Vesicles formed from block copolymers, and novel block copolymers

Abstract

Vesicles formed from a block copolymer comprising at least one (poly)2-C.sub.1-3alkyl-2-oxazoline block and at least one polybutadiene block; and membranes comprising such vesicles. Block copolymers comprising at least one (poly)2-C.sub.1-3alkyl-2-oxazoline block and at least one polybutadiene block, provided that the copolymer is not the diblock copolymer consisting of 40 butadiene units and 190 2-methyl-2-oxazoline units terminated by a hydroxy group, are novel, and also form part of the invention.

Claims

1. A block copolymer comprising at least one (poly)2-C.sub.1-3alkyl-2-oxazoline block and at least one polybutadiene block, which is a diblock copolymer AB, in which (poly)2-C.sub.1-3alkyl-2-oxazoline forms the A block and polybutadiene forms the B block, and which comprises an end group at the end of a (poly)2-C.sub.1-3alkyl-2-oxazoline block wherein the end group is selected from carboxy, activated carboxy, amine, methacrylate, thiol, azide, and alkyne and wherein the B block contains 5 to 15 butadiene units.

2. A block copolymer as claimed in claim 1, in which the number of butadiene units in the B block is at least half the number of 2-C.sub.1-3alkyl-2-oxazoline units in the A block.

3. A block copolymer as claimed in claim 2, in which the number of butadiene units in the B block is at least twice the number of 2-C.sub.1-3alkyl-2-oxazoline units in the A block.

4. A block copolymer as claimed in claim 1, which contains from 5 to 180 2-C.sub.1-3alkyl-2-oxazoline units in the A block.

5. A block copolymer as claimed in claim 1, in which said end group is selected from carboxy, activated carboxy, or an amine end group having the formula NHR in which R represent an alkyl group having from one to 6 carbon atoms substituted by at least one NH.sub.2 group.

6. A block copolymer as claimed in claim 1, in which the (poly)2-C.sub.1-3alkyl-2-oxazoline is (poly)2-methyl-2-oxazoline.

7. Vesicles formed from a block copolymer of claim 1.

8. Vesicles as claimed in claim 7, in which CC double bonds present in polybutadiene blocks of different block copolymer chains are cross-linked together.

9. Vesicles as claimed in claim 7, having transmembrane proteins incorporated therein.

10. Vesicles as claimed in claim 9, in which the transmembrane protein is an aquaporin.

11. Vesicles as claimed in claim 7, containing a drug or a cosmetic agent.

12. A filtration membrane comprising vesicles as claimed in claim 9.

13. A filtration membrane as claimed in claim 12, which comprises a porous support and, covalently bonded to a surface thereof, a layer comprising a plurality of vesicles formed from the block copolymer, and having transmembrane proteins incorporated therein; and in which within said layer, vesicles are covalently linked together to form a coherent mass.

14. A process for the preparation of a membrane as claimed in claim 13, which comprises providing an aqueous suspension of vesicles formed from the block copolymer, and having transmembrane proteins incorporated therein; depositing said suspension of vesicles on a surface of a porous support; and providing reaction conditions such that covalent bonds are formed between different vesicles and between vesicles and said surface.

15. A process as claimed in claim 14, which comprises either: (a) providing an aqueous suspension of vesicles formed from the block copolymer, and having transmembrane proteins incorporated therein, said vesicles being formed from block copolymers having reactive end groups X; (b) providing a multifunctional linking agent having at least two reactive groups Y which are reactive with polymer end groups X; (c) depositing said suspension of vesicles and said multifunctional linker on a support having a surface which is reactive with either polymer end groups X or reactive groups Y; and (d) causing reaction of end groups X with groups Y, and either end groups X or groups Y with the surface of the support; or (aa) providing a first aqueous suspension of vesicles formed from the block copolymer, and having transmembrane proteins incorporated therein, said vesicles being formed from block copolymers having reactive end groups X; (bb) providing a second aqueous suspension of vesicles formed from the block copolymer, and having transmembrane proteins incorporated therein, said vesicles being formed from block copolymers having reactive end groups Y which are reactive with polymer end groups X; (cc) depositing said suspensions of vesicles on a support having a surface which is reactive with either polymer end groups X or Y; and (dd) causing reaction of end groups X with end groups Y, and either end groups X or end groups Y with the surface of the support.

16. A process as claimed in claim 15, in which said multifunctional linking agent of step (b) is N-sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino)hexanoate.

17. A filtration membrane as claimed in claim 12, in which the transmembrane protein is an aquaporin.

18. A membrane as claimed in claim 13, in which the transmembrane proteins are aquaporins.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A and 1B show LSM imaging micrographs of vesicles according to the invention.

(2) FIGS. 2 and 3 show the results of the stopped-flow experiments of Example 2.

(3) FIG. 4 shows the results of the DLS experiments of Example 2.

(4) FIG. 5 shows the effect of incorporating Aquaporin Z protein into vesicles according to the invention as described in Example 3.

(5) FIG. 6 is a micrograph of the membrane of Example 5.

(6) FIG. 7 shows the effect of internally cross-linking the polybutadiene in the membrane of Example 5.

(7) FIG. 8 shows the results of the encapsulation efficiency experiment described in Example 6.

(8) The following Examples illustrate the invention.

EXAMPLE 1: POLYMER PREPARATION

(9) Step (a): PB Synthesis

(10) Polybutadiene was synthesized following the protocol of Hillmyer, M. A.; Bates, F. S. 1996, 9297, 6994-7002 with some modifications. The anionic polymerization of butadiene was carried out in THF at 60 to 50 C. using sec-butyl-butyllithium as the initiator. A dry 2 neck flask was dried in the oven overnight and a line was attached to one port with a septum to another. The flask was flame dried and a stir bar was added. 30 ml of Dry Solv THF was added to the 2 neck flask using a cannula. 11 ml butadiene (0.13 mol) was condensed in a condensing flask. Liquid nitrogen was first used to condense polybutadiene and then melted using a dry ice-acetone bath. This was transferred to the 2 neck flask using a cannula. 7 ml (0.0098 moles) of 1.4 M sec-butyl lithium initiator was swiftly added. The polymerization was allowed to proceed for 3 h. End capping was accomplished by adding 2 ml (0.051 moles) of ethylene oxide at 60 C. upon complete conversion of the butadiene. Acidic methanol (5 ml HCl:50 ml methanol) was then used to liberate the polybutadiene alcohol which was isolated by evaporation of the solvent. Inorganic salts were removed by extraction of a cyclohexane solution of the polymer with distilled water. Polymer was left on high vacuum to remove water. Further drying was achieved by refluxing the polymer in dry hexane using molecular sieves in soxhlet extractor.

(11) Step (b): PB-PMOXA Synthesis

(12) 20 g (0.0260M) of polybutadiene (Mn 769 g/mol) were functionalized with 7.33 g (0.0260M) triflic acid anhydride (SigmaAldrich 176176-5G) in the presence of 2.63 g (0.0260M) of triethylamine (SigmaAldrich T0886) at 10 deg C. under argon. Organic salts were further filtered out. Triflate-functionalized PB served as a macro-initiator of cationic ring opening polymerization of 2-methyl-2-oxazoline (SigmaAldrich 137448).

(13) Polymerisation was allowed to proceed in anhydrous ethyl acetate (SigmaAldrich 270989) at 40 deg C. for 12 h. Reaction was terminated with ethylene diamine 0.4 g (SigmaAldrich 03550). This provided primary- and secondary-amine terminated PB-PMOXA polymer.

(14) Polymer Characterization:

(15) PB.sub.12OH

(16) NMR

(17) 5.45 ppm CHCH.sub.2 (repeating unit), 4.94 ppm CHCH.sub.2 (repeating unit), 2.12 ppm CH (repeating unitbackbone), 1.27 ppm CH.sub.2 (repeating unitbackbone), CH.sub.2 and CH.sub.3 3.65 ppm 0.82 ppmend groups.

(18) TABLE-US-00001 Polymer Solvent Mn Mw PDI PB.sub.12 CHCl.sub.3 526 602 1.14 PB.sub.12PMOXA.sub.5 CHCl.sub.3 632 738 1.19

(19) PB.sub.12-PMOXA.sub.5-NH(CH.sub.2)NH.sub.2

(20) NMR

(21) PB: 5.45 ppm CHCH.sub.2 (repeating unit), 4.94 ppm CHCH.sub.2 (repeating unit), 2.12 ppm CH (repeating unitbackbone), 1.27 ppm CH.sub.2 (repeating unitbackbone), CH.sub.2 and CH.sub.3 3.65 ppm 0.82 ppmend groups. PMOXA: 3.45 ppm (CH.sub.2CH.sub.2N), 2.11 ppm (NCOCH.sub.3)

EXAMPLE 2: VESICLE PREPARATION

(22) PB.sub.12-PMOXA.sub.5-NH(CH.sub.2).sub.2NH.sub.2 polymer (50 mg) was dissolved in 1 ml chloroform in a round bottom flask (Pyrex 200 ml). Solvent was evaporated on a rotary evaporator under reduced pressure producing a thin film of polymer. Subsequent 3 h high vacuum treatment removed the traces of chloroform. 5 ml of water was further added and stirred at 600 rpm. This way a 10 mg/ml suspension of vesicles was prepared. Upon sampling for characterization (LSM, Stopped-Flow, DLS), the suspension was extruded successively through polycarbonate Track ached filters (Millipore) of 1 m, 800 nm, 400 nm, 200 nm. At each of the extrusions, the suspension was sampled for characterization.

(23) The vesicles were characterised as follows. Cryogenic transmission electron microscopy (cryo-TEM) was used for particle imaging, and surface functionalization was studied using LSM imaging.

(24) For the cryo-TEM, the microscope was FEI TecnaiG2, TF20. Samples were vitrified using a vitrification robot, Vitrobot FEI. Magnification used was 25000 (calibrated 31625)=scale bar 200 rm.

(25) For the LSM imaging, the amine end groups present on the surface of the vesicles prepared as above were allowed to react with tetramethylrhodamine isothiocyanate fluorescent dye (1:1000 molar ratio) and dialyzed against deionized water. Dialysis was performed until dialysate showed no signs of fluorescence, followed by additional change of DI water to eliminate unspecific binding. The vesicles were visualized using a Zeiss LSM 710 Inverted Confocal Microscope with Apochromat 63/1.4 Oil DIC M27 objective and 561 nm Laser line. Pinhole was varied from 50 um to 70 um. This allowed for the confocal plane to see through the vesicles, which thus appear as rims of light (center of vesicle in the center of confocal point) or discs of light (top of the vesicle in confocal point) in suspension where a vesicle floated in and out of focus dynamically. FIGS. 1A and 1B show two sample micrographs clearly showing vesicles.

(26) A secondary approach to investigating vesicle structure is the use of stopped-flow experiments to measure permeability. A stopped flow spectroscope was utilized to mix vesicle suspensions with either hypertonic or hypotonic solutions and the light scattering signal was collected upon deployment of stop syringe. This leads to gradient of concentration across polymer bilayer resulting in water permeation in direction of the gradient. Shrinkage of the polymer vesicles in case of hypertonic and swelling of the polymer vesicles in case of hypotonic solutions is observed. The change in size can be monitored by means of light scattering and the rate at which the change occurs can be attributed to the permeation of water at given size of vesicles. This has been shown also to validate aggregate morphology (A framework for accurate evaluation of the promise of aquaporin based biomimetic membranes M. Grzelakowski, M. F. Cherenet, Y. Shen, M. Kumar Journal of Membrane Science doi:10.1016/j.memsci.2015.01.023.

(27) Vesicle suspensions were rapidly mixed with osmotic solutions, 1:1 ratio at 16 C. and light scattering signal was collected at 365 nm and at 8 mL/s flow rate. Osmotic water permeability (P.sub.f) is calculated from:

(28) $P_f=\frac{k}{\left(\frac{S}{V_0}\right)V_W{}_{\mathrm{osm}}}$
where k is the initial slope of the light scattering curve corresponding to the change of vesicle diameter with time, S is the initial surface area of the vesicles, V.sub.0 is the initial volume of the vesicles, V.sub.w is the molar volume of water, and .sub.osm is the osmolarity difference driving the shrinking of the vesicles.

(29) A range of hypertonic solutions was prepared by adding given a given amount of NaCl to hydration buffer (0.1M NaMOPS) resulting in gradients of 600 mM, 300 mM, 150 mM 75 mM, 0 mM (hydration buffer) and 100 mM (pure DI water). As expected and shown in FIG. 2 shrinkage due to hyportonicity was reversed to swelling with hypotonic solution, thus confirming vesicular morphology.

(30) Additionally, stopped flow was also used to confirm the vesicular morphology at every step of extrusion process. The time of the water permeation was proportional to the size of the vesicles exposed to hypertonic condition. The permeability of the bilayer remains constant therefore larger amount of water permeating through larger area of membrane produces longer timeframes. FIG. 3 shows stopped the flow graphs for vesicles extruded through filters with decreasing pore-sizes.

(31) Malvern ZetraSizer Nano-S dynamic light scattering (DLS) was used to determine particle diameter and size polydispersity index. Vesicle suspensions were left for equilibration overnight before DLS measurements. Afterwards, samples were extruded through membranes with pore sizes 800, 400, 200 and 100 nm. DLS measurements were carried out 6 hours after extrusion. Polystyrene latex was used as the reference material (RI: 1.590; absorption: 0.010 at 633 nm), and water as the dispersant (viscosity: 0.9781 cP; RI: 1.330). The measurements were carried out in Science Brand disposable microcuvettes with a sample volume of 100 L, at 21 C. Each sample was measured 5 times, each measurement was the average of 11 runs. The results are shown in the table below and in FIG. 4.

(32) TABLE-US-00002 Mean Size Peak (d .Math. nm) Extrusion PDI Intensity Volume Number Pre 0.337 1521 1327 931 0.8 um 0.203 585.7 750.8 439.8 0.4 um 0.169 433.8 520.1 312.3 0.2 um 0.15 238.1 239.2 137.1 0.1 um 0.093 165.4 151.7 107.2

EXAMPLE 3: INSERTION OF PROTEIN INTO VESICLES

(33) Water permeability of polymer vesicles was enhanced by reconstitution of water channel membrane proteinaquaporin Z. Film hydration procedure was modified to accommodate addition of protein at PoPr 400. Shortly: to the hydrating vesicles protein solution is added at PoPr 400. Next steps follow the protocol of standard vesicles formation.

(34) PB.sub.12-PMOXA.sub.5-NH(CH.sub.2).sub.2NH.sub.2 polymer (50 mg) was dissolved in 1 ml chloroform in a round bottom flask (Pyrex 200 ml). Solvent was evaporated on a rotary evaporator under reduced pressure producing a thin film of polymer. Subsequent 3 h high vacuum treatment removed the traces of chloroform. 5 ml of 100 mM Na-MOPS buffer containing 0.1245 mg of aquaporin Z (Applied Biomimetic) and 0.5% octyl glucoside (O311-n-Octyl--D-Glucopyranoside, Anagrade, Anatrace) and was further added and stirred at 600 rpm. 10 mg/ml suspension of proteo-vesicles was extruded trough 200 nm polycarbonate Track ached filter (Millipore). Permeability measurements were performed using stopped-flow spectrometer.

(35) Stopped flow spectroscopy was used to evaluate protein insertion. This is measured as increase in water permeability of vesicles reconstituted with aquaporin water channel. With the amount of protein added as little as PoPR (polymer to protein ratio) of 400 the increase in water permeability over control vesicles was measured to be 46 times. The results are shown in FIG. 5.

EXAMPLE 4: CORE CROSS-LINKING OF PB-PMOXA VESICLES USING FREE-RADICALS

(36) PB-PMOXA vesicles prepared as in Example 2 were subjected to known amounts of free-radical generating solutions under isotonic conditions in order to cross-link the PB cores and produce less permeable/more rigid structures.

(37) PB-PMOXA was prepared as in Example 1 and made into vesicles using thin-film rehydration in 100 mM NaMOPS pH7.5. Vesicles were then extruded through Millipore Isopore membranes of pore size 0.2 m and tested on a Kintek SFLS and ZetasizerNANO DLS (Malvern). All chemicals and buffers were purchased from Sigma Aldrich.

(38) Prepared PB-PMOXA vesicles were aliquoted (250 L) into four 4 mL clear glass vials and set aside to equilibrate at room temperature. Three solutions were prepared in NanoPur water for the cross-linking procedure. 100 mM Potassium Persulfate (K.sub.2SO.sub.4) was prepared by dissolving 100 mg in 3.699 mL NanoPur water, 100 mM Sodium Metabisulfite (Na.sub.2S.sub.2O.sub.5) by dissolving 100 mg in 5.26 mL NanoPur water, and 100 mM Iron(II) Sulfate Heptahydrate (FeSO.sub.4.7H.sub.2O) by dissolving 100 mg in 3.597 mL NanoPur water. 2 vials of vesicles were given 50 L of 100 mM Potassium Persulfate, 25 L of 100 mM sodium metabisulfite, and 1 L of 100 mM Iron(II) sulfate heptahydrate while the other two were given 100 L, 50 L, and 2 L respectively. These pairs were then split; one reacting at room temperature, while the other reacted at 70 C. Samples were allowed to react for one hour and then tested on DLS and SFLS for size and permeability after equilibrating to room temperature.

(39) Effectiveness of the cross-linking of the hydrophobic core was manifested by change in the solubility of cross-linked polymer vesicles in organic solvents. Prior to cross-linking, the polymer was soluble in both ethanol and chloroform. After cross-linking, the vesicles had reduced solubility in both. The amount of radical initiating solution added was directly proportional to the decreased solubility of cross-linked material in chloroform.

EXAMPLE 5: MEMBRANE PREPARATION

(40) In this Example, the concentration of deposited vesicles was kept constant and monitored by matching the count rate (250 kcps) in Dynamic Light Scattering (Malvern Zetasizer Nano) with static attenuator.

(41) Sulfo-SANPAH (SS) solution (10 mM in 100 mM NaMOPS pH 7.5) was allowed to react with previously prepared PB-PMOXA-NH(CH.sub.2).sub.2NH.sub.2 vesicles in the absence of light (250 L of vesicle solution combined with 50 L SS for 15-minutes). A series of 47 mm polysulfone membranes (hand casted) were cut by punch press and placed into Teflon membrane holders and rinsed with deionized water. Excess water was removed by compressed air and 300 L (each) of SS-activated vesicle suspensions were placed onto the polysulfone support membranes. The membrane holders were then placed under UV light approximately 5 cm from the source and covered with foil for protection for 30 minutes. Excess reactants were then removed from the membrane surface using a 1 ml pipette without touching the membrane surface. The above steps were repeated three times, following which the membranes were removed from the holders and 25 mm diameter membrane samples were cut from the coated area using a punch press. These were then rinsed in excess 100 mM NaMOPS ph7.5 on a shake table for at least one hour before testing.

(42) FIG. 6 is a micrograph of the resulting membrane, showing a coherent mass comprising a plurality of vesicles cross-linked on the surface of the support membrane.

(43) Membranes prepared in the step described above were subject to treatment with either 10 or 150 L of free radical initiating solution composing of:

(44) 25 mM Iron(II) Sulfate Heptahydrate,

(45) 25 mM Sodium Metabisulfite,

(46) 25 mM Potassium Persulfate

(47) The treatment resulted in crosslinking of the PB hydrophobic core.

(48) The resulting membrane samples were tested for pore size distribution using a standard molecular weight cut-off analysis technique. The 25 mm samples prepared in the previous step were tested for their ability to retain high molecular weight materials, by measuring their molecular weight cut-off, i.e. the point at which at least 90% of molecules of a given molecular weight are retained by the membrane. Phosphate buffer (0.03M Na.sub.2HPO.sub.4+0.03M KH.sub.2PO.sub.4) was pre-filtered using a 0.2 um membrane and the pH was adjusted to 7.2 prior to use for preparation of solutions. Dextran (DXT) standards were dissolved in phosphate buffer (DXT 165 kDa, 325 kDa, 548 kDa, 1300 kDa, and 5000 kDa, DXT 0.505 kDa, 4 kDa, 6 kDa, 11 kDa, 20 kDa, and 28 kDa). All of the dextran solutions were diluted to 0.5 mg/ml with phosphate buffer and pre-filtrated using a 0.2 um polyethersulfone membrane prior to use. All filtration experiments were conducted in a 10 ml Amicon stirred ultrafiltration cell (Model 8010, Millipore Corp.) All samples were evaluated according to the protocol described below: Filtered 10 ml volume of deionised water at 20 psi to wet the pore structure and the whole system. Connected the feed line with dextran solution feed to a digital peristaltic pump (Thermal Fisher Science Inc.), re-pressurized the cell to 20 psi, set the filtrate flux to 5 m/s. Obtained 800 L samples of the filtrate solution after filtration of 2,000 L of water for equilibration and washing out the dead volume downstream of the membrane. Obtained 1 ml permeate samples directly from the cell after filtration. Cleaned and rinsed the whole system with deionised water. The stirring speed was kept at 600 rpm and all experiments were performed at room temperature (223 C.)

(49) Permeate was further evaluated using high-pressure liquid chromatography (HPLC columns PL1149-6840, MW 10,000 to 200,000, PL1120-6830, MW 100 to 30,000, PL1149-6860, MW 200,000 to >10,000,000). Comparison of the feed to the permeate chromatograms allowed for calculation of retention coefficients and membrane molecular cut-off. The results are shown in FIG. 7, which shows that molecular cut-off of the control membrane was reduced to half when coated with vesicles. Molecular weight cut-off of the vesicle-coated membrane decreased to 4000 Ka upon core-crosslinking of the polybutadiene using initiator. Reduction in molecular cut-off is shown to be dependent on the amount of the cross-linker used.

EXAMPLE 6: ENCAPSULATION EFFICIENCY OF VESICLES

(50) Fluorescein (Sigma-Aldrich F6377) solution 1 mM was prepared in 100 mM sodium-MOPS (GFS 5440). Polymer vesicles were prepared according to Example 2 using fluorescein Na-Mops solution as hydration media. Polymer vesicles were further extruded through polycarbonate Track ached filters (Millipore) at 200 nm. Un-encapsulated dye was removed by dialysis (Thermo Fisher Scientific 66383 10 kDa) against 100 mM sodium-MOPS (1:1000 volume ratio, three changes).

(51) Fluorescence Correlation Spectroscopy (FCS) was used to quantify the number of encapsulated dye molecules per vesicle. The experiments were performed on a time correlated single photon counting (TCSPC) module (Becker-Hickl GmbH, Berlin, Germany). The detailed instrumentation setup is described in Gullapalli et al, Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology, J. Biomedical Optics, 2007 January-February; 12(1):014012. PubMed PMID: 17343487. Pubmed Central PMCID: PMC3251961. Epub 2007/03/09. eng. The light source utilized was a Nd:YAG pulsed laser with an emission maximum of 532 nm. A 60 water immersion objective with a numerical aperture of 1.2 was used to focus the laser beam into a diffraction-limited focal point 40 m above the cover slip within the sample. Laser power was set to 30 W/m.sup.2 by measuring light intensity at the back of the objective aperture.

(52) In an FCS experiment, time-dependent changes in fluorescent intensity within a small observation (confocal) volume (1 femtoliter) are monitored and the fluctuations are fit to an autocorrelation function described below (Equation 1).

(53) $\begin{array}{cc}G\left(\right)=\frac{.Math.F\left(t\right).Math..Math.F\left(t+\right).Math.}{{.Math.F\left(t\right).Math.}^2}& \left(1\right)\end{array}$

(54) Here, G() is the normalized autocorrelation function; F(t) is the fluorescence intensity fluctuation at time t; F(t+) is the fluorescence intensity fluctuation after a time lag , and F(t) is the average fluorescence intensity at time t. When =0, the term on the right side of the Equation 1 equals the variance of the fluorescence intensity fluctuation, which yields G(0)=1/N. N represents the average number of fluorophores in the confocal volume. These principles were used to obtain the concentration of fluorescein in aqueous solutions and polymer vesicles by fitting observed autocorrelation curves from FCS experiments to a 3D diffusion model shown below in Equation 2 and described in Gullapalli et al, above.

(55) $\begin{array}{cc}G\left(\right)=\frac{1}{N}\underset{i=1}{\overset{M}{.Math.}}{f_i\left[\frac{1}{1+\left(/{}_{D_i}\right)}\right]\left[\frac{1}{1+{\left(r/z\right)}^2\left(/{}_{D_i}\right)}\right]}^{1/2}& \left(2\right)\end{array}$

(56) Here, r and z are radius and half height of the confocal volume, which is often assumed to have 3D Gaussian illumination profile, see Maiti et al, Proc. Nat. Acad. Sci., 1997; 94(22): 11753-7. .sub.D.sub.i is 2D lateral diffusion time of fluorescent species i across the confocal volume. f.sub.i is the fraction of fluorescent species i. We used a single species fitting for both free fluorescein dye and polymer vesicles with encapsulated dye.

(57) Molecular brightness was measured using method based on FCS as described in Rigler et al, Correlation Spectroscopy, 2006 (9):367-73, to calculate the number of fluorescein molecules per vesicle, N.sub.encap-fluor. In this method, free fluorescein molecules in 0.1M Na MOPS buffer (pH=7.5) was used as a standard. Molecular brightness (photons emitted per molecule at standard excitation rate) of fluorescein, .sub.fluorescein, was measured by dividing total number of collected photons throughout the experiment by the duration of the measurement and dividing that number by the number of fluorescein molecules in the confocal volume, N.sub.free-fluor, as obtained by fitting of the time-shifted autocorrelation curve to a 3D diffusion model (Equation 2). The molecular brightness of the fluorescein encapsulated polymer vesicles, .sub.polymersome, was determined likewise. The ratio of the molecular brightness of the polymer vesicles to the molecular brightness of the free fluorescein, (.sub.polymersome/.sub.fluorecein), yielded an estimate of the number of fluorescein molecules per polymersome, N.sub.encap-flour.

(58) The results obtained are shown in FIG. 8. The molecular brightness of fluorescein, .sub.fluorescin, was 29 photons/molecule/second. The molecular brightness of the polymersomes at 0.2 m, .sub.polmersome, was 11244 photons/molecule/second. Hence the number of encapsulated fluorescein molecules per polymersome, N.sub.fluorescein=.sub.polmersome/.sub.fluorescein, was 387 molecules. The radius of 200 nm vesicle was corrected for wall thickness (cryo-TEM, R effective=9.09E.sup.08 m) and the encapsulation volume of vesicle was calculated to be 3.14E.sup.18 dm.sup.3. 1 mM solution was used in the hydration of polymer vesicles and concentration of fluorescein measured inside of vesicles was 0.204196992 mM. Therefore, these results show a 25% encapsulation efficiency, which is a high figure.