F.
Mastrotto
a,
A. F.
Breen
a,
G.
Sicilia
a,
S.
Murdan
b,
A. D.
Johnstone
c,
G. E.
Marsh
a,
C.
Grainger-Boultby
a,
N. A.
Russell
c,
C.
Alexander
a and
G.
Mantovani
*a
aSchool of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK. E-mail: giuseppe.mantovani@nottingham.ac.uk; Fax: +44 (0)115 846 6296; Tel: +44 (0)0115 823 2348
bDepartment of Pharmaceutics, UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London, WC1N 1AX, UK
cFaculty of Engineering, University of Nottingham, Nottingham NG7 2RD, UK
First published on 20th September 2016
Rapid and simple routes to functional polymersomes are increasingly needed to expand their clinical or industrial applications. Here we describe a novel strategy where polymersomes are prepared through an in-line process in just a few hours, starting from simple acrylate or acrylamide monomers. Using Perrier's protocol, well-defined amphiphilic diblock copolymers formed from PEG acrylate (mPEGA480), 2-(acryloyloxy)ethyl-3-chloro-4-hydroxybenzoate (ACH) or 2-(3-chloro-4-hydroxybenzamido)ethyl acrylate (CHB), have been synthesised by RAFT polymerisation in one-pot, pushing the monomer conversion for each block close to completion (≥94%). The reaction mixture, consisting of green biocompatible solvents (ethanol/water) have then been directly utilised to generate well-defined polymersomes, by simple cannulation into water or in a more automated process, by using a bespoke microfluidic device. Terbinafine and cyanocobalamine were used to demonstrate the suitability of the process to incorporate model hydrophobic and hydrophilic drugs, respectively. Vesicles size and morphology were characterised by DLS, TEM, and AFM. In this work we show that materials and experimental conditions can be chosen to allow facile and rapid generation drug-loaded polymersomes, through a suitable in-line process, directly from acrylate or acrylamide monomer building blocks.
An increasingly large area of research is focussed on the use of polymersomes as drug nanovectors in drug delivery.7–10 Recently, Battaglia and coworkers showed delivery of an IgG model cargo to the Central Nervous System (CNS) parenchyma and CNS cells mediated by poly(2-diisopropylamino)ethyl methacrylate)-based pH-responsive polymersomes functionalized with LRP-1-targeting Angiopep-2 and Rabies Virus Glycoprotein (RVG) peptide ligands to facilitate transport through the blood–brain barrier.11
Polymersomes have also been utilised to create nanosized reactors, where entrapped (bio)molecules – e.g. enzymes – are part of multicompartmental arrangements mimicking those found in cell organelles. In an early work Meier and coworkers incorporated β-lactamase within PDMS-PMOXA triblock copolymer vesicles.12 The introduction of a channel-forming protein from the outer cell wall of Gram-negative bacteria (OmpF) into the vesicle membrane allowed passive transport of ampicillin, an enzyme substrate, which was converted in the polymersome aqueous lumen into ampicillinoic acid. Lecommandoux and van Hest engineered multicompartimentalised polymersomes-in-polymersomes systems, mimicking artificial cells and cell organelles.13 These studies, in conjunction with the potential for self-propulsion,14 make polymersomes ideal candidates for the development of polymeric protocells.15,16
In terms of their manufacturing, polymersomes are often assembled from amphiphilic block copolymers synthesised through long synthetic procedures – typically at least two sequential polymerisations, requiring purification of both polymer intermediates and final amphiphilic macromolecules from unreacted monomers and traces of other low molecular weight impurities. Importantly, polymerisation reactions often need to be stopped at moderate monomer conversions to ensure high proportion of chain-end fidelity, which is required for subsequent polymer chain-extension.17–19
In addition to being not fully efficient, this approach invariably results in waste of functional monomers utilised for the polymerisation step, which is undesirable especially when these are particularly expensive, not commercially available, or both. Finally, the processes for the assembly of polymersomes are not always trivial.20 Although a range of techniques have been developed,21 most methods still suffer from low reproducibility and poor scalability, which may complicate industrial application of polymersomes.6 Whilst chemists are increasingly focussing on developing more efficient and greener chemical transformations – i.e. as described by Sharpless and coworkers’ click chemistry philosophy – one could envisage that analogous approaches could be applied not only to individual chemical reactions, but also to entire processes. A clear move in this direction is the work on polymerization-induced self-assembly (PISA) by Armes’ group and others,22–30 where block copolymers nano-objects can be assembled with predictable shape and size during the polymerisation process, by judicious choice of monomers and variation of polymer physicochemical characteristics (e.g. packing parameter P).31
In the present work we aimed to develop a route to self-assembled drug nanovectors which would complement the range of applications inherent to PISA. In this case, drug-loaded nanovesicles are engineered by sequential and quantitative polymerisation of appropriate monomers in one pot, followed by self-assembly of the resulting amphiphilic block copolymers into vesicles, by directly adding the final reaction mixture to a suitable aqueous phase (Chart 1). Key advantages of this approach include the avoidance of intrinsically inefficient purifications of polymer intermediates, the potential for incorporating a wide range of drugs – including those not compatible with radical polymerisation conditions, and, similarly to PISA, facile assembly of nanovectors directly from the polymerisation solution, but without having to remove excess unreacted monomers.
![]() | ||
Chart 1 In line engineering of mPEGA12a-b-ACH36 or mPEGA12a-b-CHB36 polymeric vesicles via one-pot RAFT and in line post-polymerisation assembly. |
For the polymerisation step, recent studies by Perrier and co-workers showed that well-defined multiblock copolymers can be obtained by RAFT polymerisation in one pot by sequential and quantitative polymerisation of suitable monomers in only a few hours18,32,33 or even minutes.34 The monomers were selected based on previous studies, where we showed that block copolymers based on novel pH-responsive monomers bearing substituted aromatic alcohols could be effectively assembled into well-defined nanovesicles, whilst incorporating specific drugs with high efficiency.35,36
Polymer | Code | [M]/[CTA]/[I] | Conv.%a | M n, NMR (kDa) | M n, SEC (kDa) | Đ |
---|---|---|---|---|---|---|
a Obtained from 1H NMR analysis. b Number of repeating unit of ACH or CHB monomers, DPACH and DPCHB respectively, were calculated by 1H NMR by comparing the integral of monomer aromatic proton peak at 6.93 (I6.93) ppm, and the methoxy peak of mPEGA repeating units at 3.23 ppm (I3.23) set as 3. DPACH (or DPCHB) = 12 × (I6.93/(I3.23/3). Mn, NMR was calculated as: (−DPACH (or DPCHB) × MWmonomer) + Mn, macroCTA). c Obtained from SEC analysis in DMF + 0.1% LiBr (PMMA standards). d Obtained from SEC analysis in CHCl3 (polystirene standards). | ||||||
mPEGA 12a | 4 | 12![]() ![]() ![]() ![]() |
95 | 5.7 | 6.5c | 1.05c |
mPEGA 12b | 5 | 12![]() ![]() ![]() ![]() |
98 | 5.3 | 6.1d | 1.13d |
mPEGA 12a -b-ACH 36 | 6 | 36![]() ![]() ![]() ![]() |
94 | 15.5b | 14.9c | 1.18c |
mPEGA 12b -b-CHB 36 | 7 | 36![]() ![]() ![]() ![]() |
95 | 15.2b | 15.1d | 1.17d |
In a separate Schlenk tube ACH monomer (803 mg, 2.96 mmol) was dissolved in 1.5 mL of water:
EtOH (1
:
2 vol/vol); the tube was placed in an ice bath, stirred for 5 min to allow temperature equilibration, and deoxygenated by gentle Argon bubbling for 15 minutes. This solution was then cannulated under Argon into the polymerisation tube containing mPEG12a macro-CTA, and the resulting solution heated at 70 °C under stirring. The polymerisation was started by addition of a previously degassed VA-044 solution in HPLC-grade water (96 μL, 0.54 μmol) via gas-tight syringe. After 2 h at 70 °C ACH monomer conversion was checked by 1H NMR in DMSO-d6. Three sequential additions of degassed fresh initiator solution (96 μL), each followed by 2 h stirring at 70 °C were performed, until conversion finally reached 94%.
The polymersomes formation occurred instantaneously, as confirmed by DLS. To remove the residual ethanol, the vesicles suspension was transferred into a dialysis bag (3.5 kDa MWCO) and dialyzed against 5 L of DI water for 4 hours at room temperature with at least 4 water exchanges. Alternatively, the beaker containing the polymersomes suspensions were left to stir overnight to allow the organic solvent to evaporate. Samples were analysed by DLS and TEM.
Drug loading was then quantified via a RP-HPLC method (see ESI†). Loading reported for each polymer is the average of two independent experiments, each prepared and analysed in triplicate.
Loading Capacity (LC) and Encapsulation Efficiency (EE) were calculated as:
To successfully prepare multiblock copolymers via this one-pot RAFT process, a number of parameters – e.g. choice of the solvent, initiator, monomer concentration, temperature and [CTA]/[I] ratio – are key for maintaining near-perfect chain-end fidelity, thus ensuring “livingness” of the polymerisation.18
A mixture of water/EtOH was chosen as a suitable “green” solvent39 for the subsequent polymerisation step. Water is known to increase the propagation rate coefficient (Kp), and thus the polymerisation rate (Rp), of vinyl monomers,40 which is critical for efficient in situ RAFT synthesis of multiblock copolymers,18 whilst EtOH was required to solubilise the monomers ACH (2) and CHB (3), and the (methyl 2-((((2hydroxyethyl)thio)carbonothioyl)thio)propanoate), (MHP) chain-transfer agent. The latter was chosen due to the higher hydrolytic stability of trithiocarbonates compared to other suitable CTAs – e.g. dithioesters – and their ability to minimise initial rate retardation sometimes observed in RAFT polymerisations.41 In the one-pot protocol optimised in this work a macro-CTA was first synthetised using commercially available poly[ethylene glycol(480)]methyl ether acrylate, mPEGA(480) and 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044) as the water soluble initiator [I], at 70 °C (Scheme 1). VA-044 is characterised by a high decomposition rate (t1/2 2 h at 70 °C), which again is key to speeding up the polymerisation without losing the chain livingness.32,33 mPEGA(480) was chosen for the synthesis of the first hydrophilic block because of its aqueous solubility, biocompatibility and ability to generate polymers with prolonged plasma half-life and low immunogenicity.42–44 After an initial series of experiments required to identify suitable reaction conditions (i.e. monomer concentration and [CTA]/[I] molar ratio) which would allow to reach near-quantitative monomer conversion whilst retaining chain-end fidelity, a [CTA]/[I] ratio of 250:
1 and a monomer concentration of 2.0 M were chosen. The degree of polymerisation (DP) for the p(mPEGA) macro-CTA was targeted to 12. After 2 h the reaction reached a monomer conversion >95%, as assessed by 1H NMR, by comparing the decrease of the signal for the vinyl proton at ∼5.96 ppm to that of the mPEG terminal methoxy group at 3.24 ppm (see ESI, Fig. S9 and S10†). SEC analysis of the macro-CTA (mPEG12a, mPEG12b, Table 1 and Fig. 1), using DMF + 0.1% LiBr as the mobile phase showed unimodal, symmetric, and narrow molecular weight distributions (Đ < 1.13), with Mn, SEC in good agreement with the theoretical one (Table 1, polymers 4 and 5). Two different macro-CTAs, mPEGA12a and mPEG12b, both with DP = 12 were synthesised and directly utilised without isolation or purification for the synthesis of mPEGA12a-b-ACH36(6) and mPEGA12b-b-CHB36(7), respectively. Importantly, both reactions led to the desired mPEGA12 macro-CTAs with near-quantitative monomer conversion, thus validating the reproducibility of this process. Both acrylate ACH (2) and acrylamide CHB (3) monomers have limited solubility in water, hence for the second polymerisation step they were first dissolved in EtOH, then the resulting solutions were diluted with water to a final EtOH/water volume ratio of 2
:
1. To ensure full monomer conversion in the one-pot RAFT with higher EtOH content, a larger amount of radical intitiator was required;33 in this work for the second block a [macro-CTA]/[I] ratio of 150
:
1 was initially chosen in both cases. A DP of 34 for both ACH and CHB was targeted, to provide final copolymers with a hydrophilic block weight fraction (f = 35% ± 10%)45 suitable for self-assembling into vesicles.2 Indeed, when considering the Mn provided by 1H NMR analysis the calculated f values were 0.358 and 0.357 for mPEGA12a-b-ACH36 and mPEGA12b-b-CHB36, respectively.
![]() | ||
Fig. 1 SEC traces and 1H NMR spectra (DMSO-d6) of non-purified mPEGA12 macro-CTA and final block copolymers for A. mPEGA12a-b-ACH36(6), and B. mPEGA12b-b-CHB36(7). |
Following the addition of ACH (2) or CHB (3) monomers and VA-044 radical initiator, a monomer conversion of 80–90% was observed after 2 hours. Additional radical initiator was necessary to achieve >95% of monomer conversion (Table 1, polymer 6 and 7, see ESI Fig. S11 and S12†), resulting in a final [macro-CTA]/[I] ratio of 1:
50 or 1
:
30 for mPEGA12a-b-ACH36 and mPEGA12b-b-CHB36, respectively. A possible explanation for this is that the second block grown here is longer than most of those prepared by Perrier and coworkers for which near-full conversion could be achieved under the conditions reported,18,27,32–34 although other side-processes – e.g. very minimal H transfer from ACH (2) or CHB (3) monomers – cannot in theory be ruled out at this stage. Nevertheless, pleasingly, SEC analysis (Table 1 and Fig. 1) showed that very well defined block-copolymers with a final Đ of 1.18 (mPEGA12a-b-ACH36) and 1.17 (mPEGA12b-b-CHB36), were synthesised indicating good control over the polymerisation process. The DP of the second block was determined by 1H NMR, by comparing the integral of the OCH3 signal of mPEGA repeating units in the hydrophilic block at 3.24 ppm, with that of the aromatic proton of ACH and CHB repeating units at 6.93 ppm (ESI, Fig. S11 and S12†). A DPNMR of 36 was found for the second block of both mPEGA12a-b-ACH36(6), and mPEGA12b-b-CHB36(7), slightly higher than the expected 34. This can be explained considering that a minimal amount of mPEGA12 macro-CTA was withdrawn at the end of the first polymerisation step for 1H NMR and SEC analysis, thus very marginally increasing the monomer
:
macro-CTA molar ratio.
Polymersomes assembly was confirmed by DLS analysis of the water/ethanol mixture immediately after the cannulation process. DLS traces showed the presence of nanoassemblies with an average diameter of 136 ± 2 nm and 89 ± 11 nm for mPEGA12a-b-ACH36, and mPEGA12b-b-CHB36, respectively.
Ethanol was removed by either dialysis or slow evaporation at room temperature, and the resulting assemblies were characterised by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in line polymersomes assembly process led to homogeneous dispersions of polymeric vesicles in water, (Fig. 2A), with hydrodynamic diameters estimated by DLS of 122 ± 20 nm or 41 ± 0.18 nm, for mPEGA12a-b-ACH36(6) and mPEGA12b-b-CHB36(7), respectively, with narrow PDI (<0.23). Similar results in terms of vesicle size and polydispersity were obtained regardless of the method used for removal of traces of ethanol in the polymersomes suspension (data not shown).
To test the efficiency of the in-line process in assembling well-defined vesicles, conventional nanoprecipitation of purified mPEGA12a-b-ACH36(6) and mPEGA12b-b-CHB36(7) was also performed. Accordingly, polymers were dissolved in DMSO (5.0 mg mL−1) and water was added dropwise to the resulting solution until a 1.0 mg mL−1 concentration was reached.
This procedure led to the formation of very similar size vesicles in the case of mPEGA12a-b-ACH36 (122 ± 9 nm), but larger assemblies in the case of mPEGA12b-b-CHB36 (172 ± 2 nm ESI, Fig. S13†), a difference that could be ascribed to the different procedures and conditions used for their assembly.46
Analysis of the TEM images performed (ESI Fig. S14 and S15†) on polymersomes prepared via the in-line procedure and stained with 3% uranyl acetate solution gave results consistent with the size estimated by DLS for both mPEGA12a-b-ACH36, with an average core diameter of 126 ± 75 nm, and mPEGA12b-b-CHB36 (44 ± 5 nm).
To gain further insight into the morphology of these vesicles in solution, mPEGA12a-b-ACH36 and mPEGA12a-b-CHB36 polymersomes were analysed by AFM in PeakForce tapping mode (Fig. 3). A major advantage of this method is that the sample can be imaged in solution, thus obtaining a more realistic representation of the vesicles morphology in comparison to microscopy techniques performed on dry samples (i.e. TEM). Furthermore, the use of tapping instead of contact mode avoid the flattening caused by the pressure exterted, allowing a more accurate estimation of vesicle size and three-dimensional shape.
Immobilisation of the polymersomes on the mica surface, a pre-requisite to perform the analysis, was achieved by electrostatic interactions.47 As described by Li et al.,48 negatively charged polymers can be easily immobilized on a mica surface, also negatively charged, via electrostatic interaction mediated by Mg2+.
Our previous studies35 showed that at physiological conditions (pH 7.4) polymers containing 3-chloro-4-hydroxybenzoate repeating units are negatively charged due to the deprotonation of part of their aromatic alcohol pendant groups. Accordingly, 1.0 mg mL−1 polymersome suspensions were prepared by nanoprecipitation in PBS buffer. The average sizes detected were around 80 nm for mPEGA12a-b-ACH36 and 45 nm for mPEGA12a-b-CHB36 (Fig. 3). The height of both polymeric aggregates was found to be around 14–17 nm, thus smaller than their diameter, which can be ascribed to a partial collapse of their hollow structure, thus confirming their vesicular morphology.49 Partially collapsed structures with typical ‘deflated’ balloon’ morphology could also be visualized (Fig. 3, insert 1D), thus further confirming the vesicular nature of these supramolecular assemblies.
Larger sizes were estimated by DLS analysis of the polymersomes in PBS (170 and 70 nm for mPEGA12a-b-ACH36 and mPEGA12a-b-CHB36, respectively, Fig. S16 ESI†) in comparison to those observed by AFM analysis. It has already been shown that the vesicles hydrodynamic diameter estimated by DLS can be larger than the geometrical diameter because of the hydration layer of the polymer shell.50 Liang et al. have discussed a similar discrepancy observed when analysing EggPC/cholesterol vesicles by both DLS and AFM and ascribed it to a potential partial fragmentation of larger vesicle upon adsorption on mica, and/or the intrinsic higher sensitivity of DLS for larger vesicle, which results in an overestimation of the mean hydrodynamic diameter.51 The latter phenomenon has also been described when AgNPs53 and polystyrene nanoparticles52 were utilised to compare these two analytical techniques.
To establish an automated and robust process to generate polymersomes reproducibly and with very low polydispersity, a microfluidic system was then engineered (Fig. 4). Indeed, microfluidic strategies have been successfully employed to fabricate soft micro- and nano-assemblies54,55 To this aim, polytetrafluoroethylene (PTFE) tubing were secured with gaskets inside the screw-tight caps of a 3-way connector. An ethanolic solution (12 mg mL−1) of mPEGA12b-b-CHB36 maintained at 50 °C was injected via pump syringe at 10 μL min−1 (Flow A, Fig. 4) into a junction where DI water entered via another ID 0.5 mm tube at a flow rate of 120 μL min−1 (Flow B, Fig. 4). The mixed liquids (Flow C, Fig. 4) exited the junction via an ID 1 mm tube and were collected for size analysis. A range of A and B flow rates were tested in preliminary experiments to optimize the solvent mixing inside the connector. Polymersomes were obtained with this methodology at a rate of 0.12 mg min−1, and were analysed by DLS before and after ethanol evaporation.
![]() | ||
Fig. 4 Automated assembly of mPEGA12b-b-CHB36 polymersomes: schematic representation of the microfluidic device utilised in this study. |
The average size in the two cases was found to be very similar, with a diameter for the vesicles in pure water of 37 ± 7 nm and a PDI of 0.15 ± 0.04. One of the major advantages offered by this technique is the possibility of finely tuning the ratio of the two solvents in the device by independently modifying the solvent flow rates via syringe pumps, and to minimise the use of organic solvent required to induce the formation of homogeneous vesicles. The accurate control of the flow rate also resulted in very consistent batch to batch reproducibility (Fig. S17 ESI†), in a process that could easily be scaled up for large scale production.
Finally, the efficiency of the in-line assembly process to incorporate hydrophilic and hydrophobic drugs within the vesicle structures was then investigated. In general, therapeutics are entrapped within polymersomes either during their preparation, or by introduction of drug molecules into pre-formed vesicles.21 For the latter, injection56 or electroporation57 techniques are typically employed. In the former case, hydrophobic molecules can be incorporated into polymeric films utilised to prepare polymersomes, whilst hydrophilic drugs can be introduced into the aqueous phase used to rehydrate these films.58 Other commonly used techniques include solvent exchange mediated encapsulation,59 and the use of microfluidics to load small molecules or proteins while forming vesicles.60
In this present study two model molecules, terbinafine, a hydrophobic antifungal drug which is very poorly soluble in water in its free base form (<2 μg mL−1, as assessed by RP-HPLC, data not shown), and hydrophilic cyanocobalamine (Vit B12, solubility in water ca. 10–11 mg mL−1)61 were chosen as model drugs for incorporation within vesicle lipophilic membrane and aqueous lumen, respectively. In addition to its hydrophobic nature, terbinafine was chosen due to the presence of a tertiary alkyl amine in its structure, which by forming ionic pairs with the weakly acidic 3-chloro-4-hydroxybenzoic ester repeating units (pKa 7–7.5)36 in the hydrophobic blocks of mPEGA12a-b-ACH36 and mPEGA12b-b-CHB36 could lead to more efficient drug entrapment. This phenomenon was observed in our previous studies using analogous block copolymers, where we utilised tamoxifen, a hydrophobic drug with a basic tertiary amine group, which gave drug loading capacity as high as 18%.36 In typical experiments, at the end of the polymerisation reaction an ethanolic solution of terbinafine free base (35 mg mL−1) was added via syringe to the polymerisation mixture to give a 10% w/w drug/polymer solution, at 40 °C. Cyanocobalamine was dissolved in the aqueous medium used for the subsequent assembly of mPEGA12a-b-ACH36(6) and mPEGA12b-b-CHB36(7)-based polymersomes, again in a 10% w/w drug/polymer ratio. The polymer–terbinafine solutions were then cannulated into water–cyanocobalamine solutions, to generate the desired drug-loaded mPEGA12a-b-ACH36 and mPEGA12b-b-CHB36-based polymersomes. As observed in previous experiments, rapid self-assembly into vesicles with no visible precipitation was observed, suggesting that the method was robust, and that rapid formation of polymersomes under these conditions was not negatively affected by the presence of loaded molecules. The excess of cyanocobalamine was removed by dialysis against water using a 6–8 kDa MWCO membrane, while traces of unentrapped terbinafine were subsequently removed after the dialysis step by centrifugation, followed by filtration through a 0.45 μm filter. Drug loading was quantified by RP-HPLC (see ESI†) on the resulting purified vesicles.
Both mPEGA12a-b-ACH36 and mPEGA12b-b-CHB36, block copolymers displayed a higher cyanocobalamine loading capacity (LC) (Table 2). Interestingly, the acrylamide based polymer mPEGA12b-b-CHB36 showed better drug encapsulation for both the hydrophilic cyanocobalamine and lipophilic terbinafine. Vesicles were analysed by DLS (Fig. 5) to investigate how drug loading affected their size and size distribution. As we observed earlier with similar polymers,36 loaded polymersomes showed a smaller hydrodynamic radius than the empty assemblies, decreasing to ∼72 nm for mPEGA12a-b-ACH36 while an increase in size to ∼80 nm was observed for mPEGA12b-b-CHB36, as assessed by DLS analysis.
One possible explanation for this phenomenon is a different membrane packing caused by the presence of terbinafine and potential ion pairing between the drug and the weakly acidic 3-chloro-4-hydroxybenzoate of ACH and 3-chloro-4-hydroxybenzamide of CHB polymer repeating units. The observed high entrapment efficiency of cyanocobalamine may be due, at least in part, to additional electrostatic interactions between the partially deprotonated 3-chloro-4-hydroxybenzoate/benzamide repeating units and positively charged CoIII centres.
Similarly to click techniques where sub-families of chemical reactions are selected based on a range of favourable features, and PISA, where monomers and macromolecular parameters are chosen to direct supramolecular assembly, our results suggest that when appropriate reagents and conditions are utilised, in line processes could become a viable general route for efficient, scaleable and cost-effective assembly of polymersome nanovectors.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6py01292b |
This journal is © The Royal Society of Chemistry 2016 |