CN102573814B - Polymer vesicles of asymmetric membrane - Google Patents

Abstract

The present invention describes a novel polymersome system with an asymmetric membrane structure formed from amphiphilic diblock (AB type) copolymers of two different hydrophilic blocks , or formed by triblock copolymers (ABC type) with different hydrophilic blocks attached to both ends. Biomolecules or macromolecules can be encapsulated in this system at the nanoscale or submicron scale by thermodynamic partitioning and are thermodynamically stable. This system can be used for micronization and delivery of biopharmaceuticals. Equally important in the present invention is the method used to form polymersomes with asymmetric membranes: phase-directed self-assembly. The method involves a hydrophilic two-phase system for directing the above block copolymers to align on a hydrophilic interface in a designed orientation. This method has great application in the functionalization or biofunctionalization of particle surfaces.

Description

Polymeric vesicles with asymmetric membranes

Cross References to Related Applications

This application claims priority to US Patent Serial No. 61/221,022, filed June 26, 2009, the contents of which are incorporated herein by reference.

Various publications are referenced throughout this application. The disclosures of these publications in their entirety are incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

field of invention

The present invention describes a novel particle system called polymersomes with asymmetric bilayer membranes, the vesicle membrane of which is formed from two types of diblock copolymers, one forming The inner leaflet and the other form the outer leaflet.

Background of the invention

Polymer vesicles, also known as polymersomes, have a similar structure to lipid vesicles (or liposomes), except that the membrane enclosing the aqueous core is composed of amphiphilic Formation of permanent block copolymers rather than phospholipids. Polymersomes have attracted much attention due to their better mechanical stability compared to liposomes and the flexibility of chemical modification of membrane-forming copolymers. A large number of amphiphilic block copolymers, including poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PBD), poly(ethylene oxide)-block-poly(ethylene ethylene oxide) (PEO-b-PEE), poly(ethylene oxide)-block-poly(lactic acid) (PEO-b-PLA), poly(ethylene oxide)-block-poly(caproic acid ester) (PEO-b-PCL), and poly(ethylene oxide)-block-polystyrene (PEO-b-PS), have been used to prepare polymersomes. On the other hand, unlike other polymer particles, polymersomes are unique in having a watery interior in which sensitive biopharmaceuticals such as protein drugs, nucleotides, and vaccines can be encapsulated in a preserved natural state. These properties make polymersomes a promising biopharmaceutical delivery system.

Compared with the advancement of biotechnology and the rapid development of the biotherapy market, the pace of development of biological delivery systems is far behind. Decades of research into sustained-release versus non-invasive delivery of proteins has yet to lead to a product in these categories. For example, although the discovery of siRNA gene silencing has great potential to guide the production of revolutionary drugs ^[6-9] , its application in disease treatment lacks a safe and effective delivery system for target cells and different regions of cells while being restricted ^{[10, 11]} . Attempts to assemble such systems have encountered a series of difficulties. Particularly challenging is the incorporation of biomolecules into particle systems of a size capable of being engulfed by target cells. Technical obstacles include limited loading rate, the stability of the carrier system before reaching the target site, denaturation of biomolecules during the preparation process, degradation after phagocytosis, and the complexity of preparation ^[1-5] .

The fact that many microorganisms can efficiently transport their nucleotide or peptide cargos to host cells suggests that chemical mechanisms for intercellular and intracellular transfer of biological substances exist in nature. Most microorganisms consist of a hydrophilic interior separated by a stabilized and functionalized shell that allows them to perform various biological functions. The membranes of polymersomes have a liquid crystal structure, a structure similar to that of many microorganisms that can be flexibly modified to mimic these functions. However, so far, the reported polymersomes lack a mechanism to encapsulate biomacromolecules with sufficient capacity and stability, while lacking a mechanism to facilitate endosomal escape after phagocytosis by cells. These limitations encouraged us to develop a new polymersome system for improvement.

It has been reported that the phospholipid compositions of the inner and outer layers of cell membranes are different from each other ^[12-14] . In the plasma-facing membrane of eukaryotic cells, the outer layer is dominated by lecithin and sphingomyelin, while the cytoplasmic side is dominated by aminophospholipids ^[12] . The asymmetric structure of the cell membrane maintains the different composition and fluid environment inside and outside the cell. The asymmetric membranes of biological cell membranes inspired us to develop polymersome structures. With an asymmetric membrane, the chemical environment of vesicles is significantly different from that in the continuous phase, allowing therapeutic molecules to be encapsulated in vesicles through thermodynamic partitioning. In addition, if the distribution tends to the interior of the vesicle, the biomolecules loaded in the vesicle will reduce the Gibbs free energy (ΔG=-RTLnK _P , where K _P is the biomolecule between the two aqueous phases. partition coefficient) and can be thermodynamically stabilized.

To form polymersomes with asymmetric membranes, a method that enables two different amphiphilic diblock copolymers to form each layer of the vesicle bilayer separately is necessary. The method should also be able to direct the triblock copolymer to form a monolayer with a designed orientation. Therefore, creating/using a targeting system is a key factor in the assembly of vesicles with asymmetric membranes.

Summary of the invention

The present invention solves the above-discussed technical problems by polymersomes with asymmetric bilayer membranes and their phase-guided self-assembly method. It combines polymersome technology with an aqueous two-phase system to provide polymersomes with a protein-friendly interior of dextran polymers. During preparation, two amphiphilic diblock polymers with polysaccharides and polyethylene glycol (PEG) as hydrophilic blocks were added to an aqueous two-phase system consisting of a polysaccharide dispersed phase and a PEG continuous phase . Due to the incompatibility of the two phases of polysaccharide and PEG, the block copolymer with polysaccharide block is arranged at the interface of the aqueous two-phase system, and its hydrophilic block faces the dispersed phase of polysaccharide, while the block copolymer with PEG block The block copolymers of are aligned at the interface with their hydrophilic blocks facing the PEG continuous phase. If targeting molecules or other functional molecules are incorporated at the end of the PEG block, the polymersomes will assemble surrounded by functional groups. In addition, since most water-soluble proteins and other biological reagents tend to be more polysaccharide phase than PEG phase, these reagents, when added to the system, can be easily encapsulated in the polysaccharide dispersion by thermodynamic partitioning. in phase. Encapsulation by thermodynamically facilitated partitioning will greatly enhance the loading efficiency and stability of the reagents to be encapsulated. For protein encapsulation, the loading efficiency in the experiment reached 90%.

The polysaccharide core of the new polymersome serves several functions. In addition to preferential partitioning of bioburden, the mechanical stability of polymersomes can be greatly improved if the polysaccharide core can be cross-linked by grafting certain groups (eg glycidyl methacrylate binding). In addition, the grafted structures can be designed to be pH-sensitive so that the cross-linked polysaccharide core can be degraded and dissociated in endosomes of target cells. This property can be used to design delivery systems that facilitate endocytic escape of their cargo.

Finally, the material used in the present invention can be any biocompatible copolymer capable of forming self-assembled and fully biodegradable polymersomes for human in vivo applications. As the hydrophobic block of the aliphatic polyester, such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-polyglycolic acid (PLGA) and polyε-caprolactone (PCL), or Polymers of similar structure. The biologically active agents suitable for loading in polymersomes with asymmetric bilayer membranes can be protein, DNA, siRNA and the like.

Brief description of the drawings

Figure 1. Schematic depiction of the preparation procedure and structure of polymersomes with asymmetric bilayer membranes. a) Preparation procedure by adding two block copolymers, namely DEX ₂₂ -PCL ₆₆ and PEG ₄₅ -PCL ₃₀ , into a dextran/polyethylene glycol aqueous biphasic system. b) Polymeric vesicle structure whose dextran core is loaded with biomacromolecules preferentially partitioned therein, while its asymmetric block copolymer double shell consists of dextran blocks facing the dextran core and PEG The continuous phase is composed of PEG blocks. Two AB-type diblock copolymers (DEX-PCL and PEG-PCL) can be replaced by ABC-type triblock copolymers (DEX-PCL-PEG), and the dextran dispersed phase of the hydrophilic two-phase system It can be replaced by solid particles such as polyplex (particles condensed from polycations and nucleotides).

Figure 2. Photographic and microscopic images demonstrating polymersome formation of asymmetric bilayers. a) Photograph of FITC-dextran labeled dextran/PEG aqueous biphasic system, in which (i) no block copolymer was added, (ii) only PEG ₄₅ -PCL ₃₀ was added, (iii) only DEX was added ₂₂ -PCL ₆₆ , and (iv) adding both DEX ₂₂ -PCL ₆₆ and PEG ₄₅ -PCL ₃₀ . b) Microscopic image of Nile Red-labeled dextran/PEG aqueous two-phase system, in which (ii) only PEG ₄₅ -PCL ₃₀ was added, (iii) only DEX ₂₂ -PCL ₆₆ was added, and (iv) simultaneously Add DEX ₂₂ -PCL ₆₆ and PEG ₄₅ -PCL ₃₀ . c) Microscopic image of FITC-dextran labeled dextran/PEG aqueous biphasic system in which DEX ₂₂ -PCL ₆₆ and PEG ₄₅ -PCL ₃₀ were added at the following copolymer/dextran ratio: (iv -1) 0.25; (iv-2) 0.5; (iv-3) 1.0; and (iv-4) 3. Scale bar: (b) is 20 μm, (c) is 10 μm.

Figure 3. 2D- ^1H -NOESY-NMR spectrum of polymersomes in _D2O recorded at 500 MHz with a mixing time of 150 ms. No correlation role between DEX chains and PEG chains was identified.

Figure 4. Confocal laser microscopy images of polymersomes with asymmetric bilayer membranes and lateral mobility of the membranes. a) Polymersomes labeled with Nile Red and FITC-dextran, wherein (i) is a monochrome image scanned by a 559nm laser beam, and (ii) is a combined image of successively scanned beams of 559nm and 488nm. b) Schematic of an experiment to bleach a portion of a Nile Red-labeled polymersome surface and display lateral diffusion. c) Confocal images of Nile Red-labeled vesicles at 0s, 3s, 14s and 28s after bleaching; d) The relationship between the relative fluorescence intensity (I _t /I ₀ ) and the time after bleaching, where, ○ Represents the measured value, -- represents the theoretical calculation value. Scale bar: 10 μm.

Figure 5. Microscopic images of polymersomes with and without inner core crosslinking. a) Fluorescence microscopy images of polymersomes when the continuous phase was diluted 0, 3, and 6-fold without crosslinking the inner core. b) Microscopic image of a polymersome with a crosslinked inner core. c) Microscopic image (i) and fluorescent microscopic image (ii and iii) of a 10-fold dilution of the polymersomes in b). d) Molecular structure of GMA-dextran used to form the cross-linked inner core. Scale bar: 10 μm.

Figure 6. a) In vitro cumulative release profile of EPO from polymersomes with asymmetric bilayer membrane. b) Cumulative bioactivity curve of released EPO determined by UT-7 cell proliferation assay. ○ in each graph indicates the average value of two measurements.

Figure 7. TEM images (scale bar: 200 nm) of dried polymersomes with asymmetric bilayer membranes with cross-linked cores prepared in water by PEG-PCL and DEX-PCL.

Figure 8. Particle size distribution of asymmetric bilayer membrane polymersomes formed by phase-directed self-assembly of diblock copolymers PEG-PCL and DEX-PCL.

Detailed description of the invention

The present invention describes a method (phase-directed self-assembly) for the formation of polymersomes (polymersomes with asymmetric bilayer membranes) of unique composition with diameters ranging from submicron to micron. These polymersomes and their composition provide convenient encapsulation of soluble proteins and other biomolecules in submicron-sized particle systems for drug therapy, immunotherapy, gene therapy and other applications.

The amphiphilic molecules used to form these polymersomes by phase-directed self-assembly can be two different amphiphilic diblock copolymers (AB type) or have two different hydrophilicity at the two ends Block triblock copolymers (ABC type). Some examples are: hydrophilic polyethylene oxide (PEG) conjugated to hydrophobic poly(caprolactone), hydrophilic dextran (DEX) conjugated to hydrophobic poly(caprolactone), Or a block combined with PEG and DEX at its two ends. These blocks were chosen for several favorable properties, such as their non-toxicity, biodegradability and known metabolic pathways in vivo. Due to known safety and strong hydrophilicity, PEG is used as the hydrophilic block of various block copolymers to form polymersomes. Dextran is widely used as a plasma substitute, a drug carrier and an aqueous material for protein purification because of its good biocompatibility, biodegradability, good hydrophilicity, and high affinity for biological macromolecules ^{[ 20, 21]} . Due to its good biocompatibility and biodegradability, PCL has been widely used as a drug delivery material ^[23,24] . An example of an ABC type copolymer may be DEX-PCL-EPG. DEX can be replaced by other polysaccharides or oligosaccharides.

The polymersomes of the present invention are prepared by a unique phase-directed self-assembly method. Regarding phase-directed assembly (also known as self-assembly), a hydrophilic two-phase system consisting of a dispersed phase and a continuous phase is required as a "template". The continuous phase must be in liquid form, while the dispersed phase can be in liquid or solid form. The materials selected for forming the dispersed and continuous phases of the hydrophilic two-phase system should each have selective/biased affinity for the different hydrophilic blocks of the block copolymer (defined above). Asymmetric membrane polymersomes are formed by adding amphiphilic block copolymers (with blocks of different hydrophilicity) to an aqueous (or hydrophilic) two-phase system. An example of a hydrophilic two-phase system may be a hydrophilic two-phase system composed of an aqueous dextran solution as a dispersed phase and an aqueous PEG solution as a continuous phase. The DEX dispersed phase can be solidified by cross-linking treatment, or replaced by other aqueous solutions or solid particles such as polyplexes formed by cationic polymers and nucleotides. Intra-core crosslinking can be achieved through covalent or ionic (electrostatic) interactions between the polymer chains of the core (dispersed phase) material. The selective/biased affinity between the dispersed and continuous phases of the template two-phase system and the hydrophilic block of the copolymer is based on various intermolecular interactions including electrostatic interactions , hydrogen bonding, or hydrodynamic repulsion (eg, PEG with other macromolecules). As an example of electrostatic interactions, the sugar blocks of polysaccharide (or oligosaccharide)-PCL-EPG copolymers can be modified with charge-generating molecules such as succinic anhydride.

In our example, the selected dextran-PEG biphasic system was effectively used in protein purification due to the preference for the preferential partitioning of the dextran phase and the stabilizing effect of the dextran hydroxyl groups on the protein ^{[15 ]} . In the preparation process, under the direction of phase separation, the copolymers with dextran blocks and copolymers are arranged along the surface of the dextran droplets to form the inner layer of the bilayer; while the copolymers with PEG blocks form the outer layer , where the PEG chains face the PEG continuous phase. Encapsulated by an asymmetric bilayer, the interior of the new polymersome has a different chemical environment than the continuous phase, and thus can efficiently encapsulate biomolecules via thermodynamic partitioning. By incorporating targeting molecules to the ends of the hydrophilic blocks of the copolymers that form the outer layer, it is possible to assemble polymersomes capable of interacting with specific cells. Figure 1 schematically depicts an example of this phase-directed self-assembly, and the structure of an asymmetric bilayer polymersome.

The phase-directed strategy illustrated in this invention is very effective in the preparation of polymersomes with asymmetric bilayer membranes formed from two different diblock copolymers, and asymmetric bilayer membranes formed from ABC triblock copolymers. Polymersomes with a single membrane. In phase-directed assembly, hydrophilic-hydrophobic-hydrophilic triblock (ABC) copolymers are arranged in an "A to A" and "C to C" pattern to form an asymmetric monolayer through engineered phase selection. The portion and precision of phase selection can be determined by selecting the phase diagram of the system or by partitioning experiments. There are hundreds of reported phase diagrams for so-called aqueous two-phase systems in the literature including several textbooks ^[15] . Although as previously reported, the asymmetric monolayer can also be tuned by the relative hydrodynamic sizes of the A and C blocks (blocks with smaller hydrodynamic sizes tend to align inward, while larger sized blocks tend to align outward), however, size selection is much less efficient than phase selection. The phase-directed assembly set forth in this invention may provide a more efficient and better defined assembly for block polymer orientation, the underlying phase diagram summarizing the separation of two hydrophilic phases can be found in textbooks turn up. For example, for highly concentrated forms of dextran/PEG biphasic systems, the amount of dextran dissolved in the PEG phase, or the amount of PEG dissolved in the dextran phase, is less than 1.0 wt% ^[15] . Moreover, the phase-directed assembly enables two well-selective amphiphilic diblock copolymers to form the inner and outer layers of the bilayer, respectively, with defined distributions.

Example

The purpose of the following examples is to enable those skilled in the art to better understand the present invention. This embodiment should not be used to limit the application and rights of the present invention.

Example 1. Preparation of polymersomes with asymmetric bilayer membranes

To confirm the feasibility of the proposed phase-directed assembly method in forming polymersomes with asymmetric bilayer membranes, we synthesized two block copolymers, such as PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ , separately Added to the dextran/PEG aqueous two-phase system separately and jointly, followed by visual and microscopic observation. The footnote to the molecular formula for each block copolymer indicates the number of repeating units of the block. The aqueous two-phase system was prepared by mixing dextran (70KDa) solution (concentration 10%) and PEG (8KDa) solution (concentration 10%), and added 0.1% by weight of FITC-labeled dextran to the dextran solution. Glycans for easy observation. Besides PCL, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-polyglycolic acid (PLGA) can also be used as the hydrophobic block.

For direct observation, the prepared dextran/PEG two-phase system solution was then divided into four glass tubes: one tube without any block copolymer, one tube with only PEG ₄₅ -PCL ₃₀ , one tube with Only DEX ₂₂ -PCL ₆₆ was added to the tube, and both PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ were added to the last tube. The aqueous two-phase system without block copolymers easily separated into two clear bulky phases after the stirring was stopped, with the fluorescently labeled dextran phase at the bottom and the PEG phase at the top (Fig. 2a-i). For the samples with only PEG ₄₅ -PCL ₃₀ or DEX ₂₂ -PCL ₆₆ added, the system separated into two bulk phases (Fig. 2a-ii and 2a-iii). Moreover, when PEG ₄₅ -PCL ₃₀ was added, the bulk phase of PEG became cloudy (Fig. 2a-ii); and when DEX ₂₂ -PCL ₆₆ was added, the bulk phase of dextran became cloudy (Fig. 2a-iii) . Apparently, the two block copolymers preferentially partition as aggregates in each of the phases with the same content as their hydrophilic blocks. Interesting observations emerged when two block copolymers, namely PEG ₄₅ -PCL ₃₀ or DEX ₂₂ -PCL ₆₆ were added together in the dextran/PEG aqueous biphasic system. The sample remained in its "emulsion" form for at least 60 minutes after stirring was stopped (Fig. 2a-iv). This result indicates that when the two block copolymers are added together, they are thermodynamically preferential to aggregate at the interface between dextran and PEG.

To directly observe the copolymer incorporated into the dextran/PEG biphasic system, we repeated the above experiment by substituting the hydrophobic fluorescent dye Nile red for FITC-dextran, and then observed under the microscope. For the aqueous two-phase system with only PEG ₄₅ -PCL ₃₀ added, the background (ie, the PEG continuous phase) showed stronger fluorescence emission, while the fluorescence in the dextran dispersed phase was weaker (Fig. 2b-ii). This result is consistent with the experiment of FITC-dextran labeling with _PEG45 - _PCL30 dispersed in the PEG phase. Similarly, when only DEX ₂₂ -PCL ₆₆ was added to the dextran/PEG biphasic system, the dextran dispersed phase became stronger in fluorescence (Fig. 2b-iii). For the samples added with two copolymers, PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ , Nile Red was distributed on the surface of the dispersed phase (Fig. 2b-iv). Nile red labeling experiments further proved that when PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ were added simultaneously, they would aggregate at the interface between the dextran dispersed phase and the PEG continuous phase.

The relationship between the particle size of the dextran dispersed phase and the amount of PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ added together into the biphasic system is that the block copolymer at the interface of the dextran/PEG biphasic system The specific distribution of provides further evidence. The dextran dispersed phase was again labeled with FITC-dextran. As shown in Fig. 2c–iv, as the ratio of copolymer to dextran (w/w) increased from 0.25 to 0.5 and 1.0, the average diameter of the dispersed phase particles decreased from 9.2 μm to 5.3 μm and 2.2 μm, respectively . Further increasing the copolymer to dextran ratio to 3.0 resulted in the dextran dispersed phase not being visible under the light microscope. Clearly, both copolymers, when added together into a dextran/PEG biphasic system, thermodynamically promote the dextran-PEG interface and produce more interfacial films (resulting from the reduced particle size of the dextran dispersed phase). That is reflected by the increased specific surface area).

The above three experiments (Figure 2) collectively indicated that the two copolymers, PEG ₄₅ -PCL ₃₀ and DEX ₂₂ -PCL ₆₆ , formed an asymmetric bilayer structure at the interface of the dextran/PEG aqueous two-phase system, in which the dextran The sugar and PEG blocks are in contact with their respective analogs.

Example 2. Cross-interaction between DEX-block and PEG-block after formation of polymersomes

2D- ¹ H-NOESY-NMR spectra were reported as a reference method for indicating/suggesting the presence or absence of PEG blocks and glycosyl groups of polymersomes formed from ABC triblock molecules. According to literature reports ^[16-18] , 2D- ¹ H-NOESY-NMR spectroscopy can detect the interaction between individual molecules stacked at a physical distance equivalent to the physical distance of adjacent groups in the molecule through cross peaks. Figure 3 shows the chemical shift peaks of the proton signal of the methylene group in the PEG unit located at 3.57ppm, and the chemical shift peaks of the dextran unit located at 3.60-3.65ppm and 3.53-3.35ppm, respectively. No cross-interaction peaks were observed between dextran and PEG. The absence of hydrogen-bonding interactions between PEG and dextran blocks indicated that PEG blocks and DEX blocks were not mixed together in bilayer membranes ^[16] . Although NMR spectroscopy cannot detect a small amount of mixing of DEX blocks and PEG blocks in the opposite phase, the results (Fig. 3) are convincing in that most of the DEX blocks and PEG blocks are not Mix together. Therefore, the structure of the polymersome can only be that of an asymmetric bilayer membrane, as described in FIG. 1 . This result supports our previous conclusion that the PEG block and the dextran block of the two amphiphilic diblock copolymers are arranged separately, facing both sides of the dextran/PEG interface.

Example 3. Lateral Diffusion of the Vesicular Membrane of Polymersomes with Asymmetric Bilayers

In the present invention, the lateral mobility of an asymmetric copolymer bilayer is determined using time-based confocal imaging. Hydrophobic and hydrophilic fluorescent dyes (Nile red and FITC-dextran) were used to label the hydrophobic shell and hydrophilic core of the vesicle membrane, respectively. Figures 4a-i and 4a-ii show confocal images of Nile Red-labeled surfaces and composite images of surfaces and nuclei labeled with each of the dyes, respectively. Apparently, Nile red and FITC-dextran are distributed on the surface and core of the dispersed dextran phase, respectively. This result is consistent with our previous conclusion that the two block copolymers form a bilayer around the dispersed dextran. To examine the lateral mobility of surface membranes, the laser confocal beam of a laser confocal microscope (LSCM) was positioned on a portion of the polymersome membrane to induce localized photobleaching of Nile red, thereby showing the Gaps were created in the rings (Fig. 4b, 4c-i). After the bleaching process, confocal images across the blanched region were acquired at predetermined intervals. The gapped ring gradually closed within 28 s (Figures 4c-ii, 4c-iii and 4c-iv), indicating the diffusion of unbleached Nile Red molecules from the surroundings into the bleached area. Figure 4d shows the time course of fluorescence recovery in the bleached region. Although the diffusion rate of dye molecules is not necessarily the same as that of block copolymers, the rapid diffusion of hydrophobic nylon red can only occur in a moving and continuous polymer bilayer.

Because the vertical extension of the bleached region is significantly longer than its horizontal extension, and the confocal plane is thin (Fig. 4b), the lateral diffusion rate of Nile Red can be estimated using a one-dimensional model of Fick's law (along the confocal plane). The diffusion coefficient D of Nile Red along the surface can be calculated by substituting the measurements of the fluorescence intensity and time of the bleached region into the earlier state algorithm of Fick's second equation:

${\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; t</span>}}<span\; class="notranslate">\; /</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 4</span>}{<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; Dt</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; \&pi;</span>}{\mathrm{<span\; class="notranslate">\; L</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; )</span>}^{\frac{\mathrm{<span\; class="notranslate">\; I</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; -</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 1</span>}<span\; class="notranslate">\; )</span>$

where L is the length of the gap of the fluorescent ring in cm _; t is the time after bleaching in seconds; I and _I are the measured fluorescence intensity above background at time t and before bleaching, respectively. Based on the measurement data, D was calculated using the least square method, and it was 2.67×10 ⁻⁹ cm ² ·s ⁻¹ . Substituting the values of D and L into the equation yielded a curve consistent with the measured data (Fig. 4d). This value is comparable to that of phospholipid bilayers (10 ^-8 cm ² ·s ^-1 ) ^[20] .

Example 4. Modification of the core structure of polymersomes with asymmetric bilayer membranes

The inner core of the polymersome can be endowed with the ability to respond to intracellular pathways. If the core is immobilized by a pH-sensitive cross-linking mechanism, it can retain shape in a neutral environment, but at low pH endosomes dissociate and rupture by acid-driven fragmentation of intranuclear linkages. The microscopic and fluorescent images shown in Figure 5 describe the nuclear properties based on which desired functions can be incorporated. When the PEG continuous phase was removed or diluted, polymersomes whose core matrix was not cross-linked were enlarged and ruptured (Figures 5a-i, 5a-ii and 5a-iii). When the core matrix was formed from methacrylate-grafted dextran and thus by using ammonium peroxodisulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) (concentrations of 0.2% by weight and 0.4% by weight) as the free radical polymerization crosslinking of the initiator system, it can withstand the sudden change of osmotic pressure, and the dilution of the PEG continuous phase no longer causes the expansion and rupture of the polymer vesicles (Fig. 5b, 5c-i, 5c-ii and 5c-iii). The cross-linked core matrix can effectively maintain the bilayer membrane. Breaking the matrix crosslinks will result in immediate breakdown of the particles. This key feature is particularly useful for the delivery of biomolecules into the cytoplasm of target cells by phagocytosis (endosomal escape).

Example 5. Encapsulation of biomolecules in and release of biomolecules from polymersomes in asymmetric bilayers

Further provided is the encapsulation of fragile biomolecules in polymersomes of asymmetric bilayer membranes and the controlled release of said biomolecules therefrom. For example, erythropoietin (EPO) was added to a phase-directed assembly method, followed by testing for protein content, release profile, and bioactivity (Fig. 6a). The encapsulation efficiency of the protein measured by the micro-BCA method was 89%, which is significantly higher than the 5% reported for polymersomes with symmetrical bilayers ^[1] . In addition, Figure 6 shows that the release kinetics of EPO from polymersomes (with a cross-linked core) is consistent with the cumulative bioactivity profile measured in the UT-7 cell proliferation assay (Figure 6), thus indicating that the protein activity in the particle system has been well maintained. In addition, since the proteins loaded in the interior of the polymersomes are stabilized by dextran, they could withstand 40°C for 6h with no increase in protein aggregation as observed by the HPLC method.

Example 6. Preparation of nanosized polymersomes with asymmetric bilayer polymersomes

The procedure for the preparation of nanosized polymersomes with asymmetric bilayer membranes was derived from the method in Example 1 by increasing the copolymer/dextran ratio. Briefly, 1 mL diblock copolymer solutions at a concentration of 5 mg ^mL were prepared by incubating at 60 °C for 12–24 h with or without magnetic stirring. Then, PEG/GMA-dextran (400 mg/100 mg) and protein were dissolved into 4 ml of KCl solution (0.22 M) purged with nitrogen for 10 minutes, and then transferred to the above diblock copolymer solution, followed by Vortex to mix for 1 min. Then, ammonium peroxodisulfate (180 μl, 50 mg/mL) and N,N,N′,N′-tetramethylethylenediamine (100 μl, 20% v/v, adjusted to pH 7 with 4M HCl) were added, The system was incubated at 40-50°C for 1 h to cross-link the internal dextran of the polymersomes. Polymersomes with cross-linked cavities were characterized by TEM and DLS (see Figure 7 and Figure 8).

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Claims (20)

1. for the preparation of a method for the polymer vesicle of asymmetric membrane, described method comprises:

A) prepare hydrophilic two-phase system, described hydrophilic two-phase system is made up of polysaccharide decentralized photo and Polyethylene Glycol or poly(ethylene oxide) continuous phase, and wherein said polysaccharide decentralized photo is formed by following aqueous solution: glucosan, starch, inulin or carboxymethyl cellulose;

B) two kinds of amphiphilic diblock polymer respectively with polysaccharide and Polyethylene Glycol hydrophilic block are joined step a) in the described hydrophilic two-phase system of preparation, wherein said two kinds of amphiphilic diblock polymer respectively with polysaccharide and Polyethylene Glycol hydrophilic block are two kinds of amphiphilic diblock copolymers with different hydrophilic block; With

C) by step b) in the sample stirring, shake, ultrasonic or adopt other method to mix of preparation.

2. method claimed in claim 1, the described decentralized photo of wherein said hydrophilic two-phase system is in liquid form or in solid form.

3. method claimed in claim 1, wherein said polysaccharide and Polyethylene Glycol hydrophilic the block respectively described decentralized photo to described hydrophilic two-phase system and described continuous phase have selection affinity.

4. method claimed in claim 2, wherein liquid form decentralized photo passes through via crosslinked solidifying in the core of covalency and ionic interaction.

5. method claimed in claim 1, wherein said hydrophilic two-phase system is made up of polysaccharide decentralized photo and PEG continuous phase.

6. method claimed in claim 4, wherein crosslinking Treatment comprises that the molecule using with having crosslinkable key is grafted on polysaccharide.

7. method claimed in claim 1, wherein said hydrophilic two-phase system is made up of solid hydrophilic decentralized photo and PEG continuous phase.

8. method claimed in claim 7, wherein said solid hydrophilic decentralized photo is complex.

9. method claimed in claim 1, wherein adds biomolecule in any step of preparation technology, to be encapsulated in described polymer vesicle.

10. method claimed in claim 9, wherein said biomolecule is selected from albumen, peptide, DNA and RNA.

The polymer vesicle system of 11. 1 kinds of asymmetric membranes of preparing by method described in claim 1, described polymer vesicle system comprises hydrophilic polysaccharide core and the asymmetric block polymer film take polysaccharide as one of hydrophilic block, described asymmetric block polymer film is inside according to polysaccharide block by block polymer, and the outside mode of Polyethylene Glycol block forms in the self assembly of polysaccharide core surface.

Polymer vesicle system described in 12. claim 11, wherein said hydrophilic polysaccharide core is in liquid form or solid form.

Polymer vesicle system described in 13. claim 12 is wherein polysaccharide solution in the hydrophilic polysaccharide core of liquid form.

Polymer vesicle system described in 14. claim 12 is wherein crosslinked polysaccharide or complex particle in the hydrophilic core of solid form.

Polymer vesicle system described in 15. claim 11, wherein said two kinds of amphiphilic diblock polymer respectively with polysaccharide and Polyethylene Glycol hydrophilic block are two kinds of amphiphilic diblock copolymers with different hydrophilic block.

Polymer vesicle system described in 16. claim 15, wherein said amphiphilic diblock copolymer is the polysaccharide block of being combined with hydrophobic polymer block and the PEG block of being combined with hydrophobic polymer block.

Polymer vesicle system described in 17. claim 16, wherein said hydrophobic polymer block is biodegradable polymers.

Polymer vesicle system described in 18. claim 17, wherein said biodegradable polymers is selected from polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-altogether-polyglycolic acid (PLGA) and poly-epsilon-caprolactone (PCL).

Polymer vesicle system described in 19. claim 11, wherein said polymer vesicle system is sealed biomolecule.

Polymer vesicle system described in 20. claim 19, wherein said biomolecule is selected from albumen, peptide, DNA and RNA.

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