Criterios para identificar el rol del Estado en la industrialización de la minería

Polymersome functionalization is useful to develop active targeting via specific receptor- ligand interaction for enhanced therapeutic efficiency as well as a diagnostic ability for especially drug delivery purposes.55 _{In addition, the integrated functional moieties can be}

favored in specific immobilization of the polymersomes onto solid substrates19, 87-88 _which

is important for designing microfluidic devices, e.g biosensors. In this respect, two common routes are available to incorporate functional groups to the polymersome surface. The first approach is the pre-functionalization method in which the desired molecules are incorporated to the block copolymer structures before the self-assembly process. Herein, the functional groups are equally placed at both inner and outer region of the bilayer membrane after the polymersome formation. In the second approach named as post-functionalization method, the functional moieties are conjugated to the polymersome surface after their formation (Figure 1.13).17, 54-55, 89-90 _{Depending on the polymersome structure, functionality}

(a) ( b)

20

can be integrated either inside or outside within this method, e.g. when stimuli-responsive vesicles are used.

In this regard, attachment of many recognitive ligands such as antibodies,91-93

peptides,94-95 _{folic acids,}90, 96 _{and sugars}89, 97 _{have been studied by using either pre-}

functionalization or post-functionalization methods. It is possible to conjugate the functionalities through covalent binding approaches like azide-alkyne click chemistry,17, 89, 98-99 _{and attachment via bis-aryl hydrazine bond.}34 _{Another way of conjugation is using non-}

covalent binding approaches including biotin-streptavidin binding,100-102 _adamantane-

cyclodextrin binding,103-104 _{and nitrilotriacetic acid (NTA) metal complexations.}105-106

Figure 1.13 Scheme showing the principle of pre/post-functionalization routes The group of van Hest has shown the use of click chemistry in polymersome field that illustrated both pre- and post- functionalization routes. In this study, azido terminated poly(styrene)-b-poly(acrylic acid) block copolymers were self-assembled to form the corresponding polymersomes. The further surface functionalization was carried out by conjugating different alkyne-modified ligands such as fluorescent dansyl probe, biotin, and enhanced green fluorescent protein (EGFP) in the presence of a copper catalyst (CuSO4.5H2O), sodium ascorbate and tris-(benzyltriazolymethyl) amine (TBTA) as a

stabilization ligand for copper species (Figure 1.14).17 _{The same group has also developed}

alkyne-functionalized polymersomes self-assembled from the mixture of polystyrene-b- Pre-functionalization

Post-functionalization

Functional Groups Self-assembly of functionalized BCs

21

poly(ethylene glycol) (PS-b-PEG) bearing an acetylene moiety and polystyrene-b-poly[L- isocyanoalanine(2-thiophen-3-yl-ethyl) amide] (PS-b-PIAT) block copolymers. The conjugation of azido-modified Candida Antarctica Lipase B (CalB) was subsequently performed which retained its activity while attached on this hybrid polymersomes.98

Figure 1.14 Clickable polymersomes self-assembled from azido-terminated PS-b-PAA block copolymers (pre-functionalization) and further post-functionalization with different molecules.17

Figure 1.15 Scheme illustrating the immobilization of biotin-functionalized PMOXA-b- PDMS-b-PMOXA based polymersomes onto a glass substrate through streptavidin binding for realizing a nanoreactor platform.87

As an example of non-covalent conjugation approaches, Hunziker and Meier et al. have reported biotin-functionalized triblock copolymers having the structure of [poly(2-

methyloxazoline)–b-poly(dimethylsiloxane)–b-poly(2-methyloxazoline)] (PMOXA-b-

PDMS-b-PMOXA) which was then self-assembled to form the respective polymersomes. In

CuSO4.5H2O, TBTA Sodium ascorbate Self-assembly

R = Biotin, dansyl probe, enhanced green fluorescent protein

ELF 97 substrate Streptavidin Acid phosphatase OmpF channel Biotin ELF 97 alcohol

22

this study they also linked the biotinylated ligands to these functionalized polymersomes through streptavidin coupling for cell-targeting purposes.100 _{In another study, the same}

conjugation chemistry was utilized to immobilize the biotin-possessing polymersomes onto glass substrates to realize a nanoreactor platform by performing enzymatic conversions for applications in the field of analytics like sensors. The glass surface was modified with biotin bearing bovine serum albumin (BSA) through microcontact printing followed by exposure with streptavidin to prepare the relevant conjugation platform for biotinylated polymersomes. As a model enzymatic reaction, acid phosphatase was encapsulated within the polymersomes to trigger the dephosphorylation of the fluorogenic substrate ELF 97. The enzyme was able to access the substrate through the protein F channels (OmpF) embedded

in the polymersome membrane that became permeable afterwards (Figure 1.15).87

Figure 1.16 Surface modification of polymersomes with β-cyclodextrin molecules by pre- functionalization and further post-conjugation of adamantane-modified PEG having different molecular weights.104

Another type of non-covalent conjugation is the host-guest complexation of adamantane and β-cyclodextrin (β-CD) molecules. This type of binding has been widely used in polymer chemistry since adamantane groups tightly fit into the cavity of β-CD molecules with a high association constant between 104 _{to 10}5 _M-1 _{leading to a strong host-guest interaction.}107-109

In this regard, Felici et al. has reported polymersomes decorated with β-cyclodextrin which was further conjugated to an adamantane modified enzyme, horseradish peroxidase (HRP),

CD-PI-CD

β-CD

Ada-PEGn

v

23

through the host-guest interaction mechanism. Although the non-modified enzyme was also able to interact with polymersomes, the adamantane bearing HRP showed increased affinity and retained its activity while conjugated to the polymersomes. In this study, the vesicle formation was performed by the self-assembly of polystyrene having the permethylated β- cyclodextrin compounds.103 _{Similar approach was also applied by Guo et al. by preparing a}

cyclodextrin-capped polyether imide (CD-PI-CD) that was self-assembled into polymersomes in water. As a guest molecule, polyethylene glycol with adamantane groups was utilized for conjugation to the CD-functionalized polymersomes. The successful host- guest complexation at both inner and outer region of polymersome corona was proven by using isothermal titration calorimetry (ITC) as well as static light scattering (SLS) measurements (Figure 1.16).104