Extraction of Plant-based Capsules for Microencapsulation Applications

Summary

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Abstract

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.

Introduction

There is significant interest in natural plant-based capsules obtained from plant spores and pollens for use in microencapsulation applications.^1-15 In nature, spores and pollen provide protection for sensitive genetic materials against harsh environmental conditions. The basic structure of plant spores and pollen typically comprises an outer shell layer (exine), an inner shell layer (intine), and the internal cytoplasmic material. The exine is comprised of a chemically robust biopolymer^1,9,10,13,16 referred to as sporopollenin and the intine is comprised primarily of cellulosic materials.^16-18 Empty capsules can be isolated by various....

Protocol

1. Extraction of Sporopollenin Exine Capsules (SECs) from L. clavatum Spores

Note: The SEC extraction process involves a flammable powder (L. clavatum), hot corrosive acids, and flammable solvents, hence proper personal protective equipment (goggles, face mask, gloves, lab coat), approved risk assessment on usage, and disposal of chemicals by authorized laboratory personnel is essential.

1. Spore Defatting
   1. Prepare a reflux set-up in a fume hood by using a glass condenser, water circulation system, and water bath (Figure 1).
   2. Weigh 100 g L. clavatum spores without creatin....

Results

Streamlined Extraction Process for Sporopollenin Exine Capsules

The L. clavatum SEC extraction was achieved by three main steps: (1) Defatting using acetone; (2) Acidolysis using phosphoric acid 85% (v/v); and (3) Extensive SEC washing using solvents. The flow of the streamlined SEC extraction process is presented in Figure 1 A - I. Briefly, the process involves a defatting step with a.......

Discussion

In this work, a systematic analysis of SEC extraction from L. clavatum spores is presented and this report shows that it is possible to produce higher quality capsules while also achieving a significant streamlining of the pre-existing commonly used protocol.¹¹ In contrast to the existing protocol requiring a high process temperature (180 °C) and a long process duration (7 days),¹¹ the current SEC extraction processing optimization was primarily focused on reducing the temperature and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lycopodium clavatum spores (s-type)	Sigma	19108-500G-F
Bovine serum albumin	Sigma	A2153-50G
FITC-conjugated BSA	Sigma	A9771-250MG
Phosphoric acid (85% w/v)	Sigma	438081-2.5L
Hydrochloric acid	Sigma	V800202
Sodium hydroxide	Sigma	S5881-1KG
Acetone	Sigma	V800022
Ethanol	AcME	C000356
Deionized water	Millipore purified water
Qualitative filter paper (grade No. 1, cotton cellulose)
Polystyrene microspheres (50 ± 1 µm)	Thermoscientific (CA, USA)	4250A
Vectashield	Vector labs (CA, USA)	H-1000
Sticky-slides, D 263 M Schott glass, No.1.5H (170 μm, 25 mm x 75 mm) unsterile glass slide	Ibidi GmbH (Munich, Germany)	10812
Commercial Lycopodium SECs (L-type)	Polysciences, Inc. (PA, USA)	16867-1
Heating plates	IKA, Germany
Scanning electron microscope	Jeol, Japan	JFC-1600
Elemental analyzer	Elementar, Germany	VarioEL III
FlowCam: The benchtop system	Fluid Imaging Technologies, USA	FlowCamVS
Confocal laser scanning microscope	Carl Zeiss, Germany	LSM710
Freeze dryer	Labconco, USA
UV Spectrometer	Boeco, Germany	S220