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.

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.

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.

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.

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 Masson's 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.

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.

Poly(N-vinylcaprolactam-co-methacrylic acid) hydrogel microparticles for oral insulin delivery.

pH-sensitive copolymeric hydrogels prepared from N-vinylcaprolactam and methacrylic acid monomers by free radical polymerization offered 52% encapsulation efficiency and evaluated for oral delivery of human insulin. The in vitro experiments performed on insulin-loaded microparticles in pH 1.2 media (stomach condition) demonstrated no release of insulin in the first 2 h, but almost 100% insulin was released in pH 7.4 media (intestinal condition) in 6 h. The carrier was characterized by Fourier transform infrared, differential scanning calorimeter, thermogravimetry and nuclear magnetic resonance techniques to confirm the formation of copolymer, while scanning electron microscopy was used to assess the morphology of hydrogel microparticles. The in vivo experiments on alloxan-induced diabetic rats showed the biological inhibition up to 50% and glucose tolerance tests exhibited 44% inhibition. The formulations of this study are the promising carriers for oral delivery of insulin.

pH-Sensitive oral insulin delivery systems using Eudragit microspheres.

In this paper, we present in vitro and in vivo release data on pH-sensitive microspheres of Eudragit L100, Eudragit RS100 and their blend systems prepared by double emulsion-solvent evaporation technique for oral delivery of insulin. Of the three systems developed, Eudragit L100 was chosen for preclinical studies. Insulin was encapsulated and in vitro experiments performed on insulin-loaded microspheres in pH 1.2 media did not release insulin during the first 2 h, but maximum insulin was released in pH 7.4 buffer media from 4 to 6 h. The microspheres were characterized by scanning electron microscopy to understand particle size, shape and surface morphology. The size of microspheres ranged between 1 and 40 µm. Circular dichroism spectra indicated the structural integrity of insulin during encapsulation as well as after its release in pH 7.4 buffer media. The in vivo release studies on diabetic-induced rat models exhibited maximum inhibition of up to 86%, suggesting absorption of insulin in the intestine.

Randomized, controlled, single-masked, clinical study to compare and evaluate the efficacy of microspheres and gel in periodontal pocket therapy.

BACKGROUND: The aim of this randomized, split-mouth, single-masked study is to compare the efficacy of a gel and microspheres as drug-delivery systems in the treatment of periodontal disease. METHODS: Microspheres were prepared, the release patterns of the microspheres and gel formulations were analyzed using an ultraviolet spectrophotometer, and particle shapes were studied under a scanning electron microscope. A split-mouth design was followed in which 30 potential sites were identified and divided into three groups: one control group and two groups in which microspheres or a gel was placed. Patients were recalled at 1, 3, 6, and 9 months. Clinical recordings included plaque index (PI), gingival index (GI), probing depth (PD), and relative attachment level (RAL) measurements; subgingival plaque was also obtained for microbiologic examination prior to and after therapy. RESULTS: Microspheres had a more sustained release and a high initial drug concentration. There was a significant improvement in the PI and GI in the initial 3 months. The results were statistically significant at P = 0.01. The mean PD scores among scores for the three groups at baseline and follow-up visits showed a reduction of 0.4 to 1 mm. The microbiologic parameters were also statistically significant. CONCLUSION: These data suggest that the type of delivery system could significantly influence the outcome of therapy.

Developments in polymeric devices for oral insulin delivery.

BACKGROUND: Development of improved oral insulin administration is necessary for the treatment of diabetes mellitus, to overcome the problem of daily subcutaneous injections. The vast amount of literature data on oral insulin delivery prompted us to cover this area in a review. OBJECTIVE: Insulin delivery using polymeric devices is discussed, with an ultimate aim of addressing the technological development in this area. METHODS: The development of oral delivery devices for insulin using hydrogels and micro/nanoparticles is discussed with reference to polymers. These efforts must be directed to increase the residence time of insulin near the intestinal absorptive cells. RESULTS/CONCLUSION: The published results on oral insulin delivery devices, particularly on inter-polymer complexes of the grafted copolymers, are discussed in greater depth. The use of absorption enhancers like cyclodextrins, bile salts and surfactants is covered. The state-of-the-art technology and challenges in this area are discussed, with typical examples.

Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives.

Biodegradable nano/microparticles of poly(D,L-lactide-co-glycolide) (PLGA) and PLGA-based polymers are widely explored as carriers for controlled delivery of macromolecular therapeutics such as proteins, peptides, vaccines, genes, antigens, growth factors, etc. These devices are mainly produced by emulsion or double-emulsion technique followed by solvent evaporation or spray drying. Drug encapsulation, particle size, additives added during formulation, molecular weight, ratio of lactide to glycolide moieties in PLGA and surface morphology could influence the release characteristics. Encapsulation efficiency and release rates through nano/microparticle-mediated drug delivery devices can be optimized to improve their therapeutic efficacy. In this review, important findings of the past decade on the encapsulation and release profiles of macromolecular therapeutics from PLGA and PLGA-based nano/microparticles are discussed critically in relation to nature and type of bioactive molecule, carrier polymer and experimental variables that influence the delivery of macromolecular therapeutics. Even though extensive research on biodegradable microparticles containing macromolecular drugs has greatly advanced to the level of production know-how, the effects of critical parameters influencing drug encapsulation are not sufficiently investigated for nano-scaled carriers. The present review attempts to address some important data on nano/microparticle-based delivery systems of PLGA and PLGA-derived polymers with reference to macromolecular drugs.

Development of polysaccharide-based colon targeted drug delivery systems for the treatment of amoebiasis.

The main focus of this study is to develop colon targeted drug delivery systems for metronidazole (MTZ). Tablets were prepared using various polysaccharides or indigenously developed graft copolymer of methacrylic acid with guar gum (GG) as a carrier. Various polysaccharides such as GG, xanthan gum, pectin, carrageenan, beta-cyclodextrin (CD) or methacrylic acid-g-guar (MAA-g-GG) gum have been selected and evaluated. The prepared tablets were tested in vitro for their suitability as colon-specific drug delivery systems. To further improve the colon specificity, some selected tablet formulations were enteric coated with Eudragit-L 100 to give protection in an acidic environment. Drug release studies were performed in simulated gastric fluid (SGF) for 2 hr followed by simulated intestinal fluid (SIF) at pH 7.4. The dissolution data demonstrate that the rate of drug release is dependent upon the nature and concentration of polysaccharide/polymer used in the formulations. Uncoated tablets containing xanthan gum or mixture of xanthan gum with graft copolymer showed 30-40% drug release during the initial 4-5 hr, whereas for tablets containing GG with the graft copolymer, it was 70%. After enteric coating, the release was drastically reduced to 18-24%. The other polysaccharides were unable to protect drug release under similar conditions. Preparations with xanthan gum as a matrix showed the time-dependent release behavior. Further, in vitro release was performed in the dissolution media with rat caecal contents. Results indicated an enhanced release when compared to formulations studied in dissolution media without rat caecal contents, because of microbial degradation or polymer solubilization. The nature of drug transport was found to be non-Fickian in case of uncoated formulations, whereas for the coated formulations, it was found to be super-Case-II. Statistical analyses of release data indicated that MTZ release is significantly affected by the nature of the polysaccharide used and enteric coating of the tablet. Differential scanning calorimetry indicated the presence of crystalline nature of drug in the formulations.

Development and evaluation of novel biodegradable microspheres based on poly(d,l-lactide-co-glycolide) and poly(epsilon-caprolactone) for controlled delivery of doxycycline in the treatment of human periodontal pocket: in vitro and in vivo studies.

This study reports on the development of novel biodegradable microspheres prepared by water-in-oil-water (W/O/W) double emulsion technique using the blends of poly(d,l-lactide-co-glycolide) (PLGA) and poly(epsilon-caprolactone) (PCL) in different ratios for the controlled delivery of doxycycline (DXY). Doxycycline encapsulation of up to 24% was achieved within the polymeric microspheres. Blend placebo microspheres, drug-loaded microspheres and pristine DXY were analyzed by Fourier transform infrared spectroscopy (FT-IR), which indicated no interaction between drug and polymers. Differential scanning calorimetry (DSC) on drug-loaded microspheres confirmed the polymorphism of DXY and indicated a molecular level dispersion of DXY in the microspheres. Scanning electron microscopy (SEM) confirmed the spherical nature and smooth surfaces of the microspheres produced. Mean particle size of the microspheres as measured by dynamic laser light scattering method ranged between 90 and 200 mum. In vitro release studies performed in 7.4 pH media indicated the release of DXY from 7 to 11 days, depending upon the blend ratio of the matrix. Up to 11 days, DXY concentrations in the gingival crevicular fluid were higher than the minimum inhibitory concentration of DXY against most of the periodontal pathogens. One of the developed formulations was subjected to in vivo efficacy studies in thirty sites of human periodontal pockets. Significant results were obtained with respect to both microbiological and clinical parameters up to 3 months even as compared to commercial DXY gel. Statistical analyses of the release data and in vivo results were performed using the analysis of variance (ANOVA) method.

Evaluation and controlled release characteristics of modified xanthan films for transdermal delivery of atenolol.

The present study was performed to evaluate the possibility of using modified xanthan films as a matrix system for transdermal delivery of atenolol (ATL), which is an antihypertensive drug. Acrylamide was grafted onto xanthan gum (XG) by free radical polymerization using ceric ion as an initiator. Fourier transform infrared spectroscopy and differential scanning calorimetry indicated the formation of the graft copolymer. The obtained graft copolymer was loaded with ATL and films were fabricated by solution casting method for transdermal application. Various formulations were prepared by varying the grafting ratio, drug loading, and different penetration enhancers. The formulations prepared were characterized for weight, thickness uniformity, water vapor transmission rate, and uniformity in drug content of the matrix. All the thin films were slightly opaque, smooth, flexible, and permeable to water vapor, indicating their permeability characteristics suitable for transdermal studies. Fourier transform infrared spectroscopy and differential scanning calorimetry studies indicated no significant interactions between drug and polymer. Drug is distributed uniformly in the matrix but showed a slight amorphous nature. Drug-loaded films were analyzed by X-ray diffraction to understand the drug polymorphism inside the films. Scanning electron microscopic studies of the placebo and drug-loaded films demonstrated a remarkable change in their surface morphology. The skin irritation tests were performed in mice and these results suggested that both placebo and drug-loaded films produced negligible erythema and edema compared to formalin (0.8% v/v) as the standard irritant. The in vitro drug release studies were performed in phosphate buffer saline using a Keshary-Chien diffusion cell. Different formulations were prepared and variations in drug release profiles were observed. Release data were analyzed by using the Ritger and Peppas equation to understand the mechanism of drug release as well as the estimation of n values, which ranged between 0.41 and 0.53, suggesting a Fickian diffusion trend.