Towards the fabrication of biohybrid silk fibroin materials: entrapment and preservation of chloroplast organelles in silk fibroin films

Abstract

Chloroplasts extracted from spinach leaves were entrapped in B. mori silk fibroin films to investigate the maintenance of their photosynthetic activity in a dry environment. Chloroplast stability was studied under various storage conditions (different temperature and light cycles) using electron microscopy, spectroscopic analysis and measuring of their photosynthetic activity (i.e. oxygen production). When compared to other environments previously investigated, silk fibroin films enhanced the preservation of the chloroplasts' photosynthetic activity.

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Organelles are specialized subunits of cells where lipid bilayers enclose highly organized systems that carry out distinct metabolic activities (e.g. photosynthesis).¹ Finding methods to preserve and stabilize these biological entities in a dry environment (residual water <5 wt%) would result in the fabrication of high-tech, biohybrid devices. In addition, the ability to temporarily suspend the metabolic activity of organelles, by preserving their function over time in the absence of an appropriate stimulus, would result in the formation of biohybrid materials that can be activated on demand. Historically, refrigeration is the most common strategy to preserve biological entities.^2,3 However, the insulated storage space and the energy required to decrease temperature well below ambient conditions are major drawbacks that burden with the cost of this strategy.^2,4 As an alternative method, encapsulation in biomimetic environments has been proposed to preserve organelle activity by providing protection from hydrolytic and oxydative stresses. Despite its promise, the biomimetic strategy is still far from being fully exploited.^5–7 The positive results obtained by crystallization and bulk loading of biomacromolecules (e.g. enzymes and growth factors) into biomimetic materials are in fact difficult to be replicated with more complex entities.^8–11 Alternatively, preservation of the synthetic activity of organelles has been mainly pursued in wet environments (residual water > 30 wt%) at physiological pH and ionic strength, by encapsulation in synthetic (e.g. silica) hydrogels.^12–14

Chloroplasts are photosynthetic organelles that transform solar energy into chemical one.¹² The photosynthetic reaction takes place in thylakoids, membrane-bound compartments arranged in stacks called grana. The light-harvesting complexes (i.e. photocatalytic system I and II) found in thylakoid membranes is a subunit of proteins that bind chlorophyll to form chlorophyll–protein complexes.¹² Chlorophyll is a highly conjugated cyclical tetrapyrole that promotes photocatalysis by absorbing electromagnetic waves in the visible spectrum. Stabilizing this molecule – and the complex around it – is considered the key to maintain the chloroplasts' photocatalytic function outside their natural environment.¹² In fact, the photosynthetic complexes found in thylakoids rapidly (i.e. within few hours) degrade when exposed to visible light and to oxidative stresses, posing a major hindrance in the employment of these natural photocatalytic systems for technological applications.¹⁴

Structural biopolymers (e.g. collagen, silk fibroin, keratin, chitin) are the building blocks of living materials. These natural polymers can also be reinvented in high technological materials that are able to compete with the “synthetic” counterpart for biomedical and optoelectronic applications.^15,16 In addition, structural biopolymers possess distinct features (e.g. biocompatibility, edibility, compostability) that well exploit the interface between the biotic and abiotic worlds. Silk fibroin extracted from cocoons made by the Bombyx mori caterpillar, in particular, has been shown to well meet the stringent physical properties (transparency, conformability, flexibility and nanometric surface roughness) and the limited processing conditions (i.e. water-based and within physiological ranges) needed to design biofunctional, ‘green’ devices, resulting in the biofabrication of materials that liaises the biomedical, photonic and electronic worlds.^16–28 Silk fibroin can in fact be regenerated in several material formats (e.g. fiber, rope, sponge, sphere hydrogel and film), which enable the use of the protein as sustainable material in biomedical, optic, photonic and electronic applications. This material versatility is mainly due to the polymorphic nature of silk fibroin, that can be organized in several secondary and tertiary conformations ranging from amorphous random coils to highly-ordered beta-sheet structures. This allows for the processing of silk fibroin into flexible films that possess several compelling properties as transparency in the visible spectrum, controlled thickness (from nm to mm), low surface roughness (rms ≅ 2 nm), conformability, and tailored biodegradability (from seconds to months). Additionally, labile biomacromolecules bulk loaded in silk fibroin films at the point of material assembly are encaged in a unique environment that preserve their structure and function.¹⁰ This feature has then been exploited to incorporate unprecedented functions in silk materials as sensing capabilities, drug delivery, and cell-instruction.^16,29,30

Here, we pursued the incorporation of chloroplasts extracted from spinach leaves (Spinacia oleracea) in silk fibroin films to obtain a photosynthetic material that can be stored and then activated upon exposure to visible light.

Chloroplasts and silk fibroin were extracted in water suspensions,³¹ as previously reported. The biohybrid silk fibroin-chloroplast material (SF-Ch) was then formed by drop casting mixtures of silk fibroin and chloroplasts suspensions on polydimethylsiloxane (PDMS) molds. In general, this process yields an amorphous, water-soluble silk fibroin material where the protein has a random coil conformation. Films are then converted to a crystalline, water insoluble material by exposure to water vapors or polar solvents (e.g. ethanol). Remarkably, the incorporation of chloroplasts in silk fibroin material affected the polymorphic nature of the protein, driving the amorphous to crystalline conformational change of the protein, yielding water-insoluble SF-Ch biohybrids, where the beta-sheets content (amide I peak at 1621 cm⁻¹ in the FTIR spectra, Fig. 1b) was a function of chloroplast concentration (Fig. 1). The percentage of beta sheet crystals in the silk fibroin material, in fact, increased from 5 ± 1% in silk fibroin with no chloroplasts to 17 ± 3, 19 ± 4 and 28 ± 3% for silk SF-Ch materials with 2 × 10⁶, 6 × 10⁶, and 2 × 10⁷ chloroplasts per mg of silk fibroin, respectively. This interplay between silk fibroin polymorphism and chloroplasts indicated an active interaction between the phospholipidic membranes of the chloroplasts and the silk material, which resembled the conformational change of the protein in the presence of a hydrophobic, lipid-based material as palmitic acid (Fig. 1a).

Fig. 1 Interplay between silk fibroin and chloroplasts. (a) Silk fibroin molecules form a monolithic matrix around the phospholipid membrane of chloroplasts. Silk fibroin material allows for sufficient water and gas transportation while preserving chloroplast structure and function. (b) FTIR analysis of silk fibroin matrices at increasing concentrations of chloroplasts. Amide I spectra of silk fibroin depicted an enhanced protein crystallinity (increase of the alpha-helix and beta-sheet resonance peaks at 1657 and 1621 cm⁻¹, respectively) as a function of chloroplast concentration in silk fibroin material. Thus, chloroplast phospholipid membrane affected silk fibroin polymorphism by driving an amorphous to crystalline conformational change at the point of material assembly.

The normal turgid leaves contain approximately 98% equilibrium water content, which is pivotal for proper functioning of cells and their organelles. On the contrary, silk films contain a low water content (5–10%, depending on amount of beta sheets in the silk structure). We then further examined the entrapment and preservation of chloroplast as the osmotic pressure differences at the point of drying might have disrupted the delicate lipid bilayer that forms the chloroplasts' membrane, with detrimental effects on their function. In particular, we investigated the characteristics of SF-Ch biohybrid materials when compared to chloroplasts incorporated in polyvinyl alcohol (PVA-Ch) or dried in deionized water.

Fig. 2 depicts a macro- to micro-scopic morphological investigation of the biohybrid materials, analysed at the point of material fabrication. When dried on a glass slide (left column, Fig. 2a, d and g), chloroplasts maintained their characteristic green colour (due to the presence of chlorophyll). However, electron microscopy analysis showed the presence of chloroplast debris, indicating that the structure of the organelles did not survived the drying process. On the contrary, entrapment of chloroplast both in silk fibroin (central column, Fig. 2b, e and h) and in PVA (right column, Fig. 2c, f and i) preserved chloroplasts' structure. Additionally, SF-Ch films maintained the previously reported remarkable flexibility of silk films,^15,16 as shown in a simple bending test depicted in Fig. S1.†

Fig. 2 Morphological analysis of chloroplasts entrapment in silk fibroin. (a–c) Photographic images of chloroplasts (a) dried in deionized water, (b) entrapped in a silk film and (c) entrapped in a polyvinyl alcohol film. Scale bar is 4 mm. (d–f) Scanning electron micrographs of chloroplasts (d) dried in deionized water, (e) entrapped in a silk film and (f) entrapped in a polyvinyl alcohol film scale bar is 2 μm. (g–i) Transmission electron micrographs of chloroplasts (g) dried in deionized water, (h) entrapped in a silk film and (i) entrapped in a polyvinyl alcohol film scale bar is 1 μm.

Spectral absorbance is an efficient method used to determine the preservation of photosensitive molecules (i.e. chlorophyll) within chloroplasts, which are at the base of the photosynthetic process. Measuring the absorbance of chlorophyll molecules entrapped in SF-Ch and PVA-Ch biohybrid films was then used to evaluate the stability of the photosynthetic systems in presence of chloroplasts. SF-Ch and PVA-Ch biohybrid films were produced by casting on 9.6 cm² circular molds 1.5 ml of polymeric suspensions (50 mg ml⁻¹) mixed with 2 × 10⁷ chloroplasts per mg of polymer, for a total of 75 mg of polymer. Biohybrid films were stored under different conditions (4 °C no illumination, 25 °C no illumination, 25 °C with 1.1 mW white light illumination, 60 °C no illumination, and 90 °C no illumination) to evaluate the preservation of the organelles under several environments. Light absorbance measurements using the peak absorbance wavelength of chlorophyll (at 440 nm and 680 nm) was used to investigate preservation of the green pigment structure (Fig. 3). Decrease in the absorbance at 440 nm and 680 nm indicated a deterioration of the chlorophyll within the entrapped organelles, resulting in a loss of activity in the photosynthetic systems. In SF-Ch biohybrids, 93% of the initial absorption was maintained up to 200 days (last time point considered) when the samples were stored in the dark at 4 °C and 25 °C (Fig. 3b and c). In fact, samples were kept in the dark at 25 °C for 1100 days and maintained the 89% of the initial absorption at 440 nm and 680 nm. On the contrary, chloroplasts that were not encaged in silk films lost the typical absorption peaks of chlorophyll within 24 hours upon exposure to room temperature. Exposure to light decreased the preservation of the chlorophyll structure when compared to storage in dark conditions, due to the stress that light poses to the photocatalytic complexes. In particular, the 50% of the initial absorbance was lost at day 120 of continuous exposure to light at 25 °C. Exposure of SF-Ch biohybrid films to high temperatures (60 °C and 90 °C) accelerated the degradation of chlorophyll, which had a 50% decrease of its original absorbance in 24 and 7 days, respectively. There was no statistical significance difference (p > 0.05) between the decrease of absorbance of chlorophyll in SF-Ch and PVA-Ch films maintained in dark at different temperatures (4 °C, 25 °C, 60 °C, and 90 °C), indicating that preservation to oxidative stresses was similar for both the materials investigated (Fig. S2–S6†). This was observed in our samples which showed an increase in fluorescence intensity for chlorophyll and chloroplasts stabilized in both silk and PVA films (ESI†). However, preservation from light stresses was significantly increased in silk fibroin material (p > 0.05) when compared to PVA one, as chlorophyll the decrease of absorbance at 440 nm and 680 nm was significantly higher in PVA-Ch films when compared to SF-Ch ones (p > 0.05) already at day 3 (Fig. 3d and e). Chlorophyll fluorescence has been used to measure the orientation of the fluorescent molecules in the photosynthetic membrane, which is an indication of preservation of the chlorophyll photocatalytic activity.^26,27 Fluorescent measurements of SF-Ch and PVA-Ch materials stored under continuous light at 25 °C indicated a slower decrease in the fluorescence for SF-Ch materials, when compared to PVA-Ch counterpart, indicating an increased preservation of photocatalytic activity in chloroplasts stored in silk materials.

Fig. 3 Spectral absorbance of chlorophyll in SF-Ch and PVA-Ch materials. (a) Absorbance of chlorophyll suspension showing the peaks at 440 nm and 680 nm. (b) Absorbance measurements of chloroplast at 440 nm entrapped in silk films at different storage conditions. (c) Absorbance measurements at 680 nm of chloroplasts entrapped in silk fibroin films at different storage conditions. (d) Absorbance measurements at 440 nm of chloroplast entrapped in silk fibroin and PVA films at room temperature in the presence of light. (e) Absorbance measurements at 680 nm of chloroplast entrapped in silk fibroin and PVA films at room temperature in the presence of light. (f) Fluorescence measurements of chloroplast entrapped in silk fibroin and PVA films at room temperature in the presence of light.

Oxygen is a by-product of photosynthesis and a direct indicator of photosynthetic activity and energy conversion efficiency of chloroplasts (Fig. 4a). Oxygen production in SF-Ch and PVA-Ch materials were then measured in a Clark cell to corroborate the aforementioned spectral analysis of the biohybrid materials. The biohybrid materials were kept under continuous light at 25 °C and oxygen measurements were taken at days 1, 3, 5, 7, and 10. A calibration curve that correlates chloroplast density in suspension with oxygen production was used as reference for all the measurements (Fig. 4b). Fig. 4c shows the variation in photosynthetic activity of SF-Ch and PVA-Ch biohybrid materials as a function of time. Photosynthetic activity was maintained up to 5 days in SF-Ch films, while no oxygen production was measured in PVA-Ch films after 24 hours of exposure to continuous light (Fig. 4c and d). Similar results were measured for a free suspension of chloroplasts, the photosynthetic activity of which rapidly declined after one day of continuous exposure to light at 25° (Fig. S7†). In the normal living plants, stomatal conductance is crucial for photosynthesis as it relates to permeability of carbon dioxide and oxygen through stomata. Thus, an efficient encapsulant of chloroplasts needs to perform similarly to do not hinder photosynthesis by providing sufficient permeability to oxygen and carbon dioxide. A recent study¹⁷ has indicated that silk materials in the form of films possess modular permeability to oxygen and carbon dioxide, which varies with the beta-sheet content of the protein. Additional control over gas permeability may be obtained with nanofabrication procedures. Future studies will be focused to optimize diffusion of carbon dioxide and oxygen through SF-Ch films.

Fig. 4 Photosynthetic activity of entrapped chloroplasts. (a) Schematic representation of oxygen production of chloroplasts entrapped in a silk film. (b) Oxygen measurements of chloroplasts at different concentrations. Inset is intact chloroplasts in free suspension. Scale bar is 2 μm. (c) Oxygen production of silk and PVA films in an artificial electron acceptor buffer measured in a Clark vessel. (d) Amount of oxygen produced in silk and PVA films maintained at room temperature in the presence of light up to 10 days (*p < 0.05).

Conclusions

We have investigated the formation of silk-based photosynthetic biohybrid materials, based on the incorporation of chloroplasts in regenerated silk fibroin at the point of material assembly. The interplay between silk polymorphism and the chloroplasts phospholipidic membranes drive the amorphous to crystalline conformational change of silk fibroin, yielding bendable, water insoluble films. The preservation of chloroplasts' photosynthetic activity in a silk fibroin milieu was maintained for up to 5 days under continuous light condition at 25 °C, while the oxygen production rapidly was halted within 24 hours when chloroplasts were left in a buffered suspension or encapsulated in PVA. The typical spectral absorbance and fluorescence of the chlorophyll pigment found in chloroplasts was maintained in SF-Ch materials for up to 1100 days when stored in dark. Together these results indicate that silk fibroin materials preserve the photosynthetic activity of chloroplasts outside their physiological environment, posing the bases for the development of silk-based photosynthetic biohybrid materials.

Acknowledgements

The authors would like to acknowledge funding from the Office of Naval Research (N00014-13-1-0596). ANM acknowledges the Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13228f

‡ These authors contributed equally.

§ Current address: United States Military Academy West Point, 606 Thayer Road, West Point, NY, 10996 USA.

¶ Current address: Civil and Environmental Engineering Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 20139, USA.

|| Current address: Nanophysics Department, Istituto Italiano di Tecnologia, Via Morego 30, Genova, 16163, Italy.

** Current address: Department of Electrical and Computer Engineering, Duke University, 101 Science Drive, Durham, NC, 27705, USA.

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