Sporopollenin Exine Research Articles

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.

Preserving the inflated structure of lyophilized sporopollenin exine capsules with polyethylene glycol osmolyte

Extracted from natural pollen grains, sporopollenin exine capsules (SECs) are robust, chemically inert biopolymer shells that posess highly uniform size and shape characteristics and that can be utilized as hollow microcapsules for drug delivery applications. However, it is challenging to extract fully functional SECs from many pollen species because pollen grains often collapse, causing the loss of architectural features, loading volume, and bulk uniformity. Herein, we demonstrate that polyethylene glycol (PEG) osmolyte solutions can help preserve the native architectural features of extracted SECs, yielding inflated microcapsules of high uniformity that persist even after subsequent lyophilization. Optimal conditions were first identified to extract SECs from cattail (Typhae angustfolia) pollen via phosphoric acid processing after which successful protein removal was confirmed by elemental (CHN), mass spectrometry (MALDI-TOF), and confocal laser canning microscopy (CLSM) analyses. The shape of SECs was then assessed by scanning electron microscopy (SEM) and dynamic image particle analysis (DIPA). While acid-processed SECs experienced high degrees of structural collapse, incubation in 2.5% or higher PEG solutions significantly improved preservation of spherical SEC shape by inducing inflation within the microcapsules. A theoretical model of PEG-induced osmotic pressure effects was used to interpret the experimental data, and the results show excellent agreement with the known mechanical properties of pollen exine walls. Taken together, these findings demonstrate that PEG osmolyte is a useful additive for preserving particle shape in lyophilized SEC formulations, opening the door to broadly applicable strategies for stabilizing the structure of hollow microcapsules.

Bioinspired Spiky Micromotors Based on Sporopollenin Exine Capsules

The development of a micromotor with unique spiky morphology based on sporopollenin exine capsules (SECs) is reported here. A widely abundant natural material extracted from sunflower pollen grains, the SECs are physically robust, highly monodisperse microcapsules that are ornamented with spiky appendages, opening the door to exploring bubble generation on this unique biomaterial surface. Partial platinum coating on the SEC surface enables catalytic decomposition of hydrogen peroxide that leads to bubble‐propelled motion of individual SECs. Moreover, the hollow capsule architecture provides a large internal cavity for macromolecular encapsulation, as demonstrated here by the loading and transport of bovine serum albumin. Taking advantage of the sporopollenin biopolymer's capacity for heavy metal binding, it is further demonstrated that fluid mixing induced by the motion and bubble generation of the SEC micromotors dramatically improves heavy metal binding and removal. The bioinspired micromotors combine the advantageous properties of SECs with autonomous motion ability, resulting in a multifunctional platform for drug delivery and water purification applications.

Plant‐Based Hollow Microcapsules for Oral Delivery Applications: Toward Optimized Loading and Controlled Release

Efficient oral administration of protein‐based therapeutics faces significant challenges due to degradation from the highly acidic conditions present in the stomach and proteases present in the digestive tract. Herein, investigations into spike‐covered sunflower sporopollenin exine capsules (SECs) for oral protein delivery using bovine serum albumin (BSA) as a model drug are reported and provide significant insights into the optimization of SEC extraction, SEC loading, and controlled release. The phosphoric‐acid‐based SEC extraction process is optimized. Compound loading is shown to be driven by the evacuation of air bubbles from SEC cavities through the porous SEC shell wall, and vacuum loading is shown to be the optimal loading method. Three initial BSA‐loading proportions are evaluated, leading to a practical loading efficiency of 22.3 ± 1.5 wt% and the determination that the theoretical maximum loading is 46.4 ± 2.5 wt%. Finally, an oral delivery formulation for targeted intestinal delivery is developed by tableting BSA‐loaded SECs and enteric coating. BSA release is inhibited for 2 h in simulated gastric conditions followed by 100% release within 8 h in simulated intestinal conditions. Collectively, these results indicate that sunflower SECs provide a versatile platform for the oral delivery of therapeutics.

Chemical processing strategies to obtain sporopollenin exine capsules from multi-compartmental pine pollen

Abstract Pine pollen is widely used in traditional Chinese medicine and has been consumed as a food product for thousands of years. Owing to wind pollination, its pollen grains are composed of a sporoplasmic central cavity along with two empty air sac compartments. While this architectural configuration is evolutionarily optimized for wind dispersal, such features also lend excellent potential for encapsulating materials, especially in the context of preparing sporopollenin exine capsules (SECs). Herein, we systematically evaluated one-pot acid processing methods in order to generate pine pollen SECs that support compound loading. Morphological properties of the SECs were analysed by scanning electron microscopy (SEM) and dynamic imaging particle analysis (DIPA), and protein removal was evaluated by CHN elemental analysis and confocal laser scanning microscopy (CLSM). It was identified that 5-h acidolysis with 85% w/v phosphoric acid at 70 °C yielded an optimal balance of high protein removal and preservation of microcapsule architecture, while other processing methods were also feasible with an additional enzymatic step. Importantly, the loading efficiency of the pine pollen SECs was three-times greater than that of natural pine pollen, highlighting their potential for microencapsulation. Taken together, our findings outline a successful strategy to prepare intact pine pollen SECs and demonstrate for the first time that SECs can be prepared from multi-compartmental pollen capsules, opening the door to streamlined processing approaches to utilize pine pollen microcapsules in industrial applications.

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.

Inflated Sporopollenin Exine Capsules Obtained from Thin-Walled Pollen.

Sporopollenin is a physically robust and chemically resilient biopolymer that comprises the outermost layer of pollen walls and is the first line of defense against harsh environmental conditions. The unique physicochemical properties of sporopollenin increasingly motivate the extraction of sporopollenin exine capsules (SECs) from pollen walls as a renewable source of organic microcapsules for encapsulation applications. Despite the wide range of different pollen species with varying sizes and wall thicknesses, faithful extraction of pollen-mimetic SECs has been limited to thick-walled pollen capsules with rigid mechanical properties. There is an unmet need to develop methods for producing SECs from thin-walled pollen capsules which constitute a large fraction of all pollen species and have attractive materials properties such as greater aerosol dispersion. Herein, we report the first successful extraction of inflated SEC microcapsules from a thin-walled pollen species (Zea mays), thereby overcoming traditional challenges with mechanical stability and loss of microstructure. Morphological and compositional characterization of the SECs obtained by the newly developed extraction protocol confirms successful protein removal along with preservation of nanoscale architectural features. Looking forward, there is excellent potential to apply similar strategies across a wide range of unexplored thin-walled pollen species.

Macroporous natural capsules extracted from Phoenix dactylifera L. spore and their application in oral drugs delivery

Macroporous natural sporopollenin exine capsules (SEC) were extracted from date palm (Phoenix dactylifera L.) and coated by natural polymer composite (carboxymethyl cellulose with epichlorohydrin). The polymer coated exine capsules (PCEC) were used in in-vitro investigations for controlled delivery of paracetamol. SEC, PCEC, and drugs loaded capsules (PCEC-PAR) were characterized by scanning electron microscope (SEM), surface area (BET), Fourier-transform infrared (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The length of SEC was found to be 20–20.5μm, and the pore sized was 50–135nm, as measured using SEM. The studies revealed that maximum loading of the drug was at pH 6.0 (97.2%, with 50mgmL−1). The results indicate that by increasing the pH from 1.4 to 7.4, the cumulative release rates of paracetamol in physiological buffer solution (PBS) is more than two times as in simulated gastric fluid (SGF). In addition, the in-vitro toxicity of PCEC against Caco-2 cells was tested by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, and the results revealed that PCEC are biocompatible materials. The overall results encourage further studies on the clinical use of PCEC as drug carriers.

Delivery of ibuprofen by natural macroporous sporopollenin exine capsules extracted from Phoenix dactylifera L.

Sporopollenin macroporous capsules (SMCs) were extracted from date palm (Phoenix dactylifera L.) spores and coated by a natural polymer composite (chitosan with glutaraldehyde). The polymer coated macroporous capsules SMC@poly were used in the in vitro-controlled delivery of ibuprofen. The materials obtained were characterized through spectral, thermal, scanning electron microscopy (SEM), X-ray diffraction (XRD), and nitrogen adsorption–desorption isotherms. The IBU loading and releasing was studied by investigating the changes in various factors such as pH, temperature, and initial concentration. The results revealed that the loading of IBU increased when the concentration of IBU was decreased, following the Langmuir adsorption isotherm. The maximum loading of the IBU was observed at pH6.0 (97.2%, with 50mg/mL). The releasing results indicate that IBU was released faster when the pH was changed from 1.4 to 7.4. In addition, the cytotoxicity of the SMC, SMC@poly, and SMC@poly–IBU were tested against human intestinal Caco-2 cell line using MTT assay, and the results revea'led that all the materials in this study were biocompatible.

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

A simple extraction process was developed to isolate sunflower sporopollenin exine capsules (SECs). The sunflower SECs holds significant potential as biomaterial for applications in drug delivery, cosmetics, and food technology.

Sporopollenin, The Least Known Yet Toughest Natural Biopolymer

Sporopollenin is highly cross-linked polymer composed of carbon, hydrogen and oxygen that is extraordinarily stable and has been found chemically intact in sedimentary rocks some 500 million year old. It is the outer shell (exine) of plant spores and pollen and when extracted it is in the form of an empty exine or microcapsule. The exines resemble the spores and pollen from which they are extracted, in size and morphology. Also, from any one plant such characteristics are incredible uniform. The exines can be used is microcapsules or simply as micron-sized particles due to the variety of functional groups on their surfaces. The loading of the exine microcapsules into their cavities is via multi-directional nano-diameter sized channels. The exines can be filled with a variety of polar and non-polar materials. Such as enzymes can be encapsulated within the shells and still remain active. In vivo studies in humans have shown that an encapsulated active substance can have a substantially increased bioavailability than if it is taken alone. The sporopollenin exine surface possesses phenolic, alkane, alkene, ketone, lactone and carboxylic acid groups. Therefore it can be derivatised in a number of ways, which has given rise to its having been used for, such as, solid supported for peptide synthesis, catalysis and ion-exchange chromatography. Also, the presence of the phenolic groups on sporopollenin endows it with antioxidant activity.

Sporopollenin as an efficient green support for covalent immobilization of a lipase

Aminoalkyl functionalised sporopollenin exine capsules have been used to immobilizeCandida antarcticalipase B using a covalent diimine-based linker.

How does iron interact with sporopollenin exine capsules? An X-ray absorption study including microfocus XANES and XRF imaging.

Sporopollenin exine capsules (SECs) derived from plant spores and pollen grains have been proposed as adsorption, remediation and drug delivery agents. Despite many studies there is scant structural data available. This X-ray absorption investigation represents the first direct structural data on the interaction of metals with SECs and allows elucidation of their structure-property relationships. Fe K-edge XANES and EXAFS data have shown that the iron local environment in SECs (derived from Lycopodium clavatum) reacted with aqueous ferric chloride solutions is similar to that of ferrihydrite (FeOOH) and by implication ferritin. Fe Kα XRF micro-focus experiments show that there is a poor correlation between the iron distribution and the underlying SEC structure indicating that the SEC is coated in the FeOOH material. In contrast, the Fe Kα XRF micro-focus experiments on SECs reacted with aqueous ferrous chloride solutions show that there is a very high correlation between the iron distribution and the SEC structure, indicating a much more specific form of interaction of the iron with the SEC surface functional groups. Fe K-edge XANES and EXAFS data show that the FeII can be easily oxidised to give a structure similar to, but not identical to that in the FeIII case, and that even if anaerobic conditions are used there is still partial oxidation to FeIII.

Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking.

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers.

UV and visible light screening by individual sporopollenin exines derived from Lycopodium clavatum (club moss) and Ambrosia trifida (giant ragweed)

We have investigated the UV–visible light transmission of three types of micrometre-sized sporopollenin exine shells, two derived from Lycopodium clavatum (club moss) spores and one from Ambrosia trifida (giant ragweed) pollen. We have used spectrophotometer measurements of partial monolayers of exines and microscope absorbance imaging to derive the light transmission properties of individual exines. Measurements have been made for exines in air when light transmission losses are due to a combination of absorption, reflection and scattering processes and for exines dispersed in a liquid for which the refractive index (RI) is approximately equal to the RI of the exine such that reflection and scattering effects are negligible. Overall, it found that the light transmission of a single exine wall is approximately 50%. This value of the transmission is due mainly to light absorption, is similar for the three exines studied here and varies only slightly with light wavelength over the range 200–900 nm.

Origin of the Inner Intine in Pollen of Canna

Observations of development in the nonaperaturate pollen of Canna generalis Bailey, based upon light and electron microscopy, indicate that the inner intine of this species is initiated by plasmalemmasomes originating at widely separated areas of the cell surface.The intine is that part of the pollen wall located between the sporopollenin exine and the cytoplasmic surface. It is often interbedded with the exine but is not itself composed of sporopollenin. In appearance and composition, the intine is comparable with the primary plant cell wall and has been characterized by Roland as an amorphous matrix of pectins with infrequent microfibrils.