Determination of Permeability Coefficients of Polymersomal

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Determination of Permeability Coefficients of Polymersomal Membranes for Hydrophilic Molecules Sarah T Poschenrieder, Ludwig Klermund, Bettina Langer, and Kathrin Castiglione Langmuir, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 20, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Determination of Permeability Coefficients of Polymersomal Membranes for Hydrophilic Molecules

Sarah T. Poschenrieder, Ludwig Klermund, Bettina Langer and Kathrin Castiglione*

Institute of Biochemical Engineering Technical University of Munich Boltzmannstraße 15, 85748 Garching, Germany E-mail: [email protected]

Abstract Polymer vesicles, so-called polymersomes, can be applied as carrier-systems and universal reaction compartments, due to the possibility to encapsulate guest molecules. Compared to common lipid vesicles, polymersomes show an increased stability and decreased membrane permeability. Control of the mass transport across the membrane is necessary for any application, requiring the precise knowledge of the permeability. So far, data on permeability coefficients of polymersomal membranes are scarce because commonly applied release assays are confronted with the challenge of high detection limits and alternative methods developed so far are either restricted to the use of a certain permeating molecule or rely on the use of nuclear magnetic resonance measurements. In contrast, an influx assay that is broadly applicable to hydrophilic molecules and does not involve specialized equipment was developed in this work, which is based on the passive diffusion of compounds into initially empty vesicles. The method is valid for hydrophilic molecules that show no membrane retention and, thus, do not accumulate within the membrane. Using this method, the permeability of polymersomes made of poly(2-methyloxazoline)15-poly(dimethylsiloxane)681 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 40

poly(2-methyloxazoline)15 for seven model compounds was investigated under varying conditions. Permeability coefficients as low as 1.9·10-14 cm s-1 could be measured. Introduction The possibility of encapsulating molecules in the interior of vesicular compartments is of great interest for a huge variety of seminal applications1. The most common examples for self-assembled membrane vesicles are liposomes (lipid vesicles) and polymersomes (polymer vesicles)2. While the lipid vesicles consist of a phospholipid membrane entrapping an aqueous core3, polymer vesicles are made from amphiphilic block-copolymers which are likewise capable of spontaneous self-assembly in aqueous solution4. Liposomes show a comparatively low stability and the retention of the encapsulated molecules is barely controllable5. In contrast, the polymer membranes of polymersomes are usually much thicker (3-40 nm)6 than liposomal membranes (3-5 nm)7 and therefore provide higher stability and lower permeability. This leads to improved encapsulant retention2,

4-5, 8

. Potential medical

applications of polymersomes span from imaging of tissues in vivo9 to drug delivery systems10-11. Since the polymer vesicles can be loaded with enzymes, they can also be turned into biocatalytically active nanoreactors12-14. Regardless of the application, a controlled mass transport across the membrane is indispensable. For example, a controlled release of the encapsulated cargo is required in the field of drug delivery15. Systems relying on the slow release of pharmaceuticals over time are based on predictable and reliable mass transport rates16-17. In case of triggered-release systems, an ideally complete encapsulant retention in the non-triggered state is desirable10, 1819

. A major advantage of using polymersomes as biocatalytically active enzyme nanoreactors

is the possibility to mimic nature’s organizational principles of compartmentalization and selective mass transport. Compartmentalized reaction systems have great potential to improve multistep enzymatic syntheses with mutually incompatible reaction steps by spatial separation 2 ACS Paragon Plus Environment

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

of individual enzymatic transformations. For this purpose, a selective mass transport of the substrates and the products across the membrane is needed and the rates of unspecific transport of compounds should be as low as possible. A very elaborate system, for example, is the establishment of a highly selective mass transport by integration of natural or engineered membrane proteins20-25 or DNA nano-pores26 in polymer membranes with low intrinsic permeability. One of the most studied amphiphilic triblock copolymers for polymersomes used for biotechnological applications is poly(2-methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2methyloxazoline) (PMOXA-PDMS-PMOXA). This polymer does not evoke inflammatory responses in biological systems, shows low nonspecific protein binding properties and has a low cytotoxicity27-29. Moreover, since the membrane functionalization in form of the functional integration of diverse transmembrane proteins20-25, and the simple surface functionalization with immobilized proteins30 is feasible when using this polymer, it is ideally suited for the formation of polymersomes intended to serve as nano-scale reactors for enzymatic reactions. Although membranes made of PMOXA-PDMS-PMOXA were described as ‘impermeable’ for most of the investigated molecules, some of the substances such as superoxide anions, guanidinium chloride, BZiPAR (Rhodamine 110, bis-(N-CBZ-L-isoleucylL-prolyl-L-arginine amide), dihydrochloride), and fluorescein, were able to pass this particular type of membrane31-33. Numerous studies focusing on the permeability of vesicular membranes are based on the use of release assays, investigating the retention of the previously encapsulated molecules within a specified time. However, there is one crucial issue when using the release assay, which has to be considered. The judgement about a certain membrane to be (im)permeable depends on the considered time frame, since any molecule will permeate at some point due to passive diffusion if a concentration gradient across the membrane is applied. This aspect correlates 3 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

also with the detection limit of the applied assay. Compounds with very low permeability should be encapsulated in high concentrations in polymersomes to generate a detectable signal in reasonable time. However, this is not always possible since high concentrations of certain substances may disturb the polymersome formation. An aggravating factor is the dilution factor of the assay since the encapsulated volume fraction is typically in the range of 1 % or lower34-35. Taken together, the determination of reliable and comparable permeability coefficients – especially for slowly permeating hydrophilic substances – is much more meaningful than the highly assay-dependent classification of a compound as ‘(im)permeable’. Alternative methods to common release assays, which have been used to determine permeability coefficients of polymersomal membranes, are either restricted to certain permeating molecules or involve specialized pulsed field gradient nuclear magnetic resonance (PFG-NMR) measurements36-39. For example, the membrane permeability towards water can be determined by measuring the shrinking- or swelling behavior of the vesicles in hyper- or hypotonic solutions40-41. Similarly, in 2006 Battaglia et al. presented a method enabling the determination of the permeability coefficients of diverse membranes for 5,5’-dithiobis-(2nitrobenzoic acid)34. Both methods are very useful in order to compare the permeability of diverse membranes, but are not applicable to determine the permeability of a given membrane towards various molecules of interest. In contrast, PFG-NMR measurements are versatile and could be used to study the permeability of different molecules36-39. However, polymersomes have been attracting increasing attention from researchers from highly diverse fields and not all of them might have easy access to PFG-NMR measurements. Therefore, the aim of this work was to develop a simple assay for the determination of permeability coefficients that does not rely on the use of specialized equipment and is applicable for diverse molecules. In this study, the mass transport behavior of seven model compounds was investigated to show

4 ACS Paragon Plus Environment

Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

the functionality of the new assay. Methodical limitations of the assay, such as its limitation to hydrophilic molecules without significant membrane retention, are discussed.

Experimental Section Materials The

amphiphilic

ABA

triblock

copolymer

poly(2-methyloxazoline)15-b-

poly(dimethylsiloxane)68-b-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) was purchased from Polymersource Inc. (Dorval QC, Canada). It has a molecular mass of 7600 g mol-1 (1300-5000-1300 g mol-1) and shows a narrow molecular mass distribution which is given by a low polydispersity index of 1.23. The model compound caffeine (99.7 % purity) was purchased from Alfa Aesar (Karlsruhe, Germany). N-Acetylglucosamine (GlcNAc) (≥99 % purity), N-acetylmannosamine (ManNAc) (≥98 % purity), pyruvate (≥99 % purity), adenosine triphosphate (ATP) (≥98 % purity), cytidine triphosphate (CTP) (≥98 % purity) and the nicotinamide cofactors NAD(H) (≥98 % purity) were purchased from Carl Roth (Karlsruhe, Germany). N-Acetylneuraminate (Neu5Ac) (≥98 % purity) was purchased from Calbiochem (part of Merck, Darmstadt, Germany). Enzymes used for the concentration determination of the model compounds were either purchased or recombinantly expressed in Escherichia coli and purified. Recombinant lactate dehydrogenase (LDH) from E. coli and inorganic pyrophosphatase from baker’s yeast were purchased from Sigma-Aldrich (St. Louis, USA). The N-acetylmannosamine dehydrogenase from Flavobacterium sp. 141-7 (ManDH), the N-acetylneuraminate lyase from E. coli K12 and the CMP-sialic acid synthetase (CSS) from Neisseria meningitidis were recombinantly expressed and purified according to the method described by Groher and Hoelsch52. The N-


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acyl-D-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 was recombinantly expressed and purified according to the method described by Klermund et al.53. Polymersome Preparation and Evaluation Polymersomes were produced as described previously54. In brief, to get a 1 % w/v polymersome dispersion a 20 % w/v ethanolic polymer solution was injected manually to a stirred 67 mM phosphate buffer (pH 6/ pH 7/ pH 8) or 50 mM bicine buffer, pH 8. The dispersion (12 mL) was stirred for 1.5 h at 4000 rpm and 22 °C by using the so-called Sstirrer55-56 in an unbaffled miniaturized-stirred tank reactor. Afterwards, the polymersome dispersion was extruded one time using a 0.2 µm polycarbonate filter membrane (Millipore, Billerica, Massachusetts). Polymersome concentrations above 1 % w/v were obtained by concentrating the dispersion in 10 kDa Vivaspin 20 ultrafiltration units (Sartorius, Göttingen, Germany). Detection and quantification of polymersomes was carried out by absorption measurements in 384 well flat bottom UV-assay plates (Brand, Wertheim, Germany). For this purpose, a volume of 50 µL was measured in a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland) at 280 nm. Dynamic light scattering, described in detail elsewhere35, , was used to detect the polymersome size (z-average and number based mean diameter )

54

and the size distribution (polydispersity index). Polymersome Purification After discrete time intervals during the influx assay (described in the Results section ’Development of the Influx Assay (IFA)’) the polymersomes were separated from the free model compounds by size exclusion chromatography (SEC). A sample volume of 0.2 mL was applied on 2.5 mL laboratory columns (MoBiTec, Göttingen, Germany) packed with Sepharose 4B (GE Healthcare, Chalfont, UK). SEC running buffer (67 mM phosphate buffer pH 6/ pH 7/ pH 8 in case of caffeine or 50 mM bicine buffer, pH 8, in case of all other 6 ACS Paragon Plus Environment

Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

compounds) was added in 0.2 mL increments and was allowed to pass the column by gravitational force at room temperature. The elution volume was collected in 0.2 mL fractions in microcentrifuge tubes and a total of 30 fractions was collected. Polymersome Lysis The intravesicular molecules were forced to be released by detergent mediated polymersome lysis. Polymersome containing fractions were pooled (compare section ’Development of the Influx Assay (IFA)’) and the vesicles were disintegrated by the addition of 1 % v/v n-octylpoly-oxyethylene (O-POE) (Bachem, Bubendorf BL, Switzerland). Concentration Determination of Model Compounds The samples were either measured by UV absorption in 384 well flat bottom UV-assay plates (Brand, Wertheim, Germany) or by enzymatic assays in 96 well flat bottom plates (NUNC A/S, Roskilde, Denmark) or 96 well flat bottom luminescence plates (Brand, Wertheim, Germany) by using a microplate reader (Infinite M200, Tecan, Männedorf, Switzerland). Quantification of the model compound caffeine was carried out by absorption measurements at 273 nm. The concentration was determined using a standard curve with known concentrations (4-250 µM). GlcNAc, ManNAc and Neu5Ac concentrations were determined via an N-acetylmannosamine dehydrogenase assay. The ManDH converts ManNAc to N-acetylmannosaminolactone and requires NAD+ as stoichiometric cofactor. N-Acetylmannosaminolactone is readily hydrolyzed in aqueous solution, making the ManDH reaction irreversible and leading to a full conversion of the compounds57. The ManDH from Flavobacterium sp. 141-7 does not accept GlcNAc or Neu5Ac as substrates. Thus, samples containing ManNAc were measured directly by adding 50 µL samples to a 50 µL reaction mixture containing 50 µg mL-1 ManDH and 5 mM NAD+. Samples containing GlcNAc were converted to ManNAc by addition of 150 µg 7 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mL-1 of N-acyl-D-glucosamine 2-epimerase and 1 mM ATP in a coupled reaction with the ManDH. Likewise, samples containing Neu5Ac were converted to ManNAc by addition of 150 µg mL-1 of N-acetylneuraminate lyase in a coupled reaction with the ManDH. Each reaction was allowed to proceed to completion, which was reached after 20 min. The substrate conversion was determined by measuring the NADH concentration in the sample after 20 min at 340 nm. Reactant concentrations were determined from a respective standard curve with known reactant concentrations in the range of 0 - 70 µM. Pyruvate concentrations were determined via a lactate dehydrogenase assay. The LDH reduces pyruvate to lactate by oxidizing NADH to NAD+. The equilibrium lies far on the product side and thus the reaction is quasi-irreversible58. Samples containing pyruvate were added to a reaction mixture containing 10 µg mL-1 LDH and 1 mM NADH. The reaction was allowed to proceed to completion, which was reached after 20 min. The substrate conversion was determined by measuring the NADH consumption after 20 min at 340 nm. Pyruvate concentrations were determined from a standard curve with known pyruvate concentrations in the range of 0 - 70 µM. ATP concentrations were determined using the Kinase-Glo® Luminescent Kinase Assay according to the supplier’s manual (Promega, Fitchburg, USA). A standard curve with known ATP concentrations in the range of 0 – 100 µM was used. CTP concentrations were determined via a CMP-sialic acid synthetase reaction, which is quasi-irreversible59. Because O-POE interfered with the CSS reaction, the polymersomes were pretreated with 1 % w/v of the detergent ocytl-glucoside for 1 h at room temperature prior to CTP concentration measurements. The reaction mixture contained 50 µg mL-1 CSS, 3 mM Neu5Ac, 10 mM MgCl2, 0.2 mM dithiothreitol (DTT) and 4 U mL-1 inorganic pyrophosphatase. Stoichiometric production of phosphate was determined via the Phosphate Colorimetric Kit (Sigma-Aldrich, St. Louis, USA). The reaction was allowed to proceed to completion, which was reached after 8 ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

5 min. CTP concentrations were determined from a standard curve with known CTP concentrations in the range of 0 – 70 µM. All standard curves were prepared in the presence of empty polymersomes (same concentration as present in the pooled polymersome containing fractions) and 1 % v/v of the lysis detergent O-POE unless stated otherwise. Calculation of the Permeability Coefficient The permeability coefficient (cm s-1) of the permeating substance can be defined as the number of molecules (mol) passing the membrane area (cm2) per unit time (s) under a concentration gradient (mol cm-3). In general, the mass transport across the membrane is described by the flux of the permeating molecules (mol cm-2 s-1). When steady-state is reached, the change in concentration with time is zero

= 0 and the Fick’s first law of

diffusion can be applied. According to Fick’s first law, the diffusion flux is proportional to the negative of the constant concentration gradient across the membrane

(mol cm-4) in the

Cartesian coordinate system. The proportionality factor is synonymous with the diffusion coefficient (cm2 s-1) and it therefore follows: = − ∙

(1)

The change of the amount of substance over time is equal to the product of the diffusion flux and the perfused membrane area :

= − ∙

(2)

Thus, with = · the diffusion flux can also be expressed as follows:

= − ∙


(3)

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

With the membrane thickness (cm), the Nernst partition coefficient =

,

=

!,

!

(compare Figure 2, left side) and the assumption of no membrane retention of the molecules, equation (1) can be transformed to equation (4): = With =

& ∙ # $

" ∙ # $

∙ %& − '

(4)

, the diffusion flux across the membrane can also be expressed as a function of

the permeability coefficient: = ∙ %& − '

(5)

Combining equation (3) and equation (5) results in the following differential equation:

=−

() ∙

%& − '

(6)

By integrating equation (6) and with the equilibrium concentration )* =

! ∙ ! + ∙ ! +

, an

experimentally accessible expression for the permeability is received. Consequently, with the volumes of the donor and the acceptor phase ( and & ) and the relating time depending concentrations ( and & ), the permeability coefficient Pe can be calculated: = − %

! ∙ ! + '∙ ∙

∙ ,- .1 −

!

01

2

(7)

Results and Discussion The aim of this work was the development of a new method for the simple determination of polymersome permeability coefficients. Therefore, the permeability of polymersomal membranes made of PMOXA15-PDMS68-PMOXA15 was investigated. Each polymersome dispersion used had a polydispersity index < 0.2, indicating a narrow size distribution60. The z-average was approximately 175 nm and the number based vesicle mean diameter around


Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

100 nm. To study the membrane permeability, the passive diffusion of seven model compounds was investigated (Figure 1).

Figure 1: Chemical structures of the selected model compounds. Table 1 summarizes the physicochemical properties of the selected model compounds. They differ in size, charge and hydrophilicity (as expressed by the decadic logarithm of the octanolwater partition coefficient logP at the investigated pH). Caffeine was chosen due to its wellknown mass transport behavior across planar lipid membranes45, 48. The amino sugars and (deprotonated) acids were selected because they are examples for hydrophilic molecules that are involved in industrially relevant enzymatic syntheses. The determination of the corresponding permeability coefficients is of particular relevance if polymersomes are intended to be used as nanoreactors for synthetic purposes. For example, N-acetylglucosamine 11 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

(GlcNAc) and N-acetylmannosamine (ManNAc), respectively, can be used for the synthesis of sialic acids, such as N-acetylneuraminate (Neu5Ac) or derivatives thereof61-63. Pyruvate is a key metabolite of natural metabolic pathways and involved in many biotransformations in the chemical and pharmaceutical industries64, e.g. in the enzymatic (R)-phenylacetylcarbinol production65. The nucleotides adenosine triphosphate (ATP) and cytidine triphosphate (CTP) were chosen as examples for comparatively large and highly charged molecules. The low detection limits of these compounds in enzymatic assays (0.2 µM for ATP and 5 µM for CTP) are prerequisites for the determination of very low concentrations of these substances, which would in turn allow for the determination of very low permeability coefficients. For all experiments, a threefold determination was carried out. The permeability of caffeine was determined at 22 °C, the permeability of all other compounds was determined at 30°C since this temperature has a higher relevance for enzymatic syntheses.

Table 1: Size, charge (at pH 8.0) and octanol-water partition coefficient logP (at pH 8.0) of the model compounds. All data was calculated with Chemicalize Beta by ChemAxon Ltd. (www.chemicalize.com) Compound

Size, Da

Charge

logP

Caffeine

194

0

-0.55

N-Acetylglucosamine

221

0

-3.86

N-Acetylmannosamine

221

0

-3.86

Pyruvate

88

-1

-3.45

N-Acetylneuraminate

309

-1

-8.56

ATP

507

-3.9

-10.86

CTP

483

-3.9

-12.66

Development of the Influx Assay (IFA)


Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

In this work the passive diffusion of the target molecules from the extravesicular aqueous phase into initially unloaded polymersomes, hereinafter to be referred to as influx assay (IFA), was investigated. A scheme of the resulting concentration profile across the polymer membrane, assuming that there are no concentration gradients in both the extravesicular and the intravesicular phases, is shown in Figure 2 on the left side.

Figure 2. Concentration profile across the polymersomal membrane, separating the extravesicular donor compartment (index D) from the intravesicular acceptor compartment (index A). The permeating molecules pass the polymersomal membrane (thickness ) by passive diffusion, driven by the concentration gradient (& − ). The inner vesicle volume 34,5 of the polymersome (vesicle diameter ) corresponds to the acceptor compartment.

To calculate via equation (7), the volume of the acceptor phase and the donor phase ( , & ), the total membrane surface , the equilibrium concentration )* as well as the acceptor phase concentration at a specified incubation time have to be known. In the following, the determination of the named parameters is explained.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 40

Applying the IFA, the donor volume & is equal to the extravesicular volume and can be expressed as the difference between the sample volume 6 and the volume being occupied by all vesicles within the dispersion 789 . Since the latter is equal to the sum of the total membrane volume : and the total inner volume of all polymersomes 34 , the volume of the donor phase can be expressed as follows: & = 6 − 789 = ; − : − 34

(8)

Besides, the acceptor phase volume is synonymous with the total inner vesicle volume 34 , which can be calculated by the product of the number of polymersomes within the sample volume and the inner volume of a single polymer vesicle 34,5 : = 34 = ∙ 34,5

(9)

As shown in equation (10), the number of polymersomes within the sample can be calculated by the total polymer mass > representing the number of polymer chains forming one vesicle. The aggregation number of PMOXA15PDMS68-PMOXA15 polymersomes produced by the formerly developed polymersome production process was determined to 43.00054. =

?@ @

∙ !

(10)

!AA

Alternatively, according to the method developed by Battaglia et al., can be calculated by the aid of the encapsulation efficiency BB with =

CC·D 34 . E34

The encapsulation efficiency of

PMOXA15-PDMS68-PMOXA15 polymersomes produced by the named process54 was experimentally determined to 0.53 %35.


Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Knowing the membrane thickness of PMOXA15-PDMS68-PMOXA15 polymersomes ( = 14 nm, determined by cryo transmission electron microscopy54) and the outer polymersome diameter being equal to the number based vesicle mean diameter measured by dynamic light scattering, the inner volume of a single polymersome can be calculated by equation (11). F

34,5 = ∙ H ∙ % − 2 ∙ 'J

(11)

G

Applying the IFA, the total membrane area being available for the permeating molecules is equal to the total outer membrane surface of all polymersomes within the dispersion and is the product of the total number of polymersomes and the outer membrane surface of a single polymersome 789,5 : = ∙ 789,5 = ∙ H ∙ K

(12)

Table 2 shows the values needed for the determination of the permeability coefficients, exemplarily calculated for 1 cm3 of a 1 % w/v polymersome dispersion showing a number based mean particle diameter of 100 nm. Table 2: Required values for permeability coefficient calculation, exemplarily shown for a sample volume 6 of 1 cm3, a 1 % w/v polymersome dispersion and a number based polymersome diameter of 100 nm. Symbol Description

Value

Number of vesicles

1.84·1013

34,5

Inner vesicle volume of a single polymersome

1.95·10-16 cm3

,L

Outer surface of a single polymersome

3.14·10-10 cm2

Volume of the acceptor phase

3.60·10-3 cm3

&

Volume of the donor phase

9.90 ·10-1 cm3

Total outer membrane surface

5.80·103 cm2


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 40

The equilibrium concentration )* was calculated by the conservation of mass with the initial donor concentration &L , assuming that no membrane retention occurred: )* =

5 ∙ ! +

(13)

The acceptor phase concentration was determined experimentally, as will be explained in the following. To induce the passive diffusion of the molecules of interest from the extravesicular to the intravesicular space, empty polymersomes and the model compound were mixed at the beginning of the IFA % = 0', as shown in Figure 3. Due to the resulting concentration gradient across the polymersomal membrane, incubating the initially empty polymersomes for a specified time led to the mass transport of the model compound into the vesicles. By subsequently separating the compounds of the mixture via SEC, the partially loaded polymersomes were isolated from the free molecules. Finally, by detergent mediated vesicle lysis, the encapsulated molecules were forced to release and the intravesicular concentration was determined by absorbance measurements and enzymatic assays, respectively.

Figure 3. At the beginning of the IFA % = 0' empty polymersomes and a model compound containing solution were mixed. At a specified incubation time , the partially loaded polymersomes were separated from free molecules by size exclusion chromatography (SEC).

Dimensionless Concentration Profile


Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

The established IFA was carried out using all seven model compounds. To investigate the passive diffusion of caffeine, one part of a 100 mM stock solution was added to one part of a 2 % w/v polymersome dispersion. GlcNAc, ManNAc, Neu5Ac, pyruvate, ATP and CTP were investigated by adding one part of a 100 mM stock solution of each compound to one part of a 1 % w/v polymersome dispersion. The stock solutions and the polymersome dispersions were prepared in the same buffer, leading to iso-pH condition. As discussed above, subsequent to the mixing of the empty vesicles and the model compound containing solution, mass transport into the polymersomes occurred due to the concentration gradient across the membrane. To display the diffusion process over time, the dimensionless acceptor phase number , showing the ratio of the acceptor phase concentration to the equilibrium concentration )* was introduced: =

!

01

(14)

Since the mass transport, driven by passive diffusion occurs until the extravesicular concentration and the intravesicular concentration are equal to the equilibrium concentration M& = = )* N, ranges from zero ( = 0) to one ( = )* ): 0 ≤ %' ≤ 1. In Figure 4 A the resulting increase of over time when incubating initially empty polymersomes with GlcNAc and ManNAc, respectively, is shown exemplarily. The occurring mass transport of the amino sugar molecules from the extravesicular space into the interior of the vesicles is confirmed by the continuous increase of . 4270


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Dimensionless acceptor phase number during the IFA when using A) Nacetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) at pH 8 and B) caffeine at pH 6, pH 7 and pH 8 as model compound. According to equation (14) can adopt values between 0 and 1. The horizontal reference line at = 1 serves as visual aid.

The pH dependence of the measured permeability coefficients was investigated on the example of caffeine. No significant pH dependent mass transport of caffeine was observed within an incubation time of up to 1157 h, as shown in Figure 4 B. The pH-independent passive diffusion of caffeine can be explained by its unionizable molecule structure. Thus, at any pH investigated, the prevalent molecules were uncharged, leading to equally fast mass transport across the membrane, because the applied polymersomal membrane made of PMOXA15-PDMS68-PMOXA15 is also not pH-responsive. It is broadly accepted that the permeability of most polymersomes is lower than those of lipid vesicles1-2, 4-5. Due to the increased chain length of the polymers compared to lipids and due to their conformational freedom the membrane permeability is reduced4. However, because of the physicochemical versatility of block copolymers, the membrane properties of polymersomes are tunable. Battaglia et al. have shown that poly(ethylene oxide)-b18 ACS Paragon Plus Environment

Page 18 of 40

Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

polybutylene oxide polymersomes are an order of magnitude more permeable towards Ellman’s reagent (5,5'-dithiobis-(2-nitrobenzoic acid), DTNB) than lipid vesicles made from egg yolk phosphatidylcholine at comparable membrane thickness (3.4 nm)

34

. In case of

PMOXA-PDMS-PMOXA membranes, it was demonstrated that completely different molecules,

namely superoxide anions, guanidinium chloride, BZiPAR (Rhodamine 110, bis-(N-CBZ-Lisoleucyl-L-prolyl-L-arginine amide), dihydrochloride), and fluorescein are able to cross the compartment boundaries

31-33

. Indeed, since any compound permeates at some point if a

concentration gradient across the membrane is applied, the judgment about the (im)permeability of a certain membrane by measuring the release of the molecules is a question of the considered time frame. Consequently, being able to determine the permeability of a membrane towards a certain molecule also depends on the detectability of the molecule of interest in the extravesicular space. In contrast to the IFA developed in this work, commonly applied release assays are based on the previous encapsulation of the target molecules of interest66-69. A problem not to be underestimated when using the release assay is the concentration, which has to be entrapped within the vesicles. Compared to the initial intravesicular concentration, being synonymous to the initial donor phase concentration &L , the released substance might be diluted several orders of magnitude in the extravesicular space, depending on the ratio of the total inner vesicle volume to the extravesicular volume. In order to determine the permeability within a reasonable experimental time, the detection of at least 10 % release being equal to = 0.1, compare equation (14), should be feasible. The needed initial concentration, which has to be encapsulated within the vesicles can be calculated by combining equation (13) and equation (14) and it follows &L =

! ·%! + ' ·

.

Thus, the required initial intravesicular concentration &L greatly depends on the detectable extravesicular concentration, being synonymous to the acceptor phase concentration . By the example of the model compounds chosen in this work, with a detection limit of about 10 19 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

µM for GlcNAc, ManNAc, Neu5Ac and pyruvate and 5 µM for caffeine, the encapsulation of 200 mM or 100 mM, respectively, would have been required. Indeed, within this work the investigation of the release behavior of caffeine failed, since no vesicles were formed during the polymersome production process in the presence of 100 mM caffeine due to undesired interaction of the polymer and the drug (data not shown). Although polymersomes formed in the presence of the other model compounds used in this study, polymersome formation was impaired at higher concentration, leading to the appearance of undesired micelles and polymer aggregates and a substantial reduction of the polymersome quality. Because the interpretation of the release assay data strongly relies on the exact quantitation of the membrane area, which is hampered by polymersome preparations with high polydispersity, the investigation of the dimensionless concentration profiles using a release assay was not possible in any case. Permeability Coefficients To quantitatively evaluate the permeability of PMOXA15-PDMS68-PMOXA15 polymersomes, the permeability coefficient was calculated by equation (7). All values for discussed in this section refer to the unit cm s-1. When associated to a certain molecule with particular physicochemical properties, possibly depending on the pH, is a material constant. Since the permeability coefficient is thus independent of the incubation time , was calculated for all measurements where equilibrium was not yet reached and the average results were created. The resulting values (displayed as negative decadic logarithm of ) are shown in Figure 5. The approximately constant value of about − ,PQ = 13.72 ± 0.11 again illustrates the already mentioned pH-independent permeability of the investigated membrane towards caffeine. The polar but uncharged GlcNAc and ManNAc diffused across the polymer membrane with a − ,PQ of 9.98 ± 0.09 and 9.96 ± 0.11, respectively. The high consistency of the results obtained for the two epimers, which are identical in size and highly similar in structure, 20 ACS Paragon Plus Environment

Page 20 of 40

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

validates the accuracy of the assay. In contrast, the negatively charged and slightly larger Neu5Ac showed a more than 500-fold reduced permeability compared to GlcNAc and ManNAc with a − ,PQ of 12.70 ± 0.59. For pyruvate, a − ,PQ of 12.04 ± 0.26 was determined. Since both Neu5Ac and pyruvate have pKa values of 2.5 – 2.6, they are almost exclusively present in their deprotonated form at pH 8.0. Thus, permeability across the polymer membrane was dominated by the charge and only moderately affected by the size of the respective molecule. In congruence, the large and highly charged nucleotides ATP and CTP (overall charge at pH 8 of -3.9) could not be detected in the polymersome lumen even after prolonged incubation for more than 2 weeks, indicating that the polymer membrane has an extremely low permeability toward nucleoside triphosphates.

Figure 5. Negative decadic logarithm of the permeability coefficients (cm s-1) of polymersomal PMOXA15-PDMS68-PMOXA15 membranes. The mass transport of A) caffeine at 22°C and pH 6, pH 7 and pH 8 and B) N-acetylglucosamine (GlcNAc), Nacetylmannosamine (ManNAc), pyruvate and N-acetylneuraminate (Neu5Ac) at 30°C and pH 8.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In any case, the determined permeability coefficients of polymersomal PMOXA15-PDMS68PMOXA15 membranes were considerably lower than the permeability coefficients found for synthetic lipid membranes. In the context of lipid membranes, the permeability of substances is typically classified as low (− ,PQ > 7) moderate (6 < − ,PQ < 7) and high (−,PQ < 6) permeability71. With – ,PQ values of about 7.0-7.5, the permeability of lipid membranes towards uncharged molecules of comparable size (in relation to GlcNAc, ManNAc and caffeine) was at least 2.5 orders of magnitude higher than of the investigated polymer membrane72. For example, a − ,PQ = 7.3 was determined for the permeability of glucose across lipid membranes at 35°C73. This indicates the high diffusional barrier of PMOXA15-PDMS68-PMOXA15 membranes and the suitability of developed assay for the determination of very low permeability coefficients. For ATP and CTP, the inability to measure the permeability coefficients was due to the extremely low permeability of the highly charged molecules. With our assay, a theoretical permeability coefficient of − ,PQ = 14.5 would have been detectable with an ATP detection limit of 0.2 µM and an incubation time of 336 h. Likewise, the detection limit for CTP was approximately − ,PQ = 13.4. Thus, the permeability of the nucleotides was more than 3.4-4.5 orders of magnitude lower than the permeability of GlcNAc and ManNAc. Despite the huge efforts that were made in order to investigate the mass transport behavior across polymersomal membranes, only few permeability coefficients were published so far. Using stopped-flow spectroscopy, the osmotic permeability of polymersomes was investigated by measuring the shrinking or swelling behavior of vesicles in hyper- or hypotonic solutions40. In this way, the permeability coefficient of polymersomal membranes made of PMOXA15-PDMS68-PMOXA15 towards water was determined. Values in the range of – ,PQ = 2.00 to – ,PQ = 1.10 were found40. Thus, the permeability of the named membrane towards water is approximately as high as the permeability of liposomal 22 ACS Paragon Plus Environment

Page 22 of 40

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

membranes. Depending on the used lipid, values from – ,PQ = 2.67 to – ,PQ = 1.80 were published by Mathai et al.74. Moreover, Battaglia et al. presented a suitable method to determine the permeability coefficients of membrane vesicles based on the diffusion of DTNB from the extravesicular space into the vesicles. Within the vesicles, previously encapsulated 3,3’,3’’-phosphinidynetris-(benzenesulfonic acid) (PH) reacts with DTNB forming thionitrobenzoate (TNB), which can be measured photometrically. Using this method, it was shown that the permeability coefficients of the investigated membranes made of poly(ethylene oxide)-co-poly(butylene oxide) towards PH depend on the pH and the membrane thickness. Values from – ,PQ = 5.71 at pH 6 to – ,PQ = 7.60 at pH 8 (membrane thickness 2.40 nm) and decreasing permeability with increasing membrane thickness (– ,PQ = 7.31 for a membrane thickness of 4.07 nm and – ,PQ = 7.60 for a 7.56 nm thick membrane, pH 7.2) were published34. This method seems to be very suitable in order to compare the permeability of different membranes. Nevertheless, it is restricted to the determination of the membrane permeability towards only one molecule, namely DTNB, whereas the IFA can be used for the determination of the membrane permeability of various hydrophilic molecules with low to medium molecular mass (hydrophilic macromolecules, such as DNA or proteins, will not cross polymersomal membranes without assistance e.g. by electroporation75). In comparison with permeability investigations using PFG-NMR measurements, which represents an alternative method to the IFA with high versatility

36-39

,

our newly developed assay does not involve specialized equipment, which might not be easily accessible in every laboratory dealing with polymersome research. Methodological limitations The developed IFA has two methodological limitations which will be stressed in the following section:


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a) The assay has been developed for the determination of low permeability coefficients because the majority of polymersomal membranes has been described as significantly less permeable than classical lipid membranes. The determination of high permeability coefficients – in the range of – ,PQ ≤ 8 – is error-prone because significant backdiffusion of molecules out of the vesicles during the short SEC step, which takes about 5 min, cannot be excluded. However, the analytical limit of the presented IFA is not only a function of the permeability coefficient, but also of the diameter of the vesicles. The larger the vesicles are, the higher permeability coefficients can be determined reliably because the surface-area-to-volume ratio (/) of vesicles is inversely proportional to the vesicle diameter. Lower /-ratios lead to longer incubation times before the equilibrium is reached, which in turn poses less requirements on the speed of the separation step. b) The permeability equation used (eq. 7) is only valid for systems with negligible membrane retention, because the mole fraction of the investigated compound that partitions into the membrane is not considered. It has been shown that the octanolwater partition coefficient ,PQ correlates well with the membrane retention of (artificial) lipid membranes76 and also with PDMS-water partition coefficients77, which would be relevant for the exemplary block copolymer used in this study. However, for a mathematical description of the permeability with membrane retention not only the determination of the membrane-water or membrane-buffer partition coefficient is needed, but also knowledge about the lag time that is required to reach saturation of the membrane, which cannot be inferred from the IFA data. Only after this lag time, the steady-state approximation of Fick’s first law is valid76. For these reasons, the developed assay is restricted to (hydrophilic) molecules without significant membrane retention.


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Summary and Conclusion A simple method for the quantitative permeability determination of hydrophilic molecules was developed in this work, based on the passive diffusion of the target molecule into initially empty polymersomes (influx assay IFA). The main advantage of this assay, compared to commonly used release assays is its independence from the encapsulation of compounds in high concentrations. In comparison to PFG-NMR measurements, being suitable for permeability determination of diverse molecules, the IFA is of great benefit due to its independence from specialized equipment. Besides, the new assay is useful for various hydrophilic molecule quantifiable by photometric detection – either by direct measurements or by enzymatic assays. The functionality of the IFA was shown by the passive diffusion of seven hydrophilic model compounds. Acknowledgements The support of this work by Prof. Dirk Weuster-Botz and the possibility of using the outstanding technical facilities at his Institute of Biochemical Engineering in Garching (Technical University of Munich) are gratefully acknowledged. The support of Sarah T. Poschenrieder and Ludwig Klermund by the TUM Graduate School is acknowledged as well. This research project is funded by the German Federal Ministry of Education and Research (project funding reference number 031A178). References 1.

Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Block copolymer

vesicles—using concepts from polymer chemistry to mimic biomembranes. Polymer 2005, 46, 3540-3563. 2.

Discher, D. E.; Ahmed, F. Polymersomes. Annu. Rev. Biomed. Eng. 2006, 8, 323-341.

3.

Bangham, A. D., Liposomes: the Babraham connection. Chem. Phys. Lipids 1993, 64,

275-285. 25 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4.

Discher, B. M.; Won, Y. Y.; Ege, D. S.; Lee, J. C.; Bates, F. S.; Discher, D. E.;

Hammer, D. A. Polymersomes: tough vesicles made from diblock copolymers. Science 1999, 284, 1143-1146. 5.

Discher, D. E.; Eisenberg, A. Polymer vesicles. Science 2002, 297, 967-973.

6.

Palivan, C. G.; Goers, R.; Najer, A.; Zhang, X. Y.; Car, A.; Meier, W. Bioinspired

polymer vesicles and membranes for biological and medical applications. Chem. Soc. Rev. 2016, 45, 377-411. 7.

Le Meins, J. F.; Sandre, O.; Lecommandoux, S. Recent trends in the tuning of

polymersomes' membrane properties. Eur. Phys. J. E. Soft Matter 2011, 34, 1-17. 8.

Bermudez, H.; Brannan, A. K.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Molecular

weight dependence of polymersome membrane structure, elasticity, and stability. Macromolecules 2002, 35, 8203-8208. 9.

Ghoroghchian, P. P.; Frail, P. R.; Susumu, K.; Blessington, D.; Brannan, A. K.; Bates,

F. S.; Chance, B.; Hammer, D. A.; Therien, M. J. Near-infrared-emissive polymersomes: Selfassembled soft matter for in vivo optical imaging. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2922-2927. 10.

Meng, F. H.; Zhong, Z. Y.; Feijen, J. Stimuli-Responsive Polymersomes for

Programmed Drug Delivery. Biomacromolecules 2009, 10, 197-209. 11.

Cheng, Z. L.; Tsourkas, A. Paramagnetic porous polymersomes. Langmuir 2008, 24,

8169-8173. 12.

Winterhalter, M.; Hilty, C.; Bezrukov, S. M.; Nardin, C.; Meier, W.; Fournier, D.

Controlling membrane permeability with bacterial porins: application to encapsulated enzymes. Talanta 2001, 55, 965-971. 13.

De Vocht, C.; Ranquin, A.; Willaert, R.; Van Ginderachter, J. A.; Vanhaecke, T.;

Rogiers, V.; Versees, W.; Van Gelder, P.; Steyaert, J. Assessment of stability, toxicity and


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE. J. Control. Release 2009, 137, 246-254. 14.

Nallani, M.; Andreasson-Ochsner, M.; Tan, C. W.; Sinner, E. K.; Wisantoso, Y.;

Geifman-Shochat, S.; Hunziker, W. Proteopolymersomes: in vitro production of a membrane protein in polymersome membranes. Biointerphases 2011, 6 (4), 153-7. 15.

Ahmed, F.; Discher, D. E. Self-porating polymersomes of PEG-PLA and PEG-PCL:

hydrolysis-triggered controlled release vesicles. J. Control. Release 2004, 96, 37-53. 16.

Wittemann, A.; Azzam, T.; Eisenberg, A. Biocompatible Polymer Vesicles from

Biamphiphilic Triblock Copolymers and Their Interaction with Bovine Serum Albumin. Langmuir 2007, 23, 2224-2230. 17.

Meng, F.; Engbers, G. H. M.; Feijen, J. Biodegradable polymersomes as a basis for

artificial cells: encapsulation, release and targeting. J. Control. Release 2005, 101, 187-198. 18.

Giacomelli, C.; Schmidt, V.; Borsali, R. Nanocontainers Formed by Self-Assembly of

Poly(ethylene oxide)-b-poly(glycerol monomethacrylate)−Drug Conjugates. Macromolecules 2007, 40, 2148-2157. 19.

Zhou, Y.; Yan, D.; Dong, W.; Tian, Y. Temperature-Responsive Phase Transition of

Polymer Vesicles: Real-Time Morphology Observation and Molecular Mechanism. J. Phys. Chem. B 2007, 111, 1262-1270. 20.

Wong, D.; Jeon, T.-J.; Schmidt, J. Single molecule measurements of channel proteins

incorporated into biomimetic polymer membranes. Nanotechnology 2006, 17, 3710-3717. 21.

Nardin, C.; Thoeni, S.; Widmer, J.; Winterhalter, M.; Meier, W. Nanoreactors based

on (polymerized) ABA-triblock copolymer vesicles. Chem. Commun. 2000, 1433-1434. 22.

Graff, A.; Fraysse-Ailhas, C.; Palivan, C. G.; Grzelakowski, M.; Friedrich, T.; Vebert,

C.; Gescheidt, G.; Meier, W. Amphiphilic Copolymer Membranes Promote NADH:Ubiquinone Oxidoreductase Activity: Towards an Electron-Transfer Nanodevice. Macromol. Chem. Phys. 2010, 211, 229-238. 27 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23.

Kumar, M.; Grzelakowski, M.; Zilles, J.; Clark, M.; Meier, W. Highly permeable

polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20719-24. 24.

Ranquin, A.; Versees, W.; Meier, W.; Steyaert, J.; Van Gelder, P. Therapeutic

nanoreactors: combining chemistry and biology in a novel triblock copolymer drug delivery system. Nano Lett. 2005, 5, 2220-2224. 25.

Gaitzsch, J.; Huang, X.; Voit, B. Engineering Functional Polymer Capsules toward

Smart Nanoreactors. Chem. Rev. 2016, 116, 1053-1093. 26.

Messager, L.; Burns, J. R.; Kim, J.; Cecchin, D.; Hindley, J.; Pyne, A. L. B.; Gaitzsch,

J.; Battaglia, G.; Howorka, S. Biomimetic Hybrid Nanocontainers with Selective Permeability. Angew. Chem. Int. Ed. 2016, 55, 11106-11109. 27.

Tanner, P.; Egli, S.; Balasubramanian, V.; Onaca, O.; Palivan, C. G.; Meier, W. Can

polymeric vesicles that confine enzymatic reactions act as simplified organelles? FEBS Lett. 2011, 585, 1699-706. 28.

Broz, P.; Benito, S. M.; Saw, C.; Burger, P.; Heider, H.; Pfisterer, M.; Marsch, S.;

Meier, W.; Hunziker, P. Cell targeting by a generic receptor-targeted polymer nanocontainer platform. J. Control. Release 2005, 102, 475-488. 29.

Woodle, M. C.; Engbers, C. M.; Zalipsky, S. New Amphipatic Polymer Lipid

Conjugates Forming Long-Circulating Reticuloendothelial System-Evading Liposomes. Bioconjugate Chem. 1994, 5, 493-496. 30.

Klermund, L.; Poschenrieder, S. T.; Castiglione, K. Simple surface functionalization

of polymersomes using non-antibacterial peptide anchors. J Nanobiotechnol. 2016, 14, 48. 31.

Renggli, K.; Baumann, P.; Langowska, K.; Onaca, O.; Bruns, N.; Meier, W. Selective

and Responsive Nanoreactors. Adv. Funct. Mater. 2011, 21, 1241-1259. 32.

Spulber, M.; Najer, A.; Winkelbach, K.; Glaied, O.; Waser, M.; Pieles, U.; Meier, W.;

Bruns, N. Photoreaction of a hydroxyalkyphenone with the membrane of polymersomes: a 28 ACS Paragon Plus Environment

Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

versatile method to generate semipermeable nanoreactors. J. Am. Chem. Soc. 2013, 135, 9204-9212. 33.

Ben-Haim, N.; Broz, P.; Marsch, S.; Meier, W.; Hunziker, P. Cell-specific integration

of artificial organelles based on functionalized polymer vesicles. Nano Lett. 2008, 8, 13681373. 34.

Battaglia, G.; Ryan, A. J.; Tomas, S. Polymeric vesicle permeability: a facile chemical

assay. Langmuir 2006, 22, 4910-4913. 35.

Poschenrieder, S. T.; Schiebel, S. K.; Castiglione, K. Polymersomes for

biotechnological applications: large-scale production of nano-scale vesicles. Eng. Life. Sci 2017, 17, 58-70. 36.

Leson, A.; Filiz, V.; Forster, S.; Mayer, C. Water permeation through block-

copolymer vesicle membranes. Chem. Phys. Lett. 2007, 444, 268-272. 37.

Leson, A.; Hauschild, S.; Rank, A.; Neub, A.; Schubert, R.; Forster, S.; Mayer, C.

Molecular exchange through membranes of poly(2-vinylpyridine-block-ethylene oxide) vesicles. Small 2007, 3, 1074-1083. 38.

Rumplecker, A.; Forster, A.; Zahres, M.; Mayer, C. Molecular exchange through

vesicle membranes: A pulsed field gradient nuclear magnetic resonance study. J. Chem. Phys. 2004, 120, 8740-8747. 39.

Bauer, A.; Hauschild, S.; Stolzenburg, M.; Forster, S.; Mayer, C. Molecular exchange

through membranes of poly(2-vinylpyridine-block-ethylene oxide) vesicles. Chem. Phys. Lett. 2006, 419, 430-433. 40.

Wah Tong, Y.; Xie, W.; Wei Jun Low, J.; Armugam, A.; Jeyaseelan, K. Regulation of

Aquaporin Z osmotic permeability in ABA tri-block copolymer. AIMS Biophysics 2015, 2, 381-397. 41.

Carlsen, A.; Glaser, N.; Le Meins, J. F.; Lecommandoux, S. Block Copolymer Vesicle

Permeability Measured by Osmotic Swelling and Shrinking. Langmuir 2011, 27, 4884-4890. 29 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

42.

Avdeef, A.; Tsinman, O. PAMPA--a drug absorption in vitro model 13. Chemical

selectivity due to membrane hydrogen bonding: in combo comparisons of HDM-, DOPC-, and DS-PAMPA models. Eur. J. Pharm. Sci. 2006, 28, 43-50. 43.

Avdeef, A. The rise of PAMPA. Expert Opin Drug Metab Toxicol 2005, 1, 325-342.

44.

Avdeef, A.; Nielsen, P. E.; Tsinman, O. PAMPA--a drug absorption in vitro model 11.

Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates. Eur. J. Pharm. Sci. 2004, 22, 365-374. 45.

Nielsen, P. E.; Avdeef, A. PAMPA--a drug absorption in vitro model 8. Apparent

filter porosity and the unstirred water layer. Eur. J. Pharm. Sci. 2004, 22, 33-41. 46.

Ruell, J. A.; Tsinman, K. L.; Avdeef, A. PAMPA—a drug absorption in vitro model.

Eur. J. Pharm. Sci. 2003, 20, 393-402. 47.

Sugano, K.; Nabuchi, Y.; Machida, M.; Aso, Y. Prediction of human intestinal

permeability using artificial membrane permeability. Int. J. Pharm. 2003, 257, 245-251. 48.

Avdeef, A.; Strafford, M.; Block, E.; Balogh, M. P.; Chambliss, W.; Khan, I. Drug

absorption in vitro model: filter-immobilized artificial membranes. 2. Studies of the permeability properties of lactones in Piper methysticum Forst. Eur. J. Pharm. Sci. 2001, 14, 271-280. 49.

Wohnsland, F.; Faller, B., High-throughput permeability pH profile and high-

throughput alkane/water log P with artificial membranes. J. Med. Chem. 2001, 44, 923-930. 50.

Sugano, K.; Hamada, H.; Machida, M.; Ushio, H. High Throughput Prediction of Oral

Absorption: Improvement of the Composition of the Lipid Solution Used in Parallel Artificial Membrane Permeation Assay. J. Biomol. Screen. 2001, 6, 189-196. 51.

Kansy, M.; Senner, F.; Gubernator, K. Physicochemical high throughput screening:

parallel artificial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 1998, 41, 1007-1010.


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

52.

Groher, A.; Hoelsch, K. Mechanistic model for the synthesis of N-acetylneuraminic

acid using N-acetylneuraminate lyase from Escherichia coli K12. J. Mol. Catal. B: Enzym. 2012, 83, 1-7. 53.

Klermund, L.; Riederer, A.; Groher, A.; Castiglione, K. High-level soluble expression

of a bacterial N-acyl-D-glucosamine 2-epimerase in recombinant Escherichia coli. Protein Expr. Purif. 2015, 111, 36-41. 54.

Poschenrieder, S. T.; Wagner, S. G.; Castiglione, K. Efficient production of uniform

nanometer-sized polymer vesicles in stirred-tank reactors. J. Appl. Polym. Sci. 2016, 133, 43274. 55.

Riedlberger, P.; Bruning, S.; Weuster-Botz, D. Characterization of stirrers for

screening studies of enzymatic biomass hydrolyses on a milliliter scale. Bioproc. Biosys. Eng. 2013, 36, 927-935. 56.

Riedlberger, P.; Weuster-Botz, D. New miniature stirred-tank bioreactors for parallel

study of enzymatic biomass hydrolysis. Biores. Technol. 2012, 106, 138-146. 57.

Yamamoto-Otake, H.; Koyama, Y.; Horiuchi, T.; Nakano, E. Cloning, sequencing,

and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli. Appl. Environ. Microbiol. 1991, 57, 1418-1422. 58.

Brooks, G. A.; Dubouchaud, H.; Brown, M.; Sicurello, J. P.; Butz, C. E. Role of

mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1129-1134. 59.

Kean, E. L.; Roseman, S., The sialic acids. X. Purification and properties of cytidine

5'-monophosphosialic acid synthetase. J. Biol. Chem. 1966, 241, 5643-5650. 60.

Charcosset, C.; Juban, A.; Valour, J.-P.; Urbaniak, S.; Fessi, H. Preparation of

liposomes at large scale using the ethanol injection method: Effect of scale-up and injection devices. Chem. Eng. Res. Des. 2015, 94, 508-515.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

61.

Klermund, L.; Groher, A.; Castiglione, K. New N-acyl-D-glucosamine 2-epimerases

from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate. J. Biotechnol. 2013, 168, 256-263. 62.

Yi, D.; He, N.; Kickstein, M.; Metzner, J.; Weiss, M.; Berry, A.; Fessner, W. D.,

Engineering of a Cytidine 5 '-Monophosphate-Sialic Acid Synthetase for Improved Tolerance to Functional Sialic Acids. Adv. Synth. Catal. 2013, 355, 3597-3612. 63.

Luchansky, S. J.; Goon, S.; Bertozzi, C. R. Expanding the diversity of unnatural cell-

surface sialic acids. ChemBioChem 2004, 5, 371-374. 64.

Li, Y.; Chen, J.; Lun, S. Y. Biotechnological production of pyruvic acid. Appl.

Microbiol. Biotechnol. 2001, 57, 451-459. 65.

Leksawasdi, N.; Chow, Y. Y. S.; Breuer, M.; Hauer, B.; Rosche, B.; Rogers, P. L.

Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process. J. Biotechnol. 2004, 111, 179-189. 66.

Heerklotz, H.; Seelig, J. Leakage and lysis of lipid membranes induced by the

lipopeptide surfactin. Eur. Biophys. J. 2007, 36, 305-314. 67.

Litvinchuk, S.; Lu, Z.; Rigler, P.; Hirt, T. D.; Meier, W. Calcein Release from

Polymeric Vesicles in Blood Plasma and PVA Hydrogel. Pharm. Res. 2009, 26, 1711-1717. 68.

Patel, H.; Tscheka, C.; Heerklotz, H. Characterizing vesicle leakage by fluorescence

lifetime measurements. Soft Matter 2009, 5, 2849. 69.

Maherani, B.; Arab-Tehrany, E.; Kheirolomoom, A.; Geny, D.; Linder, M. Calcein

release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids. Biochimie 2013, 95, 2018-2033. 70.

Avdeef, A.; Box, K. J.; Comer, J. E.; Hibbert, C.; Tam, K. Y. pH-metric logP 10.

Determination of liposomal membrane-water partition coefficients of ionizable drugs. Pharm. Res. 1998, 15, 209-215.


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

71.

Fischer, H.; Kansy, M.; Avdeef, A.; Senner, F., Permeation of permanently positive

charged molecules through artificial membranes--influence of physico-chemical properties. Eur. J. Pharm. Sci. 2007, 31, 32-42. 72.

Alberts, B., Molecular biology of the cell. 3rd ed.; Garland Pub.: New York, 1994.

73.

Wood, R. E.; Wirth, F. P., Jr.; Morgan, H. E. Glucose permeability of lipid bilayer

membranes. Biochim. Biophys. Acta 1968, 163, 171-178. 74.

Mathai, J. C.; Tristram-Nagle, S.; Nagle, J. F.; Zeidel, M. L. Structural Determinants

of Water Permeability through the Lipid Membrane. J. Gen. Physiol. 2008, 131, 69-76. 75.

Wang, L.; Chierico, L.; Little, D.; Patikarnmonthon, N.; Yang, Z.; Azzouz, M.;

Madsen, J.; Armes, S. P.; Battaglia, G. Encapsulation of Biomacromolecules within Polymersomes by Electroporation. Angew. Chem. Int. Ed.. 2012, 51, 11122-11125. 76.

Avdeef, A. Physicochemical profiling (solubility, permeability and charge state). Curr.

Top. Med. Chem. 2001, 1, 277-351. 77.

Difilippo, E. L.; Eganhouse, R. P. Assessment of PDMS-Water Partition Coefficients:

Implications for Passive Environmental Sampling of Hydrophobic Organic Compounds. Environ. Sci. Technol. 2010, 44, 6917-6925.

Table of Contents Figure


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Chemical structures of the selected model compounds. 131x118mm (300 x 300 DPI)

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Concentration profile across the polymersomal membrane, separating the extravesicular donor compartment (index D) from the intravesicular acceptor compartment (index A). The permeating molecules pass the polymersomal membrane (thickness s) by passive diffusion, driven by the concentration gradient (c_D-c_A). The inner vesicle volume V_(V_(in,0) ) of the polymersome (vesicle diameter d) corresponds to the acceptor compartment. 121x61mm (300 x 300 DPI)


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 3. At the beginning of the IFA (t=0) empty polymersomes and a model compound containing solution were mixed. At a specified incubation time t, the partially loaded polymersomes were separated from free molecules by size exclusion chromatography (SEC). 284x70mm (96 x 96 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Dimensionless acceptor phase number Ac during the IFA when using A) N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) at pH 8 and B) caffeine at pH 6, pH 7 and pH 8 as model compound. According to equation (14) Ac can adopt values between 0 and 1. The horizontal reference line at Ac=1 serves as visual aid. 105x55mm (300 x 300 DPI)


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Negative decadic logarithm of the permeability coefficients Pe (cm s-1) of polymersomal PMOXA15-PDMS68-PMOXA15 membranes. The mass transport of A) caffeine at 22°C and pH 6, pH 7 and pH 8 and B) N-acetylglucosamine (GlcNAc), N-acetylmannosamine (ManNAc), pyruvate and Nacetylneuraminate (Neu5Ac) at 30°C and pH 8. 104x55mm (300 x 300 DPI)


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical table of contents 124x61mm (300 x 300 DPI)


Page 40 of 40

Determination of Permeability Coefficients of Polymersomal

Recommend Documents