Osmotically Induced Membrane Fission in Giant Polymer Vesicles

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Osmotically induced membrane fission in giant polymer vesicles: multilamellarity and effect of the amphiphilic block lengths Alberto P Munuzuri, Balanagu B, and Juan Pérez-Mercader Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01590 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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Osmotically induced membrane fission in giant polymer vesicles: multilamellarity and effect of the amphiphilic block lengths

Alberto P. Muñuzuri†‡§*, Balanagulu Busupalli†§, Juan Pérez-Mercader†# †Department of Earth and Planetary Sciences, Harvard University. Cambridge, MA 02138-1204 (USA). ‡Univ. of Santiago de Compostela, 15706 Santiago de Compostela (Spain). #The Santa Fe Institute, Santa Fe, NM 87501 (USA). *E-Mail: [email protected]

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ABSTRACT. Amphiphilic block copolymers are used to form large spherical vesicles. A sudden change in the osmotic pressure across the polymer membrane is used to induce the fission of the polymer vesicle. The membrane area to volume ratio, as expected, is observed to be a parameter suitable to describe the process and even mark the critical points along this transition. The effect of the length of the hydrophobic and hydrophilic chains on the fission process is analyzed. The effects of membrane lamellarity and initial polydispersity are thoroughly analyzed from the experimental data following mathematical models and the phenomenon of fission in these polymer vesicles is understood via measurements characterizing the membrane, i.e. area stretch modulus.

KEYWORDS. Polymersomes, membrane fission, area stretch modulus, multilamellarity, amphiphilic block copolymers, polydispersity, osmotic changes, membrane


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INTRODUCTION Amphiphilic block copolymers form spherical vesicle structures in aqueous solutions called polymer vesicles or polymersomes1-3. These structures are of relevance for material scientists as they exhibit interesting properties for new materials4-11 and are also suitable candidates for important applications such as drug delivery and compartmentalized chemical reactions12-13. Polymer vesicles are collectively self-organized hollow structures made up of amphiphilic molecules containing a lumen and whose surface properties can be tailored by the appropriate selection of block copolymers or even by introducing additional components at the surface. They can be used in many areas of materials’ science and technology as, for example, micro or macro reactors, for drug release and to understand or mimic basic biological mechanisms in living cells. Fission or division in liposomes and polymersomes is not common. Though fission in liposomes has been previously documented

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, the same is not well noticed in polymersomes29-31.

Polymersomes are vesicles made with highly rigid membranes. This property complicates the attempts to induce fission or division of the vesicle into two smaller almost-identical polymersomes32. Many theoretical studies have demonstrated that the shape a bilayer membrane exhibits is univocally determined by the total energy of the system which is basically given by its physical properties and those of the surrounding solution33. It is well known that the ratio between the surface area to the enclosed volume can be used to identify the membrane configuration. So, one can expect that changes in this ratio may induce changes in the membrane geometry. A simple way to change it is by controlling the solute concentration outside the polymersome. This induces a certain osmotic pressure gradient through the membrane that needs to re-equilibrate by


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diffusing the solvent through the membrane and, thus, changing the area to volume ratio and eventually the shape of the membrane. In this study, we consider the polymersomes formed by PB-PEO (poly butadiene-poly ethylene oxide) block copolymer with different degrees of polymerization. We select these polymersomes as they are relatively less rigid due to the low glass transition temperatures of PB-PEO block copolymer (-21 ºC, technical data from PolymerSource) compared to the polymersomes formed by other block copolymers. Taking advantage of the previous facts, we induce changes in the osmotic pressure across the membrane that are translated into changes in the ratio of surface area to volume in order to effectively produce changes in the membrane geometry and, eventually, induce the fission/division of the polymersome. We relate the dependence of properties such as the area stretch modulus of the amphiphilic block copolymer moieties that constitute the polymersomes with the observed fission/division phenomenon. The results observed are explained using measurements of physical properties such as the area stretch modulus, size distribution and multilamellarity. These properties were measured using techniques that did not interfere with the processes studied. Another interesting feature of the polymersomes used is their ability to form complicated membranes composed by several bilayers. These structures are very difficult to detect experimentally especially when only optical observations are used. We propose here a method to deal with this and identify the approximate number of bilayers in the vesicle membrane. The paper is organized as follows. First a detailed description of the experimental methods and materials used along this paper. It will be followed by the description of the experimental results. This section will contain a subsection with the fission experiments, followed by a subsection


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describing the experiments aimed to measure the area stretch modulus via the micro-pipette aspiration method. Finally, a discussion of the results is presented followed by the conclusions. EXPERIMENTAL SECTION Materials. Poly (butadiene-b-ethylene oxide) (PB46-b-PEO30), Poly (butadiene-b-ethylene oxide) (PB46-b-PEO31), Poly (butadiene-b-ethylene oxide) (PB46-b-PEO24), Poly (butadiene-b-ethylene oxide) (PB54-b-PEO29), Poly (butadiene-b-ethylene oxide) (PB33-b-PEO29), Poly (butadiene-bethylene oxide) (PB33-b-PEO14), Poly (butadiene-b-ethylene oxide) (PB33-b-PEO9), Poly (butadiene-b-ethylene oxide) (PB65-b-PEO35), Poly (styrene-b-ethyleneoxide) (PS18-PEO57) and Poly (dimethylsiloxane-b-ethyleneoxide) (PDMS47-PEO37) were purchased from Polymer Source, Canada. All the PB-PEO amphiphilic block copolymers were 1,2-rich polymers. Sucrose, D-(+)-Glucose and Sodium Chloride were purchased from Sigma Aldrich, USA. Toluene was purchased from Sigma Aldrich, USA. Distilled water was used throughout all the experiments. Polymersome Preparation Method. All the polymersomes were prepared following the reported literature on inverted emulsion/emulsion-centrifugation method32, 34. Briefly, 60 µL D(+)-Glucose solution (0.38M) was poured into a polypropylene Eppendorf microcentrifuge tube (1.5 mL capacity). To this, 60 µL polymer solution (3 mg/mL) was added slowly with a pipette and the interface was allowed without disturbance for 30 minutes. In a separate microcentrifuge tube 500 µL of the stock polymer solution (3 mg/mL) was taken and to it was added 5 µL Sucrose solution (0.38M). This mixture was hand agitated vigorously for 30-40 seconds to make an emulsion. Immediately after this, 120 µL of the emulsion was added slowly to the interface with a pipette and centrifugation of this mixture was performed instantly in a Beckman Coulter


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Microfuge 22R centrifuge. Conditions set for these centrifugation experiments were; the centrifugation rate was set at 500 rpm (rotations per minute) during 3-4 minutes and done at 22o C. Usually, several such microcentrifuge tubes containing the interfacial mixture were utilized at a time. After centrifugation, two distinct layers appeared, of which the bottom aqueous layer contained the polymersomes. After evaporation of the top organic layer, we obtained an aqueous solution containing the polymersomes. It has been demonstrated that Toluene evaporates with time35 and its effect is reflected in the physical properties of the membrane. We allowed evaporation for one week before using the samples. At that time, still some traces of Toluene exist35 but we found that the elastic properties of the membrane were more suitable to achieve fission than in the completely toluene-free case. This increased the range of parameters where division was observed. All the different polymersomes utilized in this study were prepared via this method keeping all the conditions, concentrations of the used solutions and other parameters identical for each case. Thus, the membrane structure (including the remnants of Toluene) was the same in all cases except for the natural dispersion intrinsic to all experimental procedures. Table 1 presents a summary of all the block copolymers analyzed that successfully produced polymersomes under these conditions. Here, the size distribution of all these polymersomes is summarized. Notice that the errors here do not reflect the experimental uncertainty but the large dispersion of the polymersomes produced with our method. Details are presented in the SI with the histogram distributions as well as examples of the different sizes and shapes observed for each case. Figure 1 shows the dependence of the mean radius versus the length of the hydrophilic (PEO) and hydrophobic (PB) chains. Increasing PEO length (Fig. 1a) results in a decrease of the mean radius while increasing PB chain (Fig. 1b) produces the opposite effect.


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Fission Time / radius (s/µm)

Ka(1) (mN/m)

Size (diameter) distribution (µm)

Fission

PB46-b-PEO24

Yes

Yes

16 ± 5

2.35 ± 0.9

30.3 ± 2.2

PB46-b-PEO30

Yes

Yes

12.2 ± 5.4

0.6 ± 0.3

24.2 ± 4.6

PB46-b-PEO31

Yes

Yes

14.9 ± 6.1

0.41 ± 0.3

21.0 ± 9.4

PB33-b-PEO09

Yes

Budding

28.3 ± 16.5

--

25.2 ± 5.9

PB33-b-PEO14

Yes

Yes

19.3 ± 5.7

0.85 ± 0.4

17.4 ± 8.7

PB33-b-PEO29

Yes

Yes

17.7 ± 7.3

0.72 ± 0.6

--

PB65-b-PEO35

Yes

Yes

12.4 ± 3.3

1.07 ± 0.4

26.6 ± 2.2

PB54-b-PEO29

Yes

Yes

36.7 ± 13.1

1.5 ± 0.8

30.5 ± 5.8

PS18-b-PEO57

Yes

No

--

--

--

PDMS47-b-PEO37

Yes

No

--

--

--

Table 1. Summary of all the block copolymers used and the properties analyzed.

R o ( m)

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

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Polymersome formation

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Figure 1. Variation of size distribution versus (a) length of hydrophilic chain (PEO) and (b) length of hydrophobic chain (PB).


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Optical Microscopy Experiments. A Zeiss optical microscope (Axio Observer A1) equipped with a CCD camera (Zeiss AxioCam HRc) connected to a Windows PC was used to observe the polymersomes’ dynamics on a flat clean glass slide. The temperature was kept constant at 25º C throughout the experiments using a Harvard Apparatus temperature controller (Medical Systems Research product, model: TC202A). A clean glass slide that was thoroughly washed and wiped with soft tissue paper was taken and kept in the groove of the Harvard Apparatus temperature controller unit. It is important to note that this observation technique does not interfere with the process being analyzed, thus, preserving the integrity of the sample analyzed. Images acquired by the CCD camera (Zeiss AxioCam HRc) connected to a Windows PC were further analyzed and processed by our own custom made programs. Fission induced experiments. A polymersome sample (a 5 µL droplet) was placed on to a clean glass slide under the microscope and then a 10 µL droplet of the 1M NaCl solution was gently put from above onto the already present polymersome droplet. The amount of NaCl solution drop cast onto the polymersome solution is always double the polymersome solution amount. The evolution of the polymersomes was recorded at a rate of 1 image/s. Experiments were performed at 10x magnification (Zeiss A-plan 10x/0.25 ph1) in the Zeiss optical microscope to obtain many such fission events from a single droplet. Several such polymersome sample droplets were employed to collect data for statistical analysis. See Supplementary Information for details. Harvard Apparatus Temperature Controller was used to fix the temperature at 25o C throughout all the experiments. Mechanical Properties of Polymersomes-Area Stretch Modulus (Ka) Experiments. The micropipette aspiration technique was used to determine the area stretch modulus for each


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polymersome type35,

36

Experiments were performed in the same Zeiss microscope (Axio

Observer A1) which was equipped with an Eppendorf micromanipulation motor system (Eppendorf Patchman NP2) for performing such experiments. In brief, a polymersome sample was put as a droplet (5 µL) onto a clean glass slide kept in the groove of the Harvard Apparatus temperature controller unit. Temperature was kept constant at 25º C controlled throughout the experiment in the Harvard Apparatus Temperature Controller. A sterile IMSE/TESE glass tip with inner diameter of 6 µm and bent angle 25º was fit into the glass pipette holder in the Eppendorf Celltram Air suction pump to aspirate the polymersomes by applying a negative suction pressure. Step-wise application of negative pressure resulted in step-wise suction of the membrane in the form of a tether into the glass pipette. The suction pressure was noted carefully at each step. After aspirating a part of the polymersome membrane into the glass pipette the piston on the suction pump used to induce the negative pressure was kept constant for at least 20 seconds. And then an incremental suction pressure was applied to aspirate more of the polymersome membrane into the glass pipette. This was done sequentially till a certain high applied suction pressure, beyond which either the membrane tether would break or the polymersome gets completely sucked into the glass pipette (this was the case for PB33-PEO29 and thus no Ka values could be recorded). These values are used to determine the area stretch modulus as explained in the Supplementary Information. RESULTS Induced Fission of Polymersomes. With the experimental protocol described in the Experimental Section we conducted extensive experiments considering the different polymersomes obtained. Different concentrations of NaCl were considered in order to induce fission in the polymersomes. Small concentrations produced almost no effect, while large


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concentrations, larger than 1M, produced a sudden change in the internal volume that resulted in the destruction of the membrane and, in turn, the destruction of the polymersome structure. We choose the particular value of [NaCl]=1M because this concentration produced a controlled fission that could be recorded for further analysis. Only the PB-PEO polymersomes were able to exhibit a controlled fission under this protocol. Table 1 summarizes the list of cases where fission events were observed. PB33-b-PEO09 is a particular case that does not divide into two but produces multiple polymersomes (presumably) via a completely different mechanism that we are not describing here. Figure 2 presents the analysis of a typical experiment (see experimental movie in SI). Panels 1a to 1f are a sequence of pictures showing the evolution of a PB46-b-PEO30 polymersome as its internal volume was forced to change as a consequence of the induced osmotic changes. At t=0 s a 1M drop (10 µL) of NaCl was added on top of the sample droplet (5 µL) containing the polymersomes. A few seconds after, several of the polymersomes started to divide. Panel (a) shows a prolate shaped vesicle that starts shrinking in the central region. This neck continues narrowing till the polymersome is split into two independent units. The digital version of the images was processed by our own custom made programs (see SI) and the physical properties were measured and shown in the three remaining panels. It is noteworthy that the area does not change too much, and the changes can be mainly attributed to an increase in the surface roughness. Several parts can be observed along this fission/division process. At first, a sudden decrease of the volume happens, caused by the osmotic pressure gradient induced by the increase of NaCl concentration outside to the polymersome in its surrounding solution. This is responsible for a dramatic increase of the area to volume ratio that induces the formation of a prolate shaped vesicle. The end of this part is marked with a black vertical dashed line. Once the prolate shape is


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clearly formed, the middle part shrinks producing a bottle neck that becomes more pronounced as time goes till two independent polymersomes appear. This is marked with a second dashed vertical line in the Figure 2. Along this second stage, the volume continues to decrease but at a much slower rate. Note that the area to volume ratio decreases along this second stage, clearly marking this different behavior.

Figure 2. (a) to (f) evolution of a PB46-b-PEO30 polymersome after a 1M drop of NaCl was added, (a) 2 s, (b) 4 s, (c) 9 s, (d) 14 s, (e) 19 s and (f) 23 s. Initial stages were not recorded. Horizontal line in each panel corresponds with a length of 10 µm. (g) Evolution of the volume with time. (h) Evolution of the surface area with time. (i) Evolution of the area to volume ratio with time. All spatial units are in µm.

For an experiment such as the one described in Fig. 2, we can measure the time needed for each vesicle to divide, which we call fission time (FT). Each of the different polymersomes considered was forced to undergo a similar transition and the experiment was repeated at least 50


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times. One example is shown in Fig. 3a. Here all the values of the measured FT are plotted versus the original size of the polymersome for the PB46-PEO30 case (the rest of the cases are depicted in the SI). A first inspection shows a great dispersion both in the FT and the initial radius (Ro). There are several reasons to explain this. The fission time should depend on the radius as the area to volume ratio strongly depends on the radius and this seems to be the parameter that triggers the mechanism. Another issue that could affect our measurements is due to the chemical process used to produce the polymersomes. As it is presented in the SI, this method produces a large polydispersity of polymersomes but also produces different structures including multilamellar polymersomes. Bizarre structures can be easily discarded while experimenting but multilamellar polymersomes are morphologically equivalent to those formed by a single bilayer and, thus, indistinguishable using optical techniques. Details and examples are in the Supplementary Material. Dependence of the fission times on the initial radius and multilamellarity. In order to understand this effect we can write some equations describing the evolution of the polymersome volume. The driving force of this process is the gradient of concentration across the membrane. Following Steudle et al37, and previous authors it is possible to write the equivalent of the Fick’s law for this case, ()

= ΔΠ = (Π − Π ) = (c − c )

(1)

where V(t) is the internal volume of the polymersome, Lw is the membrane hydraulic conductivity which is related to the water permeability of the membrane (volume of water that diffuses through a given area of the membrane surface per unit time) and it is known to be


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inversely proportional to the thickness of the membrane. A is the surface area (that we can consider almost constant for the sake of this discussion), ∆Π is the change in osmotic pressure across the membrane, R is the gas constant, T is the temperature and cin/cout is the concentration of solutes inside/outside the polymersome. The only parameter that changes with time is the internal volume (V(t)), thus we can rewrite cin=nin/V(t), where nin is the total number of solute molecules inside the polymersome (assumed to be constant). Eq. (1) becomes ()

= () − (2)

where β = Lw A R T is a constant that contains the information about the membrane and its thickness. As a first approximation, we can consider that the membrane thickness is equal to n δ, n being the number of bilayers in the membrane and δ the thickness of one single bilayer. Thus Lw ∝ 1/(nδ). Eq. (2) can be analytically integrated given the appropriate initial conditions but a direct inspection can be more helpful in this case. In fact, the right-hand side of the equation contains two terms, one describing the linear decay of V(t) and the other is a saturation term that only activates when the volume becomes very small. Thus !(") ≈ !(" = 0) − " (3) %

with !(" = 0) = ' & and Ro the initial radius of the polymersome. So, we can define a & characteristic time (τ) as the time needed for the system to significantly change its value so that fission can take place. It can be estimated as the time needed for V(τ)≈ 0, thus


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( = .

) + ,-* *

/ 0 ,1 2-34

= .

) + ,-* * 5 / % + ,- ,1 2-34

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∝ 6 7 (4)

This characteristic time and the fission time we are interested in are basically the same or at least, they should have the same dependence on the parameters. So, we can infer that the fission time should be proportional to the initial radius of the polymersome as well as to the number of bilayers the membrane is made of. In fact, we can use the fission times in order to estimate the number of bilayers a particular polymersome contains. Figure 3b presents the FT values normalized by the initial radius in each experiment (FTn = FT/Ro). It is still possible to observe a large dispersion in the data. In order to obtain values that we can compare from one type of polymersome to another, we need to consider the multilayer nature of the membranes. The details are described in the SI and in Fig. 3c we show a plot with the FTn versus the estimated number of bilayers. The linear fit of these points directly provides us with the FTn for a single bilayer polymersome (FTn(1)).


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Fission Time (s)

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

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FT/R o = FTn (s/ m)

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PB -PEO 46

6

30

3

(c)

0 0

5

10

Number of bilayers

Figure 3. Measuring fission times. (a) Raw data obtained for polymersomes made of PB46-PEO30, fission times are plotted versus the corresponding initial radius of the polymersome. (b) Normalized fission times (FTn=FT/Ro). (c) Linear fit of the FTn to the estimated number of bilayers, n. (d) Values of FTn(1) calculated for each of the polymersomes considered.

The same procedure was applied to the other polymersomes considered and the results are plotted in Fig. 3d. Note that the calculated values of FTn(1) can be compared from one type of polymersome to another as they are not dependent on the initial size neither on the number of bilayers and the experimental protocols to produce the samples were strictly the same in all cases. The actual recorded values are shown in Table 1 and details are in the SI.


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Effect of PB and PEO chain length on the normalized Fission Times. In this subsection we correlate the previous results with morphological properties of the block copolymers used. In particular, the length of the hydrophilic (PEO) and hydrophobic (PB) chains will be analyzed. Figure 4a presents the variation of FTn(1) with PEO length. As the hydrophilic chain lengthens, FTn(1) becomes smaller or, in other words, the time needed to induce significant changes in the vesicle morphology is decreased. PEO is known to have a great solubility in water38 and if it is included in the membrane of a polymersome, these hydrophilic heads try to be surrounded by water molecules39 and separate from each other. Increasing the length of PEO results in increasing the chances of separation among adjacent molecules thus destabilizing the membrane and, thus, making it easier to re-shape it. On the other hand, the effect of changes in the hydrophobic (PB) chain can be observed in Figure 4b. Here increasing the hydrophobic chain results in an increase in the FTn(1). The hydrophobic chains constitute the inner part of the bilayer and these chains entangle strongly, so increasing the length of the hydrophobic chain directly implies increasing the thickness of the membrane and, thus, the distance a water molecule needs to traverse in order to exit the polymersome. And this is directly reflected in a net increase in FTn(1). A simple dynamical model can be written describing the main features of this behavior and it is presented in the SI. Although the tendency seems to be clear from these results and consistent with general theoretical interpretations, the error bars are quite significant and additional experiments should be performed, included also different hydrophobic chain lengths, in order to reach firm conclusions.


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(a) (s/ m)

3

(1)

2

FTn

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

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1 0

Length of PEO (hydrophilic)

Figure 4. Variation of the normalized fission time (FTn(1)) with (a) the length of PEO chain and (b) the length of PB chain.

Area stretch modulus measurements. As noted from the previous results, the capability to induce fission in a polymersome is directly related to the properties of the membrane. The area stretch modulus (Ka) is an interesting membrane property that can be measured experimentally. Ka is defined as the ratio between the surface tension and the areal strain and provides information about the stiffness of the membrane. Micropipette aspiration techniques have been traditionally used to determine it40-48 (see details in SI). In brief, this technique can be described as a sequence of deformations induced at the membrane by applying a negative pressure with a micropipette. The deformations induced in the membrane can be related to the applied pressure by 0800-

9

9

9

2 : − , = ; =>

(5)

Osmotically Induced Membrane Fission in Giant Polymer Vesicles

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