycoprotein (P-gp) and multidrug resistant proteins (MRP), which can expel chemotherapeutic drugs out of the cells. In this study, the authors prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin. The results show that there was better drug delivery and efficacy even against MDR tumor cells. (C) 2015 Elsevier Inc. All rights reserved.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery

Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp(2) pi-pi interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through ``click'' chemistry to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a positive zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was observed. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.

Biofunctionalized surface-modified silver nanoparticles for gene delivery

Silver nanoparticles (AgNPs) find use in different biomedical applications including wound healing and cancer. We propose that the efficacy of the nanoparticles can be further augmented by using these particles for gene delivery applications. The objective of this work was to engineer biofunctionalized stable AgNPs with good DNA binding ability for efficient transfection and minimal toxicity. Herein, we report on the one-pot facile green synthesis of polyethylene glycol (PEG) stabilized chitosan-g-polyacrylamide modified AgNPs. The size of the PEG stabilized AgNPs was 38 +/- 4 nm with a tighter size distribution compared to the unstabilized nanoparticles which showed bimodal distribution of particle sizes of 68 +/- 5 nm and 7 +/- 4 nm. To enhance the efficiency of gene transfection, the Arg-Gly-Asp-Ser (RGDS) peptide was immobilized on the silver nanoparticles. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 +/- 4% and 30 +/- 3% in HeLa and A549 cells, respectively, and significantly higher than 34 +/- 3% and 23 +/- 2%, respectively, with the use of polyethyleneimine (25 kDa). These nanoparticles were found to induce minimal cellular toxicity. Differences in cellular uptake mechanisms with RGDS immobilization resulting in improved efficiency are elucidated. This study presents biofunctionalized AgNPs for potential use as efficient nonviral carriers for gene delivery with minimal cytotoxicity toward augmenting the therapeutic efficacy of AgNPs used in different biomedical products.

Development of cup shaped microneedle array for transdermal drug delivery

Microneedle technology is one of the attractive methods in transdermal drug delivery. However, the clinical applications of this method are limited owing to: complexity in the preparation of multiple coating solutions, drug leakage while inserting the microneedles into the skin and the outer walls of the solid microneedle can hold limited quantity of drug. Here, the authors present the fabrication of an array of rectangular cup shaped silicon microneedles, which provide for reduced drug leakage resulting in improvement of efficiency of drug delivery and possibility of introducing multiple drugs. The fabricated solid microneedles with rectangular cup shaped tip have a total height of 200 mu m. These cup shaped tips have dimensions: 60 x 60 mu m (length x breadth) with a depth of 60 mu m. The cups are filled with drug using a novel in-house built drop coating system. Successful drug dissolution was observed when the coated microneedle was used on mice. Also, using the above method, it is possible to fill the cups selectively with different drugs, which enables simultaneous multiple drug delivery. (C) 2015 American Vacuum Society.

Chitosan-dextran sulphate nanocapsule drug delivery system as an effective therapeutic against intraphagosomal pathogen Salmonella

Objectives: The ability to target conventional drugs efficiently inside cells to kill intraphagosomal bacteria has been a major hurdle in treatment of infective diseases. We aimed to develop an efficient drug delivery system for combating infection caused by Salmonella, a well-known intracellular and intraphagosomal pathogen. Chitosan dextran sulphate (CD) nanocapsules were assessed for their efficiency in delivering drugs against Salmonella. Methods: The CD nanocapsules were prepared using the layer-by-layer method and loaded with ciprofloxacin or ceftriaxone. Antibiotic-loaded nanocapsules were analysed in vitro for their ability to enter epithelial and macrophage cells to kill Salmonella. In vivo pharmacokinetics and organ distribution studies were performed to check the efficiency of the delivery system. The in vivo antibacterial activity of free antibiotic and antibiotic loaded into nanocapsules was tested in a murine salmonellosis model. Results: In vitro and in vivo experiments showed that this delivery system can be used effectively to clear Salmonella infection, CD nanocapsules were successfully employed for efficient targeting and killing of the intracellular pathogen at a dosage significantly lower than that of the free antibiotic. The increased retention time of ciprofloxacin in the blood and organs when it was delivered by CD nanocapsules compared with the conventional routes of administration may be the reason underlying the requirement for a reduced dosage and frequency of antibiotic administration. Conclusions: CD nanocapsules can be used as an efficient drug delivery system to treat intraphagosomal pathogens, especially Salmonella infection, This delivery system might be used effectively for other vacuolar pathogens including Mycobacteria, Brucella and Legionella.

Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications

In this study we have investigated a new class of cationic lipids - ``bolaamphiphiles'' or ``bolas'' - for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5 mu g/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production. Published by Elsevier B.V.

STX13 regulates cargo delivery from recycling endosomes during melanosome biogenesis

Melanosomes are a class of lysosome-related organelles produced by melanocytes. Biogenesis of melanosomes requires the transport of melanin-synthesizing enzymes from tubular recycling endosomes to maturing melanosomes. The SNARE proteins involved in these transport or fusion steps have been poorly studied. We found that depletion of syntaxin 13 (STX13, also known as STX12), a recycling endosomal Qa-SNARE, inhibits pigment granule maturation in melanocytes by rerouting the melanosomal proteins such as TYR and TYRP1 to lysosomes. Furthermore, live-cell imaging and electron microscopy studies showed that STX13 co-distributed with melanosomal cargo in the tubular-vesicular endosomes that are closely associated with the maturing melanosomes. STX family proteins contain an N-terminal regulatory domain, and deletion of this domain in STX13 increases both the SNARE activity in vivo and melanosome cargo transport and pigmentation, suggesting that STX13 acts as a fusion SNARE in melanosomal trafficking pathways. In addition, STX13-dependent cargo transport requires the melanosomal R-SNARE VAMP7, and its silencing blocks the melanosome maturation, reflecting a defect in endosome-melanosome fusion. Moreover, we show mutual dependency between STX13 and VAMP7 in regulating their localization for efficient cargo delivery to melanosomes.

Stimuli-responsive weak polyelectrolyte multilayer films: A thin film platform for self triggered multi-drug delivery

Polyelectrolyte multilayer (PEM) thin film composed of weak polyelectrolytes was designed by layer-by-layer (LbL) assembly of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMA) for multi-drug delivery applications. Environmental stimuli such as pH and ionic strength showed significant influence in changing the film morphology from pore-free smooth structure to porous structure and favored triggered release of loaded molecules. The film was successfully loaded with bovine serum albumin (BSA) and ciprofloxacin hydrochloride (CH) by modulating the porous polymeric network of the film. Release studies showed that the amount of release could be easily controlled by changing the environmental conditions such as pH and ionic strength. Sustained release of loaded molecules was observed up to 8 h. The fabricated films were found to be biocompatible with epithelial cells during in-vitro cell culture studies. PEM film reported here not only has the potential to be used as self-responding thin film platform for transdermal drug delivery, but also has the potential for further development in antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2015 Elsevier B.V. All rights reserved.

Rab9A is required for delivery of cargo from recycling endosomes to melanosomes

Melanosomes are a type of lysosome-related organelle that is commonly defective in Hermansky-Pudlak syndrome. Biogenesis of melanosomes is regulated by BLOC-1, -2, -3, or AP-1, -3 complexes, which mediate cargo transport from recycling endosomes to melanosomes. Although several Rab GTPases have been shown to regulate these trafficking steps, the precise role of Rab9A remains unknown. Here, we found that a cohort of Rab9A associates with the melanosomes and its knockdown in melanocytes results in hypopigmented melanosomes due to mistargeting of melanosomal proteins to lysosomes. In addition, the Rab9A-depletion phenotype resembles Rab38/ 32-inactivated or BLOC-3-deficient melanocytes, suggesting that Rab9A works in line with BLOC-3 and Rab38/ 32 during melanosome cargo transport. Furthermore, silencing of Rab9A, Rab38/ 32 or its effector VARP, or BLOC-3-deficiency in melanocytes decreased the length of STX13-positive recycling endosomal tubules and targeted the SNARE to lysosomes. This result indicates a defect in directing recycling endosomal tubules to melanosomes. Thus, Rab9A and its co-regulatory GTPases control STX13-mediated cargo delivery to maturing melanosomes.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Stimuli-responsive protamine-based biodegradable nanocapsules for enhanced bioavailability and intracellular delivery of anticancer agents

Enzyme-and pH-responsive polyelectrolyte nanocapsules having diameters in the range of 200 +/- 20 nm were fabricated by means of Layer-by-Layer assembly of biopolymers, protamine, and heparin, and then loaded with anticancer drug doxorubicin. The incorporation of the FDA-approved peptide drug protamine as a wall component rendered the capsules responsive to enzyme stimuli. The stimuli-responsive drug release from these nanocapsules was evaluated, and further modulation of capsule permeability to avoid premature release was demonstrated by crosslinking the wall components. The interaction of the nanocapsules with cancer cells was studied using MCF-7 breast cancer cells. These capsules were readily internalized and disintegrated inside the cells, culminating in the release of the loaded doxorubicin and subsequent cell death as observed by confocal microscopy and MTT Assay. The bioavailability studies performed using BALB/c mice revealed that the encapsulated doxorubicin exhibited enhanced bioavailability compared to free doxorubicin. Our results indicate that this stimuli-responsive system fabricated from clinically used FDA-approved molecules and exhibiting minimal premature release has great potential for drug-delivery applications.

Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-a-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.