polymers Article Purification of Hollow Sporopollenin Microcapsules from Sunflower and Chamomile Pollen Grains Jose Manuel Ageitos , Sandra Robla , Lorena Valverde-Fraga, Marcos Garcia-Fuentes and Noemi Csaba * Centre for Research in Molecular Medicine and Chronic Diseases (CiMUS), Department Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] (J.M.A.); [email protected] (S.R.); [email protected] (L.V.-F.); [email protected] (M.G.-F.) * Correspondence: [email protected] Abstract: Pollen grains are natural microcapsules comprised of the biopolymer sporopollenin. The uniformity and special tridimensional architecture of these sporopollenin structures confer them attractive properties such as high resistance and improved bioadhesion. However, natural pollen can be a source of allergens, hindering its biomedical applicability. Several methods have been developed to remove internal components and allergenic compounds, usually involving long and laborious processes, which often cannot be extended to other pollen types. In this work, we propose an abridged protocol to produce stable and pristine hollow pollen microcapsules, together with a complete physicochemical and morphological characterization of the intermediate and final products. The optimized procedure has been validated for different pollen samples, also producing sporopollenin microcapsules from Matricaria species for the first time. Pollen microcapsules obtained through this protocol presented low protein content (4.4%), preserved ornamented morphology with a nanoporous 3 Citation: Ageitos, J.M.; Robla, S.; surface, and low product density (0.14 g/cm ). These features make them interesting candidates from Valverde-Fraga, L.; Garcia-Fuentes, a pharmaceutical perspective due to the versatility of this biomaterial as a drug delivery platform. M.; Csaba, N. Purification of Hollow Sporopollenin Microcapsules from Keywords: sporopollenin; microcapsules; pollen; sunflower; chamomile; FTIR; SEM Sunflower and Chamomile Pollen Grains. Polymers 2021, 13, 2094. https://doi.org/10.3390/ polym13132094 1. Introduction Pollen grains are protective microcapsules of the male gametes of seed plants, involved Academic Editor: Alejandro Sosnik in dispersion and adhesion, as well as recognition, during pollination and fertilization processes [1]. These natural microcapsules are composed by several layers of different Received: 21 May 2021 lipidic and polymeric materials that confer them unique, plant-specific morphologies [2]. Accepted: 21 June 2021 The terminology of pollen wall layers is heterogeneous in the literature and is usually based Published: 25 June 2021 on the different staining affinity of the biomaterials [3]. Basically, three main layers can be distinguished: first, the external coating, named pollenkitt; followed by the multi-layered Publisher’s Note: MDPI stays neutral pollen-wall (exine and intine); and finally, the inner sporoplasm, where the cell gamete with regard to jurisdictional claims in published maps and institutional affil- is located [4]. The exine is mainly composed by sporopollenin, one of the most resistant iations. biopolymers found in nature, still part of 500-million-year-old sedimentary rocks [5,6]. This biomaterial is considered a keystone in the adaptation of early aquatic plant gametes to terrestrial life [7]. Sporopollenin is composed of carbon, oxygen, and hydrogen and is constituted of long, highly cross-linked biopolymer chains [7]. In addition to its unique chemical composition, pollen exine has a complex tridimensional structure with ornaments Copyright: © 2021 by the authors. and nanopores, naturally designed for interacting with different biological surfaces, such Licensee MDPI, Basel, Switzerland. as insect cuticula, animals, and plant tissues. This article is an open access article distributed under the terms and Synthetic microcapsules often present variability in size and morphology during their conditions of the Creative Commons production. In contrast, pollen grains can be considered natural, monodisperse nanostruc- Attribution (CC BY) license (https:// tured microcapsules with highly resistant and specific three-dimensional morphologies creativecommons.org/licenses/by/ that, so far, are difficult to replicate with current methods [8]. Pollen structure and compo- 4.0/). sition are dependent on the dispersal pathway of the species (wind, water, insects, animals) Polymers 2021, 13, 2094. https://doi.org/10.3390/polym13132094 https://www.mdpi.com/journal/polymers Polymers 2021, 13, x FOR PEER REVIEW 2 of 18 Polymers 2021, 13, 2094 nanostructured microcapsules with highly resistant and specific three-dimensional 2mor- of 18 phologies that, so far, are difficult to replicate with current methods [8]. Pollen structure and composition are dependent on the dispersal pathway of the species (wind, water, in- sects,in order animals) to protect in order the to male protect gametophyte the male gametophyte best from external best from environmental external environmental conditions. conditions.These unique These properties unique have properties already have drawn already researchers’ drawn attentionresearchers’ [2]; attention notwithstanding, [2]; not- withstanding,it is well known it is thatwell mostknown pollens that most contain polle allergensns contain that allergens constitute that constitute a potential a riskpoten- to tialhuman risk healthto human [1,9 health]. A few [1,9]. protocols A few protoc have alreadyols have been already established been established for the elimination for the elim- of inationsuch components of such components and/or the and/or chemical the extraction chemical ofextraction sporopollenin of sporopollenin from fern spores from fern and sporespollen grainsand pollen [10– 12grains] to encapsulate [10–12] to encapsulate drugs [13,14 drugs], lipids [13,14], [15], cellslipids [16 [15],], proteins cells [16], [17 pro-,18], teinsor chemical [17,18], catalystsor chemical [19 ,catalysts20]. Such [19,20]. procedures Such usuallyprocedures involve usually laborious involve processes laborious using pro- cessesharsh reagentsusing harsh (e.g., reagents strong acids (e.g., and/or strong bases)acids and/or and prolonged bases) and incubation prolonged times incubation that can timesalterthe that ultrastructure can alter the ofultrastructure the pollen grain of the [10 pollen,12]. grain [10,12]. InIn addition, the above-mentioned pollen pollen species species are are generally generally characterized characterized by by sim- sim- ple morphology and produce thin microcapsulesmicrocapsules with limited applicability. Looking for alternatives, sunflower sunflower ( Helianthus annuus )) pollen has been recently drawingdrawing attentionattention due to its widespread cultivation, flower flower size, size, and and high high pollen pollen production production with with low low aller- aller- genicity [10–12]. [10–12]. Chamomile (Matricaria chamomilla , sym. M. recutita ) belongs to the same familyfamily as sunflower sunflower and is one of the the most most important important medicinal plants [21,22]. [21,22]. Chamomile Chamomile pollen is smaller than that of sunflower sunflower (13–25 µmµm vs. 26–50 µm)µm) [23,24] [23,24] and has a highly porous surface. Despite its interesting size and structure, which make it an ideal candidate forfor producing sporopollenin microcapsules, this pollen has not yet been explored for this purpose. Taken together, together, the the aim aim of of our our study study has has been been the theestablishment establishment of a ofsimplified a simplified pro- tocolprotocol for forthe theobtention obtention of hollow of hollow pollen pollen microcapsules, microcapsules, employing employing sunflower sunflower pollen pollen as referenceas reference (Figure (Figure 1), 1and), and the theevaluation evaluation of its of the its suitability the suitability for chamomile for chamomile pollen pollen purifi- cation.purification. Each step Each of step the ofprocess the process has been has studied been studied in detail in detailby several by several independent independent tech- niquestechniques to characterize to characterize the pollen, the pollen, as well as as well the asextracted the extracted products. products. To the best To the of the best au- of thors’the authors’ knowledge, knowledge, this is thisthe first is the compar first comparativeative morphological morphological and physicochemical and physicochemical char- acterizationcharacterization of sporopollenin of sporopollenin microcapsules microcapsules throughout throughout their sequential their sequential chemical chemical purifi- cationpurification steps, steps,as well as as well the asfirst the report first reporton the onproduction the production of hollow ofhollow sporopollenin sporopollenin micro- capsulesmicrocapsules from chamomile from chamomile pollen. pollen. Figure 1. Processing scheme for the different pollen samples. Polymers 2021, 13, 2094 3 of 18 2. Materials and Methods 2.1. Materials Sunflower pollen (H. annuus) was obtained from Control Bio (El Alquián, Spain) and Pharmallerga (Lisov, Czech Republic). Chamomile (M. chamomilla) dried plant and flower were purchased from Soria Natural (Soria, Spain). Cyclohexane, orthophosphoric acid (H3PO4, 85%), perchloric acid (HClO4, 70%), hydrochloric acid (HCl, 37%), and sulfuric acid (HCl, 98%) were purchased from Merck KGaA (Darmstadt, Germany). Trifluoroacetic acid (CF3COOH,