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Artificial Cells for Enzyme Replacement Therapy for Phenylketonuria

Final Report Summary - AC FOR PKU TREATMENT (Artificial Cells for Enzyme Replacement Therapy for Phenylketonuria)

Phenylketonuria (PKU) is the most common genetic enzyme defect, with an overall incidence in Europe and the USA of 1:10,000-20,000 live births per year. Patients suffer from a genetic defect in the liver enzyme phenylalanine hydroxylase (PAH), which normally metabolizes the amino acid phenylalanine (Phe) into the amino acid tyrosine. The increase in the level of systemic Phe in the first few years of life, can lead to severe mental retardation. The implementation of newborn screening to detect PKU has facilitated the early use of dietary treatments. However, this diet is difficult to follow and does not prevent elevation of blood Phe during episodes of fever and infection. Alternative models of therapy are being pursued including enzyme or gene therapy with enzyme therapy being currently the most promising concept. PAH enzyme is complex, requires cofactors and has limited stability, making large scale isolation and purification very costly. Fortunately, there is a nonhuman enzyme, phenylalanine ammonialyase (PAL) that converts Phe into low toxic trans-cinnamic acid which is subsequently converted in the liver to benzoic acid, and then excreted via urine.

The aim of this project was to engineer a carrier system based on capsosomes (liposomes trapped within a polymer carrier capsule) that allows for the oral delivery of the PAL enzyme. While the PAL enzyme will be protected in the stomach, Phe in the intestine will be converted into trans-cinnamic acid and by doing so the Phe concentration will systemically lowered based on the enterorecirculation of amino acids theory. The specific aims and findings of the project are:

i) Assembly and characterization of PDA-based capsosomes
The design of compartmentalized carriers as artificial cells is envisioned to be an efficient tool with potential applications in the biomedical field. The advent of this area has witnessed the assembly of functional, bio-inspired systems attempting to tackle challenges in cell mimicry by encapsulating multiple compartments and performing controlled encapsulated enzymatic catalysis. Although capsosomes, which consist of liposomes embedded within a polymeric carrier capsule, are among the most advanced systems, they are still amazingly simple in their functionality and cumbersome in their assembly. We reported on capsosomes by embedding liposomes within a PDA carrier shell created in a solution-based single-step procedure. We demonstrated for the first time the potential of PDA-based capsosomes to act as artificial cell mimics by performing a two-enzyme coupled reaction in parallel with a single-enzyme conversion by encapsulating three different enzymes into separated liposomal compartments. In the former case, the enzyme uricase converts uric acid into hydrogen peroxide, CO2 and allantoin, followed by the reaction of hydrogen peroxide with the reagent Amplex Ultra Red in the presence of the enzyme horseradish peroxidase to generate the fluorescent product resorufin. The parallel enzymatic catalysis employs the enzyme ascorbate oxidase to convert ascorbic acid into 2-L-dehydroascorbic acid.

ii) PAL loaded PDA-based capsosomes
The PAL enzyme was successfully trapped in the above mentioned PDA-based capsosomes using temperature-sensitive liposomal subunits. The enzymatic conversion of Phe into trans-cinnamic acid was only observed for capsosomes incubated at 37 °C, but not at room temperature. These results not only confirmed the preserved activity of the enzyme, but they also showed that the enzyme was entrapped within intact liposomes due to the temperature dependence of the enzymatic conversion.

iii) Enzymatic activity in simulated gastric and intestine juice
With the aim to assess the potential of the PAL loaded capsosomes for oral delivery, their enzymatic activity after being exposed to simulated gastric (SGJ) and intestine juice (SIJ). It turned out that the above mentioned PAL loaded PDA-based capsosomes had similar activity in buffered saline solution and in SIJ, making the proposed assembly of biodegradable capsosomes using thiol-modified PDA unnecessary. On the other hand, these capsosomes exposed to SGJ lost their enzymatic activity likely due to the drop in pH. The proposed design of pH-sensitive liposomes turned out to be a non-vital approach. Instead, we explored the use of a pH-sensitive protective polymer layer. We employed a layer of commercially available Eudragit (Eu), which is an anionic and pH dependant polymer based on methyl acrylate, methyl methacrylate and methacrylic acid. At acidic pH (gastric fluids pH 2.0) the carboxylate groups will be protonated becoming into a hydrophobic polymer preventing the protons to cross it and reach the enzyme. At basic pH (small intestine fluids pH 8.0) the carboxylate groups will be deprotonated becoming into a hydrophilic polymer allowing the substrate reach the enzymes. The addition of Eu as the outermost layer of the capsosomes preserved the stability of the liposomes in SGJ. However, when considering the activity of the entrapped PAL enzyme, the results in SGJ are inconclusive at this point in time. Although the approach is promising, there are still optimizations in the assembly required.

iv) Extra-cellular activity of PAL loaded PDA-based capsosomes with applied peristaltic flow
Due to the overall success of the project, a final aim was added which was not proposed initially in the project application, but is the logical extension of the work. This last part focused on the performance of the PAL encapsulating capsosomes in the presence of intestine cells with applied peristaltic flow which mimicked the dynamic biological environment in the intestine.
First, it was confirmed that the capsosomes were not internalized by the cells that line the intestine and that no inherent toxicity was present. The enzymatic activity of the PAL loaded capsosomes was demonstrated in the presence of cells and full cell media, showing that the enzymes were sufficiently protected from proteins in the media and cell metabolite products while the amino acid Phe was converted into trans-cinnamic acid. Finally, we verified that the application of peristaltic flow did not significantly alter the extra-cellular performance of the microreactors. Importantly, the cell viability remained unaffected for all the tested conditions.


This project was the first successful attempt to employ an assembly with multiple subcompartments towards a real medical problem, namely PKU. PAL loaded capsosomes could be assembled with preserved activity in SIJ. The attempt to use a pH-sensitive protective polymer layer was found to be promising, but some optimization is still required. The overall beneficial findings in terms of assembly and function prompted us to assess the performance in cell culture mimicking the dynamic environment of the intestine. The success of this final aspect is identifying the PAL loaded PDA-based capsosomes as interesting candidates for an oral formulation of PAL towards the treatment of PKU.
These fundamental findings are of interest beyond this project and within 2 years, the system was advanced to such an extent that animal testing together with a medical collaborator will be feasible in the near future. If successful, the impact of the capsosome-based formulation of enzymes for oral delivery will become very attractive for pharmaceutical companies working in the field. Therefore, the socio-economic impact of this project in the future could be considerable, since the successful administration of enzymes is highly sought after, but remains challenging.