# Isolation of Anammoxosomes From the Aggregate Culture of Ca. Brocadia Sapporoensis and Assembly of Ladderane Liposomes

## TL;DR

This study shows how to isolate special lipids from anammox bacteria and use them to make stable liposomes for drug delivery.

## Contribution

A simplified method to isolate ladderane-rich anammoxosomes from mixed cultures and use them for liposome production.

## Key findings

- A simplified isolation protocol effectively removed debris and yielded anammoxosomes confirmed by TEM.
- Liposomes made from ladderanes showed increased colloid stability compared to DPPC-only liposomes.
- Ladderane-based liposomes were successfully characterized and demonstrated potential for drug delivery.

## Abstract

Anammox bacteria wield an energy‐efficient nitrogen metabolism enveloped in anammoxosome organelle composed of unique ladderane lipids. Thus, waste anammox biomass seems to be an attractive target for the isolation of ladderanes and subsequent production of artificial vesicles for drug delivery. This study proposed a novel method to isolate ladderane‐rich anammoxosomes from aggregate mixed culture of Ca. Brocadia sapporoensis. Compared to conventional isolation protocols, the protocol was simplified by omitting the prepurification of anammox cells, replacing Percoll® with a sucrose gradient and prolonging the application of EDTA. This enhanced and simplified procedure efficiently removed EPS and other debris, thus yielding the layer of anammoxosomes as confirmed by control experiments and TEM. For the first time, the resulting ladderane isolates were used for the preparation of liposomes, both with and without the addition of pure dipalmitoylphosphatidylcholine (DPPC). Vesicles were successfully created, characterised by TEM and DLS, and anammox‐based ladderanes were incorporated into their walls. These liposomes had interesting functional properties such as increased colloid stability at elevated concentrations, meaning a reduced tendency to form aggregates compared to model liposomes made solely of DPPC. Overall, this study offers insights into converting waste anammox biomass into a valuable resource for drug delivery.

Ladderane lipids in anammox bacteria can find use in artificial liposomes for drug delivery. This study developed the isolation of ladderane‐rich anammoxosome organelles from previously challenging aggregate mixed culture of Ca. Brocadia sapporoensis. The resulting ladderane isolates were used for the preparation of liposomes which had interesting functional properties.