## 6 PLGA Nanoparticles Functionalization
### BBB Transcytosis  
#### 6.2.1 Cell Penetrating Peptides  
Cell-penetrating peptides (CPPs), also known as membrane translocation sequences or protein transduction domains, are short cationic or amphipatible peptides with specific conserved sequences (between 7 and 30 amino acid residues) that have been widely used in drug delivery for their capacity of transporting cargoes into cells [132]. Indeed, they can traverse cell membranes and penetrate the BBB, and thus facilitate cellular uptake. However, their lack of specificity greatly restricts their application as pharmaceutical tools (unwanted peripheral effects may result), and hence methods of targeting CPPs are being investigated [97]. The incorporation of reactive maleimide groups on PLGA NPs allows efficient conjugation of CPPs to the surface of these PLGA NPs (Figure 8). Indeed, the maleimide-thiol reaction is frequently used for functionalization of particles because of its selectivity towards thiol groups at physiological pH, the formation of a thioether bond that is relatively stable, and the high reactivity of maleimide under mild conditions (i.e., at room temperature and using aqueous buffers) [133]. Besides, the thiol group of cysteine residues naturally present in peptides and proteins (or easily introduced in these molecules) facilitates this reaction [134]. Despite their wide application, there are still some limitations regarding the stability of maleimide-based linkers [135]. Indeed, it has been demonstrated that under certain conditions the Michael addition reaction is reversible [136; 137; 138]. Thiosuccinimides linkages are susceptible to undergo a retro-Michael reaction (deconjugation), which cleaves the thioether bond and thus reverts succinimide to maleimide and free thiol [139]. This leads to a lower conjugation efficacy and considerably decreased in vivo stability [140; 141], since the released maleimide may react with other thiol-reactive species, and the released thiol may react with other compounds in vivo [142]. EDC-NHS coupling can also be used to conjugate peptides to the NPs. Upon exposure of EDC/NHS to the carboxyl groups, reactive NHS esters are expected to be formed [143]. Therefore, when the peptide primary amine group comes into contact with the ester, a covalent amide bond is formed [144,145], allowing for efficient peptide conjugation. Angiopep-2 (Ang-2) is a promising ligand for targeted delivery to the brain. This peptide, composed of 19 amino acids derived from proteins known to interact with the LDL receptor related protein family, was identified and designed in 2008 [146]. Indeed, Ang-2 binds to LRP1, which is widely expressed throughout the CNS (by CNS endothelial cells, neuroblasts, microglia, astrocytes and neurons) [147, 148, 149, 150]. Moreover, this ligand is able to activate receptor-mediated transcytosis and cross the BBB due to the existence of a corresponding receptor of Ang-2 expressed on the brain endothelial cells. Therefore, this brain-targeting peptide has been conjugated to numerous nanocarriers in order to improve BBB crossing, including PLGA NPs [151]. Indeed, PLGA-PEG-Ang-2 NPs prepared by [PERSON] et al. [99] had sizes lower than 200 nm, were compatible with systemic administration, and enabled possible BBB crossing. In vivo analysis confirmed a clear brain accumulation in different areas (i.e., hippocampus and cortex), unlike nonmodified NPs and modified NPs used as controls, which were unable to overcome the BBB and enter the brain [99]. Consequently, these formulations show a promising brain drug delivery system and could be used as carriers of different drugs to the CNS, thus increasing the alternatives for the treatment of brain diseases like AD [99]. However, additional studies are needed to clarify the mechanism involved in the entry of Ang-2 NP into the brain or the cells [99]. PLGA-PEG NPs loaded with both S1 peptide (PQVGHL) and curcumin to target the damaging factors in AD development, and conjugated with the brain-targeting peptide CRT, were prepared [107].