Microcapsule degradation

We implemented microencapsulation of engineered human cells and designed the system for degradation of alginate microcapsules. Engineered HEK293T cells were successfully incorporated into alginate microcapsules and extended time viability of the encapsulated cells was demonstrated. Secretory alginate lyases from Pseudomoalteromonas elyakovii and from Pseudomonas aeruginosa were cloned by replacing the signal peptide of the alginate lyase with the eukaryotic signal peptide. Alginate lyases were successfully produced and secreted from HEK293T cells. Degradation of alginate beads was demonstrated by alginate lyase from Sphingobacterium multivorum.

Degradation of alginate microcapsules

Our biopharmaceutical delivery system is based on microencapsulated mammalian cells which produce the required therapeutics. These cells are safely sealed in the alginate microcapsules, forming an immune-privileged environment for the therapeutic cells, and implanted into the tissue such as e.g. the eye or placenta. The semi-permeable capsule allows free transport of nutrients, signaling molecules and produced protein therapeutics, while it prevents immune cells from reaching and destroying the implanted therapeutic cells. Therapeutic cells therefore do not need to be immuno-compatible for each individual patient and can therefore be optimized and mass produced to increase their efficiency and affordability of this therapy.

We designed our device to leave no trace after the therapy has been completed, by initiating secretion of an alginate-degrading en/zyme to break down the microcapsules followed by the apoptosis of therapeutic cells. This approach should increase the safety and decrease the unwanted effects of treatment and also makes the surgical removal of microcapsules or fibrotic tissue around microcapsules obsolete.

Figure 1. Schematic representation of alginate microcapsule and its degradation. Microcapsules serve as a semi-permeable membrane, allowing exchange of therapeutic and inducer molecules as well as nutrients and metabolites between encapsulated cells and the environment. On the other hand the microcapsule prevents the immune cells and immunoglobulin complexes to access the engineered cells while at the same time preventing uncontrolled dissemination of therapeutic cells throughout the body.

Alginate microcapsules

Alginate is the most widely used and clinically tested biomaterial for cell encapsulation. It is found in cell walls of brown seaweed. This polysaccharide is made of (1, 4)-linked monomeric units of β-D-mannuronate (M) and α-L-guluronate (G) (Figure 2). Consecutive M or G residues form so called M- or G-blocks, whereas the alternating M and G units constitute MG-blocks of alginate (Duan et al., 2009).

Figure 2. Structure of alginate polymer. α-L-guluronate sugar residues is shown on the left and β-D-mannuronate on the right side. Source: http://en.wikipedia.org/wiki/Alginate

Figure 3. Urban and Anže in action with Büchi BIOTECH Encapsulator.

The host immune system imposes a great threat on our therapeutic cells as the human defense system could quickly locate and destroy our drug-producing cells since they lack the signatures of the endogenous cells. Alginate microcapsules are an almost ideal solution for this problem: the encapsulated cells are protected against the cytotoxic cells while the material used is biocompatible and allows exchange of small molecules and proteins (Figure 1). It has been demonstrated that encapsulated cells remain viable for several months; therefore many therapeutic programs can be accomplished within this time frame. This sequestration technology also prevents the uncontrolled dissemination of our device throughout the patient's body (Ausländer et al., 2011).

Cell encapsulation is technically achieved by dropping a mixture of cells and liquid alginate into a calcium chloride solution which solidifies the droplets, transforming them into the hydrogel beads whose size in our system can be adjusted within the range of 300-500 μm in diameter. These beads are then coated with a polycation such as poly(L-lysine) (PLL) to form a membrane which further decreases the porosity of the wall of capsules. In the next step of cell encapsulation, an outer alginate layer is applied. This alginate coat also minimizes the attachment of patient's cells to the microcapsules due to the net negative charge of its surface (Vos et al., 2006). To allow cell growth and division within the alginate beads, the core of microcapsules is depolymerized with sodium citrate.

Encapsulation and degradation of microcapsules

Hopefully, after a few weeks or months of treatment, the patient would recover and we could terminate the genetically engineered microencapsulated cells by inducing apoptosis. However, before their programmed death, production and secretion of the alginate-degrading enzyme would be induced. Alginate lyase can break down the alginate polymer of microcapsules, ideally leaving no trace of our biopharmaceutical delivery system.

Alginate lyases are not present in the mammalian genome but have been found in bacteria, algae and marine mollusks. Alginate lyase is able to depolymerise the alginate crosslinked by calcium (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others prefer G-rich. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found from the literature the alginate lyase from bacterium Pseudoalteromonas elyakovii to be a promising candidate, since it can degrade all types of alginate (Sawabe et al., 2007). Since we could not obtain the gene for the described enzyme from the authors we decided to design a synthetic mammalian codon usage-optimized and BioBrick standard-compatible version of this bacterial gene for our iGEM project (BBa_K782059). Furthermore, because the selected alginate lyase consists of two domains, a putative chitin binding like-domain which is in bacterial expression system posttranslationally cleaved off and an alginate lyase domain representing the mature enzyme (Sawabe et al., 2007) (Figure 4), we also prepared a truncated version of P. elyakovii alginate lyase, abbreviated by chitin binding like-domain (BBa_K782062). We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of the secreted enzyme (Figure 5). The second implemented alginate lyase was from Pseudomonas aeruginosa strain PAO1 (bacteria were kindly provided by dr. Guillermo Martinez de Tejada from Univ. Navarra, Spain). This Gram-negative bacteria secretes alginate lyase to degrade the alginate that forms the mucoid matrix composing the bacterial biofilm (Schiller et al., 1993). This alginate lyase might therefore also be used for medical applications to degrade the viscous polysaccharide coat of Pseudomonas in patients with cystic fibrosis.

Figure 4. Pseudomoalteromonas elyakovii alginate lyase structure model prepared by I-TASSER server (Roy et al., 2011). Chitin binding like-domain is depicted in brown and alginate lyase domain in blue. The enzyme was engineered for secretion from HEK293T cells by the addition of preprotrypsin sequence to the N-terminus. Additionally, protein without chitin binding like-domain was also prepared (mature alginate lyase).

Figure 5. Schematic representation of the BioBrick parts (BBa_K782059 and BBa_K782062) for the alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, aly is alginate lyase from P. elyakovii coding sequence, Δcbd aly is P. elyakovii alginate lyase without chitin binding-like domain and Myc indicates Myc peptide tag for detection. E = EcoRI restriction site, X = XbaI restriction site, S = SpeI restriction site, P = PstI restriction site.

Results

Viability of microencapsulated cells

After the first successful encapsulation experiment we investigated how cells divide and if they remain viable for an extended period of time in the growth medium. Aliquots of encapsulated cells were collected at regular intervals, stained with cell viability dyes and observed with confocal microscopy (Figure 6). Results show that cells readily divide and fill up the hollow space inside the alginate microcapsule within three weeks (504 h). Furthermore, we have shown that encapsulated HEK293T cells remain viable for over a month (672 h) and show no signs of extensive necrosis. As a future alternative we could encapsulate a fixed number of cells that are deficient in cell devision which has been previously demonstrated to enhance production of therapeutic proteins (Fussenegger et al., 1998).

Figure 6. Extended viability of microencapsulated cells in the cell culture medium. HEK293T cells were encapsulated into the alginate microcapsules and stained with Hoechst and 7-AAD viability dyes. Images of stained encapsulated cells were taken with a confocal microscope. Hoechst stains both living and dead cells (depicted green), while 7-AAD stains only necrotic and dead cells (depicted purple). Double stained, i.e. dead cells are depicted with white color, demonstrating high cell viability even after four weeks (672 h).

Alginate lyase production in HEK293T cells

To estimate the production and secretion of constructed alginate lyases, HEK293T cells were transfected with vectors containing both alginate lyase parts (with and without the chitin binding-like domain) downstream of the constitutive promoter. After 48 h of protein production, supernatants were collected and cell lysates were prepared. Western blots of supernatants and lysates were obtained and alginate lyase was detected with anti-Myc antibodies. Mature alginate lyase was found in both supernatant and cell lysate, while full-length alginate lyase was not present in the collected supernatant (Figure 7).

Figure 7. Production and secretion of alginate lyase. 48 h after transfection of HEK293 cells with plasmids coding for full length and mature alginate lyase, cell lysates (A) and growth media (B) were collected. Proteins were separated on SDS-PAGE gel, blotted on nitrocellulose membrane and detected by immunoblot using anti-Myc antibodies. Arrows denote positions of alginate lyase from cell lysate (mature and full size protein) (A) and the mature (without chitin binding-like domain) alginate lyase secreted into the growth media (B).

Alginate microcapsules degradation

Next we investigated the degradation of alginate-PLL-alginate microcapsules by alginate lyase. Neither P. elyakovii alginate lyase produced by cells nor alginate lyase from Sphingobacterium multivorum were able to degrade alginate-PLL-alginate capsules. Next, we immobilized HEK293T cells in alginate beads without a PLL membrane and without desolidification of the bead's core. We observed a rapid rupture of alginate beads and their degradation by the addition of alginate lyase (Figure 8). Concentrations of alginate lyase as low as 5 µg/mL were shown to disintegrate produced alginate beads. In order to demonstrate that this degradation was due to the enzymatic activity of the alginate lyase and not caused by any other component that might sequester calcium, we heat denatured the enzyme, which abolished degradation of beads, demonstrating that the enzyme is indeed responsible for the capsule degradation and that this technology could be used for the therapeutic bead degradation.

Figure 8. Degradation of alginate beads and release of immobilised cells by the enzymatic activity of alginate lyase. Microcapsules with encapsulated HEK293T cells were incubated in the medium containing 2000 kDa FITC-dextran (depicted in green and red) and 400 µg/mL Sphingobacterium multivorum alginate lyase. Rapid penetration of the dye into the degraded capsules and release of microencapsulated cells was observed (left). On the other hand, heat-denaturation of the enzyme solution eliminated all degradation activity (right).

In addition, we demonstrated activity of secreted alginate lyases aly and Δcbd aly from Pseudoalteromonas elyakovii (BBa_K782059 and BBa_K782062) on alginate beads. As determined by Western blot analysis (Figure 7), HEK293T cells secreted low amounts of alginate lyase; therefore we concentrated the cell supernatants and incubated them with alginate capsules. The enzymatic activity was demonstrated by a decrease of the diameter of alginate beads determined by microscopy. Both P. elyakovii alginate lyases (BBa_K782059 and BBa_K782062) degraded alginate beads, decreasing the beads' diameter by 50 % in comparison to the control. Alginate lyase from Pseudomonas aeruginosa exhibited only slight decrease in the beads diameter (Figure 9), demonstrating that the type of alginate lyase should be carefully selected for the best efficiency.

Figure 9. Enzymatic activity of secreted alginate lyases. Alginate lyases from P. elyakovii and P. aeruginosa (BBa_K782059 and BBa_K782062) were produced and secreted by transfected HEK293T cells. Growth media with secreted alginate lyases was concentrated with Sartorius Vivaspin 6 concentrators. Alginate beads were incubated in concentrated supernatant. For easier observation under confocal microscope, we added 2000 kDa FITC-dextran (depicted in green) to cell supernatants. Beads' diameters were assessed using Leica LAS Image Analysis. software.

References

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Breguet, V., and Stockar, U. (2007) Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate. Biotechnol. Prog. 21, 1223–1230.

Duan, G., and Han, F. (2009) Cloning, sequence analysis, and expression of gene alyPI encoding an alginate lyase from marine bacterium Pseudoalteromonas sp. CY24. Canadian J. Microbiol. 1118, 1113–1118.

Fussenegger, M., Schlatter, S., Dätwyler, D., Mazur, X., and Bailey, J.E. (1998) Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat. Biotechnol. 16, 468-472.

Roy, A., Kucukural, A., and Zhang, Y. (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738.

Sawabe, T., Takahashi, H., Ezura, Y., and Gacesa, P. (2001) Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydrate Res. 335, 11–21.

Schiller, N., Monday, S., and Boyd, C. (1993) Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J. Bacteriol. 175, 4780–4789.

Vos, P. de, Faas, M., and Strand, B. (2006) Alginate-based microcapsules for immunoisolation of pancreatic islets. Biomaterials 27, 5603–5617.

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