Raghavendra C. Mundargi

Layer‐by‐Layer Nanoparticles as an Efficient siRNA Delivery Vehicle for SPARC Silencing

Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/μg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.

Natural Sunflower Pollen as a Drug Delivery Vehicle

In nature, pollen grains play a vital role for encapsulation. Many pollen species exist which are often used as human food supplements. Dynamic image particle analysis, scanning electron microscopy, and confocal microscopy analysis confirmed the size, structural uniformity, and macromolecular encapsulation in sunflower pollen, paving the way to explore natural pollen grains for the encapsulation of therapeutic molecules.

Eco-friendly streamlined process for sporopollenin exine capsule extraction.

Sporopollenin exine capsules (SECs) extracted from Lycopodium clavatum spores are an attractive biomaterial possessing a highly robust structure suitable for microencapsulation strategies. Despite several decades of research into SEC extraction methods, the protocols commonly used for L. clavatum still entail processing with both alkaline and acidolysis steps at temperatures up to 180 °C and lasting up to 7 days. Herein, we demonstrate a significantly streamlined processing regimen, which indicates that much lower temperatures and processing durations can be used without alkaline lysis. By employing CHN elemental analysis, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and dynamic image particle analysis (DIPA), the optimum conditions for L. clavatum SEC processing were determined to include 30 hours acidolysis at 70 °C without alkaline lysis. Extending these findings to proof-of-concept encapsulation studies, we further demonstrate that our SECs are able to achieve a loading of 0.170 ± 0.01 g BSA per 1 g SECs by vacuum-assisted loading. Taken together, our streamlined processing method and corresponding characterization of SECs provides important insights for the development of applications including drug delivery, cosmetics, personal care products, and foods.

Extraction of sporopollenin exine capsules from sunflower pollen grains

Sporopollenin exine capsules (SECs) are highly robust natural microscale capsules that can be extracted from plant spores and pollen grains, albeit through complex processing schemes. Herein, we report new insights into pollen processing by alkaline lysis and acidolysis with various process conditions. Alkaline lysis of sunflower pollen grains damages the unique pollen microstructure and acidolysis enables us to devise a simple process to extract SECs from sunflower pollen grains with a uniform particle size distribution. The SECs retain the natural morphology, offering an improved general scheme to streamline pollen processing for biomaterial applications.

5-Flurouracil microencapsulation and impregnation in hyaluronic acid hydrogel as composite drug delivery system for ocular fibrosis

Abstract Effective and sustained release formulations of antimetabolite 5-Flurouracil (5Fu) for ocular fibrosis are highly desirable for success of glaucoma filtration surgery (GFS). However, burst release and rapid clearance of carriers and/or drug still exist as major limitations. Here, we report the feasibility of encapsulating 5Fu in sustained release carrier of poly (d,l-lactide-co-glycolide) (PLGA) microspheres (MPs) and impregnating in a hyaluronic acid (HA) hydrogel as unit dose formulation. In order to optimize the encapsulation process by solid-in-oil-in-water emulsion technique, the effects of PLGA end groups, PLGA concentration, and 5Fu loading were studied systematically. The MPs were extensively characterized for surface morphology, particle size distribution, thermal properties, crystallinity, residual solvent, syringeability, and in vitro release. The optimized formulation of MPs was ~94 μm in size and released 5Fu in a sustained manner for up to two weeks. In our concept, MPs are dispersed first in HA solution and then cross-linked in situ after injection into the conjunctival space. Thus, syringeability experiments were also carried out with 5Fu-loaded MPs and 0.5% HA solution. To further prolong 5Fu release, a derivatized HA with methacrylic anhydride was photocross-linked along with 5Fu-loaded PLGA MPs to develop the composite hydrogel formulation. Our experimental results show that 5Fu release can be effectively controlled by PLGA MPs-loaded HA composite hydrogel for longer therapeutic benefits. These composite hydrogel systems improve the ocular residence time of the carrier and the drug, and hence could be potentially explored as injectable unit dose formulation for GFS.

Novel Sensor-Enabled Ex Vivo Bioreactor: A New Approach towards Physiological Parameters and Porcine Artery Viability

The aim of the present work is to design and construct an ex vivo bioreactor system to assess the real time viability of vascular tissue. Porcine carotid artery as a model tissue was used in the ex vivo bioreactor setup to monitor its viability under physiological conditions such as oxygen, pressure, temperature, and flow. The real time tissue viability was evaluated by monitoring tissue metabolism through a fluorescent indicator “resorufin.” Our ex vivo bioreactor allows real time monitoring of tissue responses along with physiological conditions. These ex vivo parameters were vital in determining the tissue viability in sensor-enabled bioreactor and our initial investigations suggest that, porcine tissue viability is considerably affected by high shear forces and low oxygen levels. Histological evaluations with hematoxylin and eosin and Massons trichrome staining show intact endothelium with fresh porcine tissue whereas tissues after incubation in ex vivo bioreactor studies indicate denuded endothelium supporting the viability results from real time measurements. Hence, this novel viability sensor-enabled ex vivo bioreactor acts as model to mimic in vivo system and record vascular responses to biopharmaceutical molecules and biomedical devices.

Extraction of cage-like sporopollenin exine capsules from dandelion pollen grains

Pollen-based microcapsules such as hollow sporopollenin exine capsules (SECs) have emerged as excellent drug delivery and microencapsulation vehicles. To date, SECs have been extracted primarily from a wide range of natural pollen species possessing largely spherical geometries and uniform surface features. Nonetheless, exploring pollen species with more diverse architectural features could lead to new application possibilities. One promising class of candidates is dandelion pollen grains, which possess architecturally intricate, cage-like microstructures composed of robust sporopollenin biopolymers. Here, we report the successful extraction and macromolecular loading of dandelion SECs. Preservation of SEC morphology and successful removal of proteinaceous materials was evaluated using scanning electron microscopy (SEM), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, elemental CHN analysis, dynamic image particle analysis (DIPA) and confocal laser scanning microscopy (CLSM). Among the tested processing schemes, acidolysis using 85% (v/v) phosphoric acid refluxed at 70 °C for 5 hours yielded an optimal balance of intact particle yield, protein removal, and preservation of cage-like microstructure. For proof-of-concept loading, bovine serum albumin (BSA) was encapsulated within the dandelion SECs with high efficiency (32.23 ± 0.33%). Overall, our findings highlight how hollow microcapsules with diverse architectural features can be readily prepared and utilized from plant-based materials.

Nanomedicine: Size-Related Drug Delivery Applications, Including Periodontics and Endodontics

In this chapter, we discuss polymer- and liposome-based nanocarriers used in the delivery of bioactive molecules, from drugs to proteins. The focus is on the enhancements in efficacy of bioactive molecules when nanotechnology is used for delivering them. The perspective centres around commercial and clinical successes and a rationalization of these successes. Microparticulate systems are also discussed in relation to their nano-counterparts, and the advantages of nano size are emphasized in relevant applications. In general, the main application of nanocarriers is in cancer therapy; however, with the ability to programme sustained release of bioactive molecules from certain types of nanoparticles, other applications in ocular, cardiovascular and periodontic/endodontic therapy may be possible.

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.