preparation-giant-vesicles-encapsulating-microspheres-centrifugation

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

National Institutes of Natural Sciences

The University of Tokyo

the university of tokyo

Population analysis of a system of self-reproducing giant multilamellar vesicles (GMVs) was carried out by means of flow cytometry. The multidimensional distribution of forward light scattering (FS), side light scattering (SS), and fluorescence (FL) intensities originating from each GMV provided information about changes in a population composed of 104 vesicles. FS-FL dot plots indicated that, after the addition of the membrane precursor, the size distribution of the newly generated vesicles was nearly the same as that of the original, but the catalyst content was reduced. This result can be interpreted as evidence for the occurrence of the self-reproduction of GMVs. Moreover, the new GMVs recovered the amount of catalyst to the initial value, keeping their size distribution constant, when a solution of the catalyst was added to the new GMVs. These results are the first experimental evidence for a novel phenomenon on GMV size distribution during their self-reproducing cycle.

Population study of sizes and components of self-reproducing giant multilamellar vesicles.

In water, phosphoric membrane molecule (V(-)) self-assembled to form an anionic giant vesicle, the surface of which served as a catalyst for the autocatalytic formation of V(-) from its membrane precursor (V*), and the amplified V(-) produced a new vesicle using the original vesicle as a scaffold.

Autocatalytic membrane-amplification on a pre-existing vesicular surface.

The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell.

Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.

Polymeric micelle, "Lactosome", is composed of amphiphilic polydepsipeptide with a hydrophobic block of helical poly(L-lactic acid) (PLLA) and a hydrophilic block of poly(sarcosine). Lactosome was labeled by incorporation of poly(lactic acid) having a near-infrared fluorescence (NIRF) chromophore, and studied on blood clearance and tumor imaging. In vivo blood clearance time of Lactosome was prolonged with incorporation of poly(D-lactic acid) (PDLA), but decreased with poly(D,L-lactic acid) (PDLLA). NIRF imaging with applying these Lactosomes to tumor-bearing mice revealed that the tumor/background intensity ratio increased with incorporation of PDLLA. Stereochemistry in the hydrophobic core of self-assemblies is thus an important factor for determining physical stability in the blood stream and consequently contrast in imaging.

Control of in vivo blood clearance time of polymeric micelle by stereochemistry of amphiphilic polydepsipeptides.

Lactosome, which is a polymer micelle composed of poly(lactic acid)-b-poly(sarcosine), was applied successfully for solid tumor imaging. Lactosome is considered to escape from the reticuloendothelial system recognition, and shows prolonged in vivo blood clearance time. In vivo disposition of Lactosome, however, changed upon multiple dosages. Lactosome at the 2nd dosage was cleared from the blood stream by trapping at liver. This accelerated blood clearance (ABC) phenomenon is explained by production of anti-Lactosome IgM and IgG(3) through the immune response related with B-lymphocyte cells. The memory effect of B-lymphocyte cells lasted nearly for six months in mouse. The epitope moiety of Lactosome is concluded to be poly(sarcosine) based on the competitive inhibition assay. Since the ABC phenomenon was also reported with PEGylated liposome, nanoparticles in general may be potential in triggering the immune system.

Pharmacokinetic change of nanoparticulate formulation "Lactosome" on multiple administrations.

PET (positron emission tomography) using FDG (¹⁸F-fluorodeoxyglucose) has been widely used in the evaluation of various malignancies, but its clinical application to leukemia remains limited. We report a case of leukemia in which diffuse bone marrow uptake of FDG was observed, and bone marrow aspiration subsequently revealed acute lymphoblastic leukemia. It is not easy to differentiate between physiological and pathologic uptake when diffuse homogeneous uptake in bone marrow is observed.

Diffuse homogeneous bone marrow uptake of FDG in patients with acute lymphoblastic leukemia.

PET/CT using FDG has been widely used for the imaging of various malignant tumours, including plasma cell malignancy (PCM), but (11)C-methionine (MET), as a radiolabelled amino acid tracer, may also be useful because PCM is able to activate protein synthesis. The purpose of this study was to evaluate the clinical value of PET/CT imaging using MET in PCM, including multiple myeloma, compared with that of FDG PET/CT.

Clinical value of ¹¹C-methionine PET/CT in patients with plasma cell malignancy: comparison with ¹⁸F-FDG PET/CT.

With the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named "Lactosome", which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the (18)F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out.

Radiosynthesis and initial evaluation of (18)F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide.

Nanoparticle of Lactosome, which is composed of poly(l-lactic acid)-base depsipeptide with diameter of 35nm, accumulates in solid tumors by the enhanced permeability and retention (EPR) effect. However, a pharmacokinetic alteration of Lactosome was observed when Lactosome was repeatedly administered. This phenomenon is named as the Lactosome accelerated blood clearance (ABC) phenomenon. In this study, the effect of Lactosome dose on the ABC phenomenon was examined and discussed in terms of immune tolerance.

Evasion from accelerated blood clearance of nanocarrier named as "Lactosome" induced by excessive administration of Lactosome.

A morphological transformation from hybrid micelles to giant vesicles was observed in aqueous dispersion associated with formation of a double-chained amphiphile as a result of the migration of dodecylamine from the amphiphilic imine to the amphiphilic aldehyde within the hydrophobic environment of amphiphilic aggregates.

Spontaneous transformation from micelles to vesicles associated with sequential conversions of comprising amphiphiles within assemblies.

The purpose of this study was to compare the sensitivity of single-photon emission computed tomography/computed tomography (SPECT/CT) using 99mTc-sestamibi (MIBI) with that of PET/CT using 11C-methionine (MET) for localization of parathyroid adenomas/hyperplasia in primary hyperparathyroidism.

A comparison between 11C-methionine PET/CT and MIBI SPECT/CT for localization of parathyroid adenomas/hyperplasia.

Self-organized lipid structures (protocells) have been proposed as an intermediate between nonliving material and cellular life. Synthetic production of model protocells can demonstrate the potential processes by which living cells first arose. While we have previously described a giant vesicle (GV)-based model protocell in which amplification of DNA was linked to self-reproduction, the ability of a protocell to recursively self-proliferate for multiple generations has not been demonstrated. Here we show that newborn daughter GVs can be restored to the status of their parental GVs by pH-induced vesicular fusion of daughter GVs with conveyer GVs filled with depleted substrates. We describe a primitive model cell cycle comprising four discrete phases (ingestion, replication, maturity and division), each of which is selectively activated by a specific external stimulus. The production of recursive self-proliferating model protocells represents a step towards eventual production of model protocells that are able to mimic evolution.

A recursive vesicle-based model protocell with a primitive model cell cycle.

We propose a protocell model in which compartments are constructed via a new process involving the formation of robust vesicles using an autocatalytic, self-reproducing oil droplet system as a 'scaffold'.

Article	Year
Population study of sizes and components of self-reproducing giant multilamellar vesicles. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 18278955	2008
Autocatalytic membrane-amplification on a pre-existing vesicular surface. Chemical communications (Cambridge, England)\| PubMed ID: 20957268	2010
Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA. Nature chemistry\| PubMed ID: 21941249	2011
Control of in vivo blood clearance time of polymeric micelle by stereochemistry of amphiphilic polydepsipeptides. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 22580110	2012
Pharmacokinetic change of nanoparticulate formulation "Lactosome" on multiple administrations. International immunopharmacology\| PubMed ID: 22841811	2012
Diffuse homogeneous bone marrow uptake of FDG in patients with acute lymphoblastic leukemia. Clinical nuclear medicine\| PubMed ID: 23242061	2013
Clinical value of ¹¹C-methionine PET/CT in patients with plasma cell malignancy: comparison with ¹⁸F-FDG PET/CT. European journal of nuclear medicine and molecular imaging\| PubMed ID: 23340595	2013
Radiosynthesis and initial evaluation of (18)F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide. Nuclear medicine and biology\| PubMed ID: 23347829	2013
Evasion from accelerated blood clearance of nanocarrier named as "Lactosome" induced by excessive administration of Lactosome. Biochimica et biophysica acta\| PubMed ID: 23545239	2013
Spontaneous transformation from micelles to vesicles associated with sequential conversions of comprising amphiphiles within assemblies. Chemical communications (Cambridge, England)\| PubMed ID: 24429723	2014
A comparison between 11C-methionine PET/CT and MIBI SPECT/CT for localization of parathyroid adenomas/hyperplasia. Nuclear medicine communications\| PubMed ID: 25244350	2015
A recursive vesicle-based model protocell with a primitive model cell cycle. Nature communications\| PubMed ID: 26418735	2015
Transformation of oil droplets into giant vesicles. Chemical communications (Cambridge, England)\| PubMed ID: 27152371	2016

Kensuke Kurihara

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