Alban Petit

Philippe Richard

Ivo Cacelli

Rinaldo Poli
email: poli@lcc-toulouse.fr

Dr ] A Petit

Prof. I Cacelli

Prof R Poli

A Two-State Computational Investigation of Methane C-H and Ethane C-C Oxidative Addition to [CpM(PH3

Keywords: Open-shell organometallics, DFT calculations, pairing energy, spin-crossover reactivity, C-H activation, C-C reductive elimination

published or not. The documents may come

Introduction

It is now well appreciated that organometallic reactions may involve intermediates in a different spin state than the reagents and products. [1][2][3][4][5] For instance, species containing two unpaired electrons (triplet state) may be generated from diamagnetic reagents and species containing three unpaired electrons (quartet state) may originate from radical-like 17-electron reagents. Therefore, these reactions are characterized by spin crossover phenomena, with important consequences in terms of reaction rates and selectivities. In some of our recent work, we have examined the oxidative addition of C-H bonds to spin triplet 16-electron fragments, notably the reaction of methane with Cp2M, Cp*2M and ansa-CH2(C5H4)2M (M = Mo, W) [6,7] and the reaction of ethylene with CpIr(PH3). [8] From these studies, it is apparent that a proper rationalization of particular experimental observations (notably the relative rates of H/D scrambling and CH4 elimination for the Group 6 metallocenes, [9][10][11] and the coordinative vs. oxidative addition selectivity for the iridium system [12] ) could be achieved only through the explicit calculation of the so-called Minimum Energy Crossing Points (MECPs), [13][14][15][16][17] namely the points along the reaction coordinate where the system can be thought of crossing from one potential energy surface (PES) to the other one.

Amongst the most thoroughly investigated systems for the oxidative addition of hydrocarbon C-H bonds are the Group 8 CpM(PR3) 16-electron fragments (M = Rh, Ir). [18,19] These species are typically generated in situ by photolysis of CpM(PR3)H2 or by thermolysis of CpM(PR3)(R)(H) precursors with elimination of H2 or RH, respectively. [20][21][22][23][24][25] This process has also been extensively investigated at the computational level for the Rh and Ir systems, [26- 29] indicating that the 16-electron intermediates undergoing the oxidative addition process adopt a triplet ground state. None of the previously reported investigations, however, have examined the details of the crossing region and how these may affect the reaction rate. We have previously presented a computational investigation of CpCoL (L = PH3, C2H4), [30] but this focused on the ligand addition processes and did not address the possible oxidative addition of C-H bonds.

A related phenomenon that has attracted our attention is the oxidatively induced decomposition of 18-electron LnM(CH3)2 (M = Rh, Ir) complexes, namely Cp*M(PPh3)(CH3)2, [31][32][33] L3Rh(CH3)3 (L3 = 1,4,7-trimethyl-1,4,7-triazacyclononane), [34] and Cp*M(Me2SO)(CH3)2, [35] and especially the remarkably different outcome for the two metals.

Whereas oxidation of the Rh complex induces the reductive elimination of ethane for all the above mentioned compounds, the corresponding process on the Ir analogues leads to a different decomposition pathway, featuring the elimination of methane. Mechanistically, the decomposition process on the Rh systems seems to be controlled by the simultaneous bond breaking of the two metal-carbon bonds, whereas the Ir systems seems to decompose by ratedetermining bond formation processes. [35] However, the oxidation of Cp*Ir(PMe3)(CH3)H results in the exclusive elimination of methane and an oxidatively induced reductive elimination pathway was suggested in order to rationalize this behaviour. [32] From the energetic point of view, the reduced system favours the oxidative addition product M III (R)(H) for both Rh and Ir, whereas the oxidized system favours the reductive elimination product [M II ] + , in combination with the formation of C-C bonds for Rh and C-H bonds for Ir. To our knowledge, the C-H or C-C activation process involving the cationic system has not been previously examined from the computational standpoint. A reductive elimination process on a 17-electron complex leads to a 15-electron intermediate, with the potential involvement of a quartet PES. We considered it of interest to investigate and understand the factors determining the different energetic preference for the neutral and cationic complexes, leading to different decomposition pathways for the cationic dimethyl Rh and Ir systems. For these reasons, we have carried out calculations on the following three

Results and Discussion

(a) Methane C-H oxidative addition to 16-electron CpM(PH3).

This is the first comparative study of this reaction for all three Group 9 metal complexes and the first one reporting all critical points along the reaction coordinate, including the MECP. Previous investigations have addressed the thermodynamics of the methane addition to the Rh and Ir fragments using LCAO-HFS, [26] RHF, [27] MP2, [27,28] and MP4(SDTQ), [28] as well as selectivity issues for the oxidative addition of propane and cyclopropane to the same fragments using B3LYP. [29] The CpCo(PH3) has also been previously examined theoretically by B3LYP, but the reaction with methane was not addressed. [30] The present study also makes use of the B3LYP functional within the DFT methodology. All relevant critical points of the reaction coordinate for both singlet and triplet PES have been calculated for the three metal systems and are shown in Figure 1. Selected geometric parameters for the optimized structures are listed in Table 1.

< Figure 1 and Table 1> The 16-electron CpM(PH3) fragment is more stable in the triplet state for all metals.

The singlet-triplet gap is very similar for the Rh and Ir systems, whereas it is much greater for the Co system. These calculated gaps are close to those previously calculated at similar computational levels. [36] We do not intend to stress on the accuracy of the various methods for the estimation of this gap, a topic that currently attracts considerable attention. Rather, we shall focus on the trends shown by this gap as the metal is changed down the group and on the physical reasons that allow this trend to be rationalized. These arguments will be presented in the next section. Neither shall we dwell on the optimized geometries of the 16-electron fragments, since these have also been extensively discussed in the previous reports cited above.

The interaction of methane with the triplet fragment is repulsive in all cases. This is quite expected, since the C-H bond of methane is a very weak donor and does not effectively compete with the ligands that are already present in the coordination sphere in terms of providing electronic saturation to the metal center. [4,5] A similar situation was demonstrated for the repulsive addition of N2 to triplet CpMoCl(PH3)2 (whereas the CO addition is attractive), [37,38] for the repulsive addition of methane to the triplet metallocenes of Mo and W, [6] for the repulsive addition of H2 to spin triplet FeL4 substrates (L = CO, phosphine), [39] and more recently for the repulsive addition of H2 to W[N(CH2CH2NSiMe3)3]H. [40] The methane addition, on the other hand, is attractive to singlet CpM(PH3) for all metals. Again, this is expected because suitable orbitals in terms of energy, symmetry and occupancy are available in the singlet fragment for the establishment of the two key bonding interactions with the methane ligand:  donation of the C-H bonding electrons into the metal empty orbital and  back bonding from a metal lone pair to the empty C-H antibonding orbital.

For the cobalt and rhodium systems, the methane addition on the singlet PES leads to a local minimum that corresponds to the -CH4 complex. Somewhat unexpectedly, the interaction energy is slightly greater for cobalt (-8.4 kcal mol -1 ) than for rhodium (-5.6 kcal mol -1 ). The latter value is close to those previously calculated at other computational levels.

Contrary to some of the previously reported investigations, [26,27] but in analogy to others, [28] we could not locate a stable -complex minimum for the iridium system. Such a local minimum, if it exists, is likely to have a very low barrier to oxidative addition (see below).

The optimized geometries of the Co and Rh -CH4 complexes are similar, with the interacting C-H bond lying approximately parallel to the Cp plane. The M-X distance (see Table 1) is slightly shorter for the Co system, in accordance with the smaller size of the metal. The M-H distances are correspondingly shorter for the Co system, but the differential is much greater for the M-C distance (by almost 0.2 Å) than for the M-H distance (ca. 0.08 Å). Thus, it would seem that the greater stabilization for the Co system has its origin in the Co-C interaction.

The C-H bond is slightly more stretched in the Rh complex.

Continuing on the singlet PES, the -CH4 complex rearranges to the final methyl hydride product via a transition state (for Co and Rh) at about the same energy differential (ca. 8 kcal mol -1 ) from the -CH4 complex. The methane fragment is located comparatively much closer to the metal center in the case of Co-TS (the M-X differential being ca. 0.18 Å).

The C-H bond is further stretched relative to the -CH4 complex, the stretching being more pronounced for the Co system.

The product of oxidative addition, the methyl hydride complex, is energetically more stable than the separated methane and singlet 16-electron fragment for all three systems, but this stabilization is much greater for Ir (almost 40 kcal mol -1 ) than for the other two metals (ca. 10 and 12 kcal mol -1 for Co and Rh, respectively). The very small energetic difference between the oxidative additions to the singlet Co and Rh complexes may seem surprising when considering that homolytic bond strengths generally increase down a group of transition metals. However, the energetics of this process can formally be split into three components: (i) homolytic rupture of the CH3-H bond; (ii) promotion energy from the lowest energy singlet to an excited singlet (and not triplet, as other authors have often proposed) where the two electrons used for M-H and M-CH3 bonding are located in two different orbitals; (iii) formation of the M-H and M-CH3 bonds. The third component is expected to be less exoergic for Co, but the second one should be comparatively less endoergic because the coulombic repulsion is greater for the less diffuse 3d orbitals. [36] The same phenomenon is responsible for the greater triplet-singlet gap for Co relative to Rh. On going down further to Ir, the strength of the Ir-H and Ir-CH3 bonds continues to increase, whereas the energetic cost of the electronic promotion remains approximately the same as for Rh, since the Rh 4d and Ir 5d orbitals do not differ greatly in size.

Relative to the -CH4 complex, the methyl hydride isomer is stabilized to a greater extent for Rh (ca. 6 kcal mol -1 ) than for Co (ca. 1.7 kcal mol -1 ). Relative to the triplet ground state of the 16-electron fragment, the oxidative addition process is highly endoergic for Co, only slightly exoergic for Rh, and greatly exoergic for Ir. For this reason, the oxidative addition of hydrocarbons occurs for the Ir and Rh systems (with a greater bond selectivity for the Rh system as previously discussed), [29] whereas it has not been observed for the Co system.

From the point of view of the reverse reductive elimination process, the transition state is located at relatively low energy (9.5 kcal mol -1 for Co, 14.6 kcal mol -1 for Rh), leading to the -CH4 complex. In this respect, the Co and Rh PES's are rather similar. The minimum energy crossing point (MECP) leading to the triplet surface, however, is located at a very different position in the two cases. For the Co system, it is located very near the -CH4 complex, the two geometries being very similar (see Figure 1 and Table 1). In fact, crossing at the MECP occurs earlier (at a Co-X distance of 1.93 Å) than the -methane minimum (Co-X = 2.06 Å) and crossing can consequently occur quite readily. The methane reductive elimination process would not in any way be affected by the need to change spin surface. The reaction is solely dependent on the low barrier for the rearrangement to the -CH4 complex along the singlet surface. According to this picture, a hypothetical CpCo(PR'3)(R)(H) species is predicted to be unstable relative to the reductive elimination of the alkane. Indeed, no stable compounds with this stoichiometry appear to be known in the literature. Any such compounds would readily decompose to the alkane and triplet CpCo(PR'3), which would then undergo further transformation to stable products. Corresponding dialkyl complexes, however, are known and do not exhibit a strong tendency to reductively eliminate ethane, presumably for kinetic reasons: the energy barrier to C-C bond formation is likely to be much higher than that associated to the C-H bond formation, shown in Figure 1 (see a comparison of C-H and C-C reductive elimination barriers for the oxidized Rh and Ir systems in later sections). Reaction intermediates of type CpCo(PR3) have been investigated, [41,42] but no evidence for activity in C-H oxidative addition processes has ever been presented to be the best of our knowledge, in contrast to similar unsaturated derivatives of type CpCo(olefin). [43- 45] For the Rh system, the MECP is located at much longer Rh-X distance and its geometry resembles much more that of the separated CpRh(PH3) and CH4 fragments. The energy of this MECP is only slightly lower than that of the sum of the singlet fragments, and lower than that of Rh-TS. Thus, even though the -CH4 complex is expected to have a longer lifetime relative to the Co case, the reductive elimination process is still energetically controlled by the transition state along the singlet PES. From the experimental point of view, alkyl hydride complexes of type CpRh(R)(H)(PR'3) are unstable and decompose rather readily by reductive elimination, though -alkane intermediates have been shown to be involved, in certain cases, during the isomerization of isotopically marked products. [22,46] Finally, reductive elimination of methane from CpIr(PH3)(CH3)(H) proceeds without intermediate (according to the calculations), through a relatively high energy MECP which is located at a long Ir-X distance and close in energy to the separated methane and singlet CpIr(PH3) fragments. This situation is topologically similar to the crossover region for the Rh case, but the optimized geometry for Ir-MECP is different than that of Rh-MECP, with one C-H bond pointing at the Ir center in an end-on fashion rather than in a side-on fashion (see Figure 1). This calculation agrees with the experimental evidence that alkyl hydride complexes are thermally much more robust for Ir than for Rh. [25] It is necessary to mention that there is some experimental evidence for the intermediacy of -alkane complexes during the reductive elimination process [notably a competitive isotope scrambling process during the thermolysis of Cp*Ir(PMe3)(D)(C6H11)]. [25] It is possible that the shape of the PES around the -alkane complexes is sensitive to the coordination sphere, so that a local minimum exists for the real compound while it does not for the model system used for the calculations. The PES shape is also likely to depend on the computational level. As mentioned above, a minimum was indeed found at other levels. [26,27] At any rate, this minimum is likely to be a rather shallow one, with very low barriers to oxidative addition and to scrambling. 2.

<Scheme 1 and Table 2> All oxidized complexes exhibit a similar structure with a bent PH3 moiety relative to the CNT-M bond. The bending is more accentuated in the doublet state than in the quartet state by ca. 10°. The Co system behaves in a different way relative to the heavier congeners,

showing the quartet state as the most stable. In addition, the doublet cobalt system exhibits a significant spin contamination [<S(S+1)> = 1.18], corresponding to about 30% contribution of the quartet wave function and about 17% contribution of the quartet density. The rhodium and iridium analogues yield essentially uncontaminated states [<S(S+1)> = 0.76]. Since the Hartree-Fock calculation leads to an even higher value, this feature can not be ascribed to an inadequacy of the DFT method.

The reverse energetic order of the spin states obtained for the cobalt complexes and the marked spin contamination for the doublet induced us to perform more detailed investigations, especially for the doublet cobalt system. Firstly, in order to feel confident that we are dealing with the absolute minimum for each spin state, we have carried out other optimizations starting from different starting geometries and guess densities. Indeed, we have shown in a recent contribution how minima of a quite different nature can be obtained by slight modifications of the guess structure for the Cp2Cr system. [36] The geometry optimizations for the Co + -d and Co + -q systems were repeated starting the SCF procedure with densities obtained by single excitations on the best Slater determinant (i.e. the one giving the best density) and with distorted geometries. However, all our attempts in each spin state led to the same minima reported in Table 2. Only for the quartet Co system a different structure with an essentially linear CNT-Co-P moiety was occasionally optimized. However, this local minimum has a much higher energy than the quartet ground state and even higher than the doublet state reported in Table 2. The similarity of the optimized geometries for the three metal complexes in each spin state gives us further confidence that no lower energy minima exist for these ions.

During the optimization procedures in the doublet state, large spin contaminations were still observed, even for distorted geometries. The calculations, initially performed with a small basis set, where repeated with polarized functions (see Computational Details), again leading to very similar results for all systems in terms of bond distances, bending angles and quartet-singlet energy differences. Only the results obtained with the larger basis set are shown in Table 2. Secondly, we inspected the shape and energy of the outer orbitals for all systems (both doublet and quartet states for all metals). All show strong mixing between the metal d orbitals the and Cp π orbitals. In addition, these mixings do not show any regularity either along the metal series nor on going from doublet to quartet. On the other hand, this mixing is not completely unexpected if one considers the difference in the Ionization Potential (IP) of the ligands (12.4 and 9.9 eV for Cp and PH3, respectively) and of the metals (7.9, 7.5 and 9.1 eV for Co, Rh and Ir, respectively). These data lead to the expectation that, as verified in our previous calculations, [36] the outer orbitals will be essentially metal based in the neutral CpM(PH3) systems. For the corresponding cations, on the other hand, one has to consider the metal second IP (17 and 18 eV for Co and Rh, respectively). Therefore, the one electron levels of the metal are predicted to lie at the same energy of (or even below) those of the ligands. This may cause a strong orbital mixing even in the presence of weak bond orders between the metal and the Cp moiety.

We then turned to an analysis of the global properties on the basis of the whole density, rather than on the composition of the outer orbitals, in particular we considered the excess spin (ES = Nα-Nβ) on the ligands and on the metals. As expected, the PH3 moiety shows a very small ES in view of its closed shell nature, whereas the metal center carries most of the ES in all cases. The metal ES is 2.4 , 2.0 and 2.2 in the Co-Rh-Ir series for the quartet systems, while these values are 1.34, 0.67 and 0.68 for the corresponding doublets. Since Nα-Nβ = 1 for the doublet systems, the Cp ES is negative (-0.34) in the case of Co + -d, whereas it is about +0.3 for Rh + -d and Ir + -d. The ES on the Co + -d agrees with so called intra-atomic Hund's rule [START_REF] Szabo | Modern Quantum Chemistry[END_REF] which states that for an atom bonded with a doubly occupied orbital to another atom carrying unpaired electrons (positive ES), the ES is expected to be negative. Scheme 2 highlights a qualitative fragment orbital analysis of the most relevant covalent interactions between the Cp radical and the 10-electron [M(PH3)] + fragment of the d 8 Group 9 metal cation. These are (i) the -type interaction between the singly occupied Cp Huckel-type 3

orbital and the appropriate metal d orbital, and (ii) the -type back-bonding involving the empty Cp Huckel-type 5 orbital. For the lighter cobalt center, there are two key differences relative to the heavier congeners. Firstly, both interactions are weaker, disfavouring full spin annihilation for the-type interaction. Secondly, the pairing energy for the smaller 3d orbitals is greater, stabilizing the S = 1 state of the Co + center to a greater extent (Hund's rule). Thus, the Co system results in ES > 1 on the metal, and a slightly negative ES on the Cp ring. For the heavier metal systems, spin annihilation is more effective for the -type interaction. The reason why the ES on the Cp ring is positive for Rh + -d and Ir + -d is related to the greater contribution of the -type back bonding interaction.

<Scheme 2>

It is interesting to analyze the reasons for the oxidized [CpM(PH3)] + complexes to prefer the lower spin state for M = Rh and Ir (and the higher spin state by a small margin for the Co system), compared to the preference of the higher spin state by all the CpM(PH3) precursors (by a larger margin for the Co system). The relative stabilization of the lower spin state upon oxidation is very similar for the three systems, i.e. (EQ-ED) for the cation is greater than (ET-ES) for the neutral system by 25.9, 28.9, and 29.7 kcal mol -1 for Co, Rh and Ir, respectively. Using the qualitative concepts of orbital splitting and pairing energy under the monoelectronic approximation (Hartree-Fock theory), the singlet-triplet gap for the neutral system can be expressed as in Eq. 1, [START_REF] Simpson | [END_REF] whereas the doublet-quartet gap for the cationic system is given by Eq. 2. Each expression contains the difference between the one-electron energies of the two orbitals implicated in the electronic promotion process (see Scheme 1), and a term corresponding to the pairing energy. The latter contains a difference between an equal number of J integrals (one for Eq. 1, two for Eq. 2) and a certain number of K integrals.

Both J and K integrals have positive values, but Jii values (Coulombic repulsion between electrons in the same orbital) are always greater than Jij values (Coulombic repulsion between electrons in different orbitals), therefore greater values for J and K integrals lead to a stronger preference for the higher spin state. Conventional wisdom tells us that the J and K values should be greater for the cationic complex relative to the neutral one, because the one-electron oxidation process is predicted to lead to a greater effective nuclear charge and thereby to a contraction of the electron density around the nucleus. Because of this factor, the preference for the higher spin state should be more pronounced for the cationic system. On the other hand, the relevant orbital gap is greater for the cationic system (1-3, see Scheme 1) and this factor has an opposite effect to that of the pairing energy. The observation of a greater preference for the lower spin ground state for the cationic systems points to the dominant role of the orbital gap.

EST = ET -ES = 12 -PIST = (1-2) -[(J22 -J12) + K12] (1) EDQ = EQ -ED = 13 -PIDQ = (1-3) -[(J23 + J33 -J12 -J13) + (K12 + K13)] (2)
In a recent contribution, we have presented the results pertaining to the neutral system, [36] which were in qualitative and even semi-quantitative agreement with the trend of triplet-singlet gap down the group (Co >> Rh < Ir). We have now attempted to extend the same analysis to the cationic systems using the formula of Equation 2. Unfortunately, as dicussed above, going from the neutral to the cationic system results in a contraction and an energy lowering of the metal-based orbitals, in such a way that they end up in the same energy region as the M-Cp  orbitals. Therefore, there is extensive mixing between the various orbital contributions, which complicates the J/K analysis. This effect is further accentuated for the related 4+ cations (removal of all unpaired electron from the system is necessary in order to perform an unbiased J/K analysis). [START_REF] Simpson | [END_REF] An attempt to apply a localization procedure similar to that of Pipek and Mezey, [49] in order to retrieve essentially metal-based orbitals, gave unexpected results. The lowest unoccupied metal based orbitals were found to be of s and p character, whereas the d ones were pushed to rather high energies. Thus, the unoccupied d orbitals are rather different in the 4+ cation from those observed in the +1 system, because of relaxation effects that upset the orbital ordering and gives rise to excessive contractions. In conclusion, this system is unsuitable to the application of qualitative oneelectron considerations based on Equation 2. Therefore, we consider ourselves satisfied with the above qualitative argument, i.e. the greater stabilization of the lower spin state for the cationic systems is essentially attributed to the greatly increased energy gap between the relevant orbitals on going from the neutral to the cationic system (Scheme 1), which overshadows the expected pairing energy increase.

(c) Methane C-H reductive elimination from 17-electron [CpM(PH3)(H)(CH3)] +

This process has been investigated only for the experimentally more relevant Rh and Ir systems. Since the 15-electron [CpM(PH3)] + fragments, like the 17-electron methane oxidative addition products, [CpM(PH3)(CH3)(H)] + , adopt a doublet ground state for M = Rh and Ir (see previous section), the process occurs entirely along the low spin PES in both cases, without crossover phenomena, see Figure 2. It can be safely assumed that no quartet intermediates are involved, because the approach of a weak donor such as the C-H -electron density to a high spin system with no empty metal orbital is expected to be repulsive (see Figure 2), as discussed above in relation to the repulsive approach of methane to triplet CpM(PH3). The calculations were carried out at various fixed points along the coordinate defined by the M-X vector, X being the midpoint of the C-H axis. Selected geometric parameters for the critical point structures are collected in Table 3.

<Figure 2 and Table 3> It is interesting to compare the results in Figure 2 with the reductive elimination of CH4 from the neutral singlet CpM(PH3)(H)(CH3) complexes, shown in Figure 1. The process is much more favourable for the cationic complexes than for the corresponding neutral ones. It is endoergic by only 14.6 kcal mol -1 for [CpIr(PH3)] + relative to 39.3 kcal mol -1 for CpIr(PH3), and exoergic by 13.5 kcal mol -1 for [CpRh(PH3)] + relative to endoergic by 11.6 kcal mol -1 for CpRh(PH3). Thus, for each metal, one electron oxidation makes the methane reductive elimination more favourable by ca. 25 kcal mol -1 .

It is also interesting to note that the reductive elimination process proceeds via a -CH4

tautomer. For the cationic Rh system, the methane complex is more stable than the methyl hydride isomer by > 20 kcal/mole (cf. less stable by 6 kcal/mole for the neutral Rh analogue, see Erreur ! Source du renvoi introuvable.). Thus, as may be expected, oxidation favours formation of the isomer where the metal is formally more reduced. In line with Hammond's postulate, the transition state leading from the methyl hydride to the -CH4 complex is much closer in energy and geometry to the starting complex (reactant-like) for the cationic system.

This can be readily appreciated by comparing the optimized geometric parameters in Table 3.

Indeed, the C-H distance shortens only slightly from 2.136 to 2.125 Å on going from Rh + (H)(CH3) to Rh + -TS, whereas it shortens much more considerably from 2.388 to 1.479 Å on going from Rh(H)(CH3) to Rh-TS (see Table 1). For the cationic Ir system, the methane complex and the methyl hydride isomer are essentially isoenergetic, whereas a -CH4 was not located for the neutral system. The transition state lies ca. 7.5 kcal/mol higher than the methyl hydride complex and its optimized geometry is about midway between those of the two isoenergetic isomers (C-H = 1.542 Å). This is again consistent with Hammond's postulate.

Under the hypothesis that the putative -CH4 complex for the neutral Ir complex enjoys a similar energetic stabilization on going from the neutral to the cationic system, like for the Rh analogue, then the high energy of this putative intermediate along the reaction coordinate shown in Figure 1 would demand a very much product-like transition state. Since this intermediate is energetically unfavourable relative to the product of further methane elimination (positive slope along the reaction coordinate), a local minimum is no longer obtained.

The geometry of the cationic methyl hydride complexes differs considerably from that of the neutral precursors. It is a quite distorted relative to the ideal three-legged piano stool, the distortion consisting of a wide opening of the P-M-CH3 angle. It could more appropriately be described as a four-legged piano stool with a missing leg, in such a way as to place the hydride ligand in a transoid position relative to the missing leg. The CNT-M-H angle is significantly smaller than the CNT-M-CH3 and CNT-M-PH3 angles.

As a conclusion to this section, the one-electron oxidation of M(H)(CH3) induces a more facile reductive elimination of methane. For the rhodium system, the barrier to rearrangement to a methane complex is very low and the latter complex, although energetically more stable than the separate methane and 15-electron metal fragment, can easily react with better donor molecules (e.g. the solvent) and by additional redox processes, leading to thermodynamically stable species. Therefore, our computational study agrees with the literature proposition of an oxidatively induced methane reductive elimination from a variety of 18-electron Rh III (CH3)(H) complexes. [31][32][33][34][35] For the iridium system, the decomposition mechanism of the oxidized methyl hydride complex is essentially the same, in agreement with the experimentally observed selective formation of methane from the oxidative decomposition of Cp*Ir(PMe3)(CH3)(H). [32] However, the calculations indicate that the higher relative energy of the -CH4 complex imposes a higher energy barrier.

(d) Ethane reductive elimination from 17-electron [CpM(PH3)(CH3)2] + .

In the last part of this study, we now turn to the process of reductive elimination of ethane from [CpM(PH3)(CH3)2] + for M = Rh and Ir. The end point of the process in question is the 15-electron [CpM(PH3)] + species already analyzed in the previous two sections. The variable M-X parameter is now defined with X as the midpoint of the C-C axis and, like in the previous section, the entire reaction takes place on the spin doublet surface for both metals.

The energetic results and views of the molecular geometries at critical points are shown in Figure 3, whereas selected geometric parameters for the [CpM(PH3)(CH3)2] + minimum and for the transition state are given in Table 4.

<Figure 3 and Table 4> The geometry of the oxidative addition product shows the same distortion as the analogous [CpM(PH3)(CH3)(H)] + complexes analyzed in the previous section (cf. Tables 3 and4), one methyl group being transoid to the phosphine ligand and the second methyl group being cisoid to the same phosphine ligand (transoid to a vacant coordination position) in a pseudo-four-legged piano stool geometry. This transoid methyl ligand exhibits a longer M-CH3 bond and a smaller CNT-M-CH3 angle. From the energetic point of view, the picture is very close to that of the reductive elimination of methane from [CpM(PH3)(CH3)(H)] + . The reductive elimination is exoergic by 17.3 kcal mol -1 for the Rh system (cf. 13.5 kcal mol -1 for the corresponding methane elimination from the methyl hydride) and endoergic by 10.8 kcal mol -1 for the Ir system (cf. 14.6 kcal mol -1 ). Thus, elimination of ethane from the dimethyl complex is more favourable (less unfavourable) than elimination of methane from the methyl hydride complex by about 4 kcal mol -1 in both cases. A major difference between the two processes, however, is the absence of a distinct -ethane (C-C) complex. A number of other computational studies have addressed C-C bond formation processes by dialkyl reductive elimination, and in no case was an intermediate -C-C complex reported as a stable minimum. The process leads in all cases to C-C bond formation through a high energy transition state, like in the present case, without stable intermediates or, at the most, through an intermediate -complex that involves one or more C-H bonds, rather than the C-C bond. [50- 52] The energy of the system raises rather quickly from the dimethyl complex to the transition state, then remains high until the ethane molecule is relatively far from the metal center. The transition state is more "early" for Ir and more "late" for Rh, as indicated both by a shorter M-X distance and a longer C-C distances in the former case. This is again in line with Hammond's postulate, given the different reaction energetics. Relative to the transition state for the methane reductive elimination from [CpM(PPh3)(H)(CH3)] + , however, the energetic barrier is much higher (ca. 12 kcal mol -1 higher for the Rh system, ca. 23 kcal mol -1 higher for the Ir system). This difference is typical and is generally attributed to the directionality of the sp 3 lobe orbitals. [53][54][55] Also, the reverse process of C-C oxidative addition is notoriously more difficult than in the case of C-H bonds. [56][57][58] It seems that an important role is also played by the energetic stabilization of the 3c-2e bonding, which is stronger when the H atom is implicated rather than a methyl group, and leads to the presence of a distinct -C-H intermediate in the case of the methyl-hydride elimination process and its absence in the case of the methyl-methyl elimination process. It is notable, however, that the barrier for the ethane elimination from [CpRh(PH3)(CH3)2] + is much lower than that calculated for the same process on the neutral precursor (in excess of 55 kcal mol -1 , depending on the computational level). [50] Although the latter calculation was carried out by a quite different method from ours (RHF and RMP2), the large difference is certainly significant.

This indicate that, in agreement with the experimental observation, one-electron oxidation kinetically promotes the dialkyl reductive elimination process. In addition, the process is also promoted thermodynamically, since it is exoergic by 17.3 kcal mol -1 for the cation and engoergic by > 5 kcal mol -1 (at the RHF and RMP2 levels) for the neutral complex. [50] Even though the energetics for the neutral system were given relative to the slightly less stable singlet state of the product, [50] and keeping in mind the different computational levels, the difference between the above numbers is to be considered significant.

The transition state exhibits a similar but less pronounced distortion relative to the dimethyl complex. The cisoid C atom of the ethane molecule is located at essentially the same position as in the dimethyl complex, whereas the transoid one has moved to approach the first one. It is interesting to note the relative disposition of the two CH3 groups in the TS, where they already feel the steric H-H repulsion leading to the preferred staggered conformation of the free C2H6 molecule.

Coming now to the reactivity of the [CpM(PPh3)(CH3)2] + (and other isoelectronic) complexes of Rh and Ir, our calculations provide a framework of understanding for the observed difference. For the Rh system, the reductive elimination is a thermodynamically favourable process requiring a relatively low activation energy, through a 3-center transition state where the two Rh-C bonds break simultaneously with the formation of the C-C bond.

Thus, the oxidation of CpRh(PPh3)(CH3)2 is followed by unimolecular loss of ethane. The absence of a strong rate dependence on the solvent nature is consistent with the absence of a dramatic charge redistribution on going from reagent to transition state. Ethane reductive elimination for the dimethyl iridium complex, on the other hand, is thermodynamically unfavourable and requires a greater activation energy (ca. 30 kcal mol -1 ). Thus, the complex finds other pathways for its thermodynamically favourable decomposition, ultimately leading to different products.

Conclusions

The major outcome of this study is the analysis of how one electron oxidation modifies ground state for the 16-electron CpM(PH3) systems, can be rationalized on the basis of the dominant role of orbital energies, in a monoelectronic analytical approach.

Computational Details

The calculations were carried out within the DFT methodology using the hybrid B3LYP functional [59] as implemented in the JAGUAR program suite. [60] The LACVP** [61] basis set, consisting of polarized double-zeta basis functions with ECP's on the heavy atoms, was used.

In addition, for the calculations involving the oxidative addition/reductive elimination of methane, all the methane H atoms were described more accurately by the extended 6-311G** base. [62][63][64][65] The starting geometries for the various complexes were built from structural for Co, Rh and Ir, respectively, according to literature optimized values. [66] These calculations were carried out using the Gaussian03 suite of programmes. [START_REF] Frisch | Gaussian 03, Revision B.04[END_REF] MECPs were optimised using an ad hoc code [17] together with JAGUAR. The MECP optimisation procedure is based on minimising a generalised gradient found at any geometry by combining the computed energies and gradients at that point on the two potential energy surfaces. The gradient contains one term pointing towards the hyperspace in which the two surfaces intesect, and one term pointing towards lower energies within this hypersurface. The mixed Fortran/sheel script code creates JAGUAR input files for both spin states at a given geometry, calls JAGUAR, extracts energies and gradients from the output files, tests for convergence, and cycles.

Legends for Figures.

Figure 1. Reaction coordinate for the C-H oxidative addition of methane to CpM(PH3) (M = Co, Rh, Ir). The M-X distance in the abscissa is the distance between the metal centre and the midpoint of the C-H bond.

  model systems: (a) CpM(PH3) + CH3-H for M = Co, Rh, Ir; (b) [CpM(PH3)] + + CH3-H for M = Rh, Ir; (c) [CpM(PH3)] + + CH3-CH3 for M = Rh, Ir. The inclusion of the experimentally less relevant Co system is interesting, from the fundamental standpoint, for a detailed examination of trends in bond energetics and metal electronic parameters (orbital gap, pairing energy).

( b )

 b Relative stability of spin states for 16-electron CpM(PH3) and for 15-electron [CpM(PH3)] + : orbital and pairing energy factors. Removal of one electron from the 16-electron CpM(PH3) complexes produces the corresponding cations in two possible spin states: doublet and quartet. Scheme1 shows the qualitative energy diagram for both spin states in the neutral and in the cationic complexes, limited to the five d-based metal orbitals for the d 8 (neutral system) or d 7 (cationic system) electronic configuration of Co I and Co II , respectively. The optimized energies and key geometrical parameters of all complexes are listed in Table

  the potential energy surfaces for the methane C-H bond and ethane C-C bond oxidative addition processes that link the model CpM(PH3) systems to CpM(PH3)(CH3)(H) and CpM(PH3)(CH3)2, respectively, for M = Rh and Ir. Whereas, expectedly, the oxidative addition becomes less favourable (or reductive elimination becomes more favourable) upon oxidation, other observed variations were less predictable a priori. Many of these variations (e.g. the position of the MECP at a much greater M-X distance for the neutral Rh and Ir systems than for the corresponding Co system; the appearance of a well defined -CH4 minimum for the oxidized Ir system but not for the reduced one) can be easily understood on the basis of Hammond's postulate. The comparison of the C-H and C-C activation processes for the oxidized Rh and Ir systems shows that, although the systems are thermodynamically quite similar, the activation barriers are profoundly different because of a much less favourable 3-center-2-electron MC2 interaction in the C-C activation process relative to the analogous MHC interaction in the C-H activation process. When combined with the metaldependent thermodynamics (reductive elimination favoured for Rh, oxidative addition favoured for Ir), a reactivity profile in complete agreement with the experimental observations emerges: whereas the CpRhL(X)(Y) systems undergo oxidatively induced reductive elimination of X-Y when X = H and Y = CH3 and also when X = Y = CH3, the same process from the corresponding CpIrL(X)(Y) system is limited to methane. The elimination of ethane from the dimethyl complex is kinetically inaccessible, favouring other decomposition pathways. Contrary to our initial prediction, these reactions occur entirely on the spin doublet surface without spin crossover, whereas spin crossover phenomena are involved for the corresponding neutral systems. The reason for the preference of a low spin (doublet) ground state for the 15-electron [CpM(PH3)] + systems, at odds with the preferred high spin (triplet)

  parameters available in the Cambridge Crystallographic Database. For the oxidative addition of CH4 to neutral CpM(PH3) (M = Co, Rh, Ir) only stationary points on the singlet and triplet PESs (local minima and transition states) and the MECP (which is a stationary point within the seam of crossing of the two PESs) were optimized. The transition state on the triplet surface was not optimized, however, as it is not relevant to the chemical process. For the methane C-H and ethane C-C oxidative additions to the cationic [CpM(PH3)] + (M = Rh, Ir), the explorations of the potential energy surfaces along the reaction coordinates were conducted by keeping a representative parameter at various fixed values and by freely optimizing all other 3N-7 internal coordinates. This fixed parameter was chosen as the distance of the bond being formed/broken in the organic molecule (C-H for methane, C-C for ethane). In the results and discussion section, however, the coordinate is represented relative to another internal coordinate, namely the distance between the metal center and the point X located at the center of the fixed C-H or C-C bond, respectively.The geometry optimization of [CpMPH3] + (M = Co, Rh, Ir) were also performed by unrestricted DFT calculations using the B3LYP functional, coupled with LANL2DZ basis set on the metal, and 6-31G* on the carbon, hydrogen and phosphorus atoms. Further polarization functions were added on the H (one p function with exponent α = 1.1) and on the metal (one f function) centers. The exponents of the latter were chosen as 2.78, 1.35 and 0.938

Figure 2 .

 2 Figure 2. Reaction coordinate for the C-H oxidative addition of methane to [CpM(PH3)] +(M = Rh, Ir). The M-X distance in the abscissa is the distance between the metal centre and the midpoint of the C-H bond. The points marked with the energy value in parentheses are the stationary points. The other points derive from partial optimizations with a constrained C-H distance.

Figure 3 .

 3 Figure 3. Reaction coordinate for the C-C oxidative addition of ethane to [CpM(PH3)] + (M = Rh, Ir). The M-X distance in the abscissa is the distance between the metal centre and the midpoint of the C-C bond. The points marked with the energy value in parentheses are the stationary points. The other points derive from partial optimizations with a constrained C-C distance.

a

  The symbol M (Co, Rh, Ir) is used to represent the CpM(PH3) fragment; s = singlet; t = triplet; TS = transition state; MECP = minimum energy crossing point. b X = midpoint of the H3C-H vector. c CNT = cyclopentadienyl ring centroid.

a

  The symbol M (Rh, Ir) is used to represent the CpM(PH3) fragment; TS = transition state. b X = midpoint of the H3C-CH3 vector. c CNT = cyclopentadienyl ring centroid.

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Table 1 .

 1 Selected distance (Å) and angles (°) for the optimized structures shown in Figure1.

	Molecule a	M-X b M-CNT c M-P M-H	M-C	C-H	CNT-	CNT-	CNT-
						M-P	M-H	M-C
	Co(CH3)(H) 1.290 1.798	2.157 1.446 1.977 2.310 133.66 123.75 125.62
	Co-TS	1.602 1.793	2.165 1.448 2.040 1.500 131.84 131.87 129.74
	Co-MECP	1.928 1.750	2.131 1.659 2.312 1.153 133.43 133.68 128.17
	Co(-CH4)	2.034 1.782	2.154 1.790 2.387 1.120 133.85 129.65 128.47
	Co-t	1.915	2.227			142.92
	Co-s	1.753	2.158			141.11
	Rh(CH3)(H) 1.412 2.023	2.247 1.553 2.104 2.388 136.17 125.59 136.17
	Rh-TS	1.786 2.023	2.246 1.587 2.224 1.474 133.97 133.63 130.45
	Rh(-CH4)	2.148 2.011	2.232 1.842 2.561 1.200 133.91 133.10 131.56
	Rh-MECP	2.691 2.027	2.274 2.384 3.067 1.101 135.67 126.05 125.89
	Rh-t	2.045	2.305			138.75
	Rh-s	1.990	2.231			138.42
	Ir(CH3)(H)	1.424 1.992	2.232 1.580 2.122 2.425 135.03 126.56 124.48
	Ir-MECP	2.838 1.967	2.222 2.346 3.350 1.106 140.80 123.01 132.43
	Ir-t	2.002	2.238			140.34
	Ir-s	1.954	2.205			144.24

Table 2 .

 2 Selected geometric parameters and relative energies (in kcal mol -1 ) for the optimized [CpM(PH3)] + systems. Values obtained by the DFT calculation with an f polarization function on the metal center (see text). b The symbol M (Co, Rh, Ir) is used to represent the CpM(PH3) fragment; d = doublet; q = quartet. c CNT = cyclopentadienyl ring centroid.

	Co + -d	1.83	2.33	134.7	0.0
	Co + -q	1.89	2.42	146.8	-7.4
	Rh + -d	1.91	2.38	132.4	0.0
	Rh + -q	2.12	2.44	143.1	22.7
	Ir + -d	1.88	2.34	135.9	0.0
	Ir + -q	2.08	2.36	144.7	21.3

a Molecule b M-CNT c /Å M-P/Å CNT-M-P/° E/kcal mol -1 a

Table 3 .

 3 Selected distance (Å) and angles (°) for the optimized structures shown in Figure 2. .925 2.467 1.145 131.28 127.43 134.64 a The symbol M (Rh, Ir) is used to represent the CpM(PH3) fragment; TS = transition state. b X = midpoint of the H3C-H vector. c CNT = cyclopentadienyl ring centroid.

	Molecule a	M-X b M-CNT c M-P M-H M-C C-H	CNT-	CNT-	CNT-
				M-P	M-H	M-C
	Rh + (CH3)(H) 1.497 2.009	2.333 1.538 2.097 2.136 135.05 115.22 128.68
	Rh + -TS	1.503 2.008	2.338 1.537 2.100 2.125 135.51 115.21 128.72
	Rh + (-CH4) 2.283 1.929	2.346 2.150 2.533 1.111 129.62 129.22 135.05
	Ir + (CH3)(H) 1.435 1.997	2.310 1.571 2.093 2.337 135.54 114.02 129.50
	Ir + -TS	1.774 1.955	2.303 1.612 2.210 1.542 132.19 124.24 132.09
	Ir + (-CH4)	2.138 1.919	2.301 1

Table 4 .

 4 Selected distance (Å) and angles (°) for the optimized structures shown in Figure3.

	Molecule a	M-X b M-CNT c M-P M-C	C-C CNT-	CNT-
				M-P	M-C
	Rh + (CH3)2	1.603	2.031 2.335 2.142	2.741 134.53 113.66
			2.075		127.75
	Rh + (CH3)2-TS 1.963	1.968 2.344 2.268	2.111 130.22 121.99
			2.189		133.35
	Ir + (CH3)2	1.514	2.012 2.302 2.148	2.950 133.18 112.57
			2.078		130.04
	Ir + (CH3)2-TS	2.058	1.947 2.298 2.324	1.983 130.18 124.49
			2.243		134.37

Acknowledgements. We are grateful to the Conseil Régional de Bourgogne for funds used to upgrade the University's computing facility. RP also thanks CINES (Montpellier) and CICT (Project CALMIP, Toulouse) for granting free CPU time.

Scheme 1

Scheme 2

Text for Table of Contents

One electron oxidation modifies the potential energy surfaces for the methane C-H bond and ethane C-C bond oxidative addition processes that link the model CpM(PH3) systems to CpM(PH3)(CH3)(H) and CpM(PH3)(CH3)2. The observed changes concern both thermodynamic and kinetic parameters. The intimate details of the reaction coordinate depend not only on the oxidation state, but also on the metal nature (Rh vs. Ir) and on the small molecule being activated (methane vs. ethane).


Chandra P Sharma

Christine Vauthier
email: christine.vauthier@u-psud.fr

Mucoadhesive Nanoparticles for Oral Delivery of Insulin

Insulin was introduced into clinical practice of diabetes treatments soon after it's discovery in 1921. It is still the most effective and safe treatment option available. However, oral insulinotherapy remains a distant reality for diabetic patients, mainly because it is degraded in the gastro-intestinal tract and it is not absorbed under its active form by the gut. Applications of technologies from the nanomedicine represent an opportunity to bypass the bottle neck of the oral administration of insulin and more generally for peptide and protein drugs. Polymeric nanoparticles seem to be the most promising candidate for oral insulin delivery. They are considered as part of the relevant technologies of nanomedicine to achieve oral delivery of insulin. Firstly, they were shown to protect peptides from degradation in the harsh conditions of the gastro-intestinal tract. Secondly, because of their very small size they are believed to have a high potential to enhance drug transport across the barriers found in the gastro-intestinal tract. Aparts form the chemical and biochemical barriers, one of the physical barriers is the mucus that needs to be crossed before absorption sites can be reached on the surface of the epithelium formed by a monolayer of cells. This chapter focuses on the mucoadhesive properties of polymer nanoparticles. These properties are important to consider for the success of nanomedicine in oral delivery of insulin as they will give the formulation suitable properties to reach absorption sites. In a first part, mucoadhesive polymers are presented with their mode of interactions with mucus prior to their use to design mucoadhesive nanoparticles. The second part presents the different mechanisms and modes of action of mucoadhesive nanoparticles that are discussed considering examples from the literature.

Introduction

Insulin is used in therapy of both type I and type II diabetes. It is the only efficient treatment for type I diabetes. As most protein pharmaceuticals, insulin are currently delivered by invasive routes of administration including subcutaneous injections (Ramezan and Sharma 2009). The need to find a non-injectable form (i.e.: non-invasive route of administration) has focused on the oral route (Ramezan and Sharma 2009). Apart from safety and patient compliance, oral administration of insulin is of clinical relevance for the treatment of diabetes [START_REF] Arbit | The physiological rationale for oral insulin administration[END_REF]. Indeed, in a healthy human, physiologically secreted insulin from pancreas enters portal circulation first and inhibits the hepatic glucose production. In this process, insulin undergoes a metabolism in the liver to a significant extent (more than 50%) and thereby the excess glucose is converted and stored in the form of glycogen. Thereafter, insulin not used by the liver finds its way to the peripheral circulation. This mechanism reduces the chance of hypoglycemic effect. This is contrast with the distribution of exogenous insulin obtained after subcutaneous injection to diabetic patients. In this case, insulin distributes first in the peripheral circulation increasing risks of hypoglycemic effects and being ineffective to deliver the required amount of insulin to the liver to maintain their normal physiological function in glucose metabolism [START_REF] Arbit | The physiological rationale for oral insulin administration[END_REF]. Although oral administration of insulin is indisputably the best route of administration, all efforts put to develop suitable delivery formulation since its discovery remained unsuccessful because peptides and protein drugs are degraded in digestive media and are not absorbed as intact molecules by the intestinal epithelium [START_REF] Lee | A Penetration and enzymatic barriers to peptide and protein absorption[END_REF]. On the basis of the physiology, challenges to oral delivery of peptide and protein drugs include enzymatic degradation and poor permeation across the gastrointestinal tract (GIT) [START_REF] Lee | A Penetration and enzymatic barriers to peptide and protein absorption[END_REF].

Oral administration of insulin required a suitable formulation ensuring protection against degradation and helping absorption of the entire protein. Nanomedicine includes 4 emerging therapeutical approaches taking advantages of the small size of material used as drug carriers to help drugs to bypass biological barriers. It is a very active field of research on the oral delivery of insulin problem [START_REF] Ramesan | Challenges and advances in nanoparticle-based oral insulin delivery Expert[END_REF] .

Association of insulin with formulations used in nanomedicine improved dramatically the resistance of the hormone against degradation by proteases [START_REF] Madsen | Complexation graft copolymer networks: swelling properties, calcium binding and proteolytic enzyme inhibition[END_REF]. This is in favor to the success of the method as the slow rate of degradation may enhance the amount of proteins available for absorption across the GIT. However, insulin absorption by the epithelium still remains a limitation to the development of oral insulino-therapy (Ramazan and Sharma 2009). To achieve the expected benefit from nanomedicine, its necessary to develop nano-systems by optimizing their mucoadhesive properties and giving them other functionalities such as permeation enhancing and protease inhibition properties. The present chapter was aimed to summarize the different approaches used to improve mucoadhesive properties of polymer nanoparticles. The first part presents polymers which were used as mucoadhesives in pharmaceutical formulations. The second part summarizes works done on mucoadhesive nanoparticles including those devoted to improve oral delivery of insulin. Transport and translocation of nanoparticles through the epithelium is the subject of another chapter of this book by Woitiski and will not be discussed in the present chapter.

Mucoadhesion.

The term 'mucoadhesion' refers to adhesion between polymeric material and mucosal surfaces. Polymers, with certain structural features, become adhesive to mucus layer upon hydration. Though exact reason for adhesion may vary with the types of polymer, formulations containing mucoadhesive polymers are interesting because they provide intimate contact with the mucosal layer and thereby introduce drugs directly on the top of the enterocytes responsible for the highly selective permeability of the gut (Figure 1). 5 Thus, the close contact of the mucoadhesive drug delivery system with the mucus allows to increase of the drug concentration gradient at the surface of the epithelium which in turn favors drug absorption. The exact mechanism prevailing in mucoadhesion is still unknown but there are four well accepted theories that describe possible interactions involved in the mucoadhesion phenomenon (i.e:electronic, adsorption, wetting and diffusion theory) (Smart 2005, Ponchel and[START_REF] Ponchel | Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract[END_REF]. All are based on very define types of interactions occurring between the mucoadhesive polymer and mucus glycoproteins which are composed of highly glycosylated proteins with a high content of sialic acid and thiol groups. [START_REF] Cone | Barrier properties of mucus[END_REF][START_REF] Dodou | Mucoadhesives in the gastrointestinal tract: revisiting the literature for novel applications[END_REF]). In the electronic theory of 6 mucoadhesion, an electron transfer occurring between the mucus and the polymer leads to the formation of a double layer of electrical charges at the mucus/polymer interface.

According to the adsorption theory, secondary forces such as hydrogen bonding and Van der Waals interactions are largely responsible for the interaction between the mucus and the adhesive polymers. The wetting theory applies mainly to adhesives in the liquid states in which the ability of the adhesive to swell and spread on the mucus layer depends on the interfacial energy between the two partners. Contact angle measurement can be used to predict interactions of the polymers with the mucus layer. Lower contact angle favors mucoadhesion. In the diffusion theory, interpenetration and physical entanglement of the mucoadhesive polymers with the mucus layer are the key factors for the adhesion process. Phenomena involved in this mucoadhesive mechanism depends on the molecular weight, the degree of cross-linking, the chain length, the flexibility and the spatial conformation of both the polymer and mucins found in the mucus [START_REF] Smart | The basics and underlying mechanisms of mucoadhesion[END_REF][START_REF] Dodou | Mucoadhesives in the gastrointestinal tract: revisiting the literature for novel applications[END_REF]. None of these theories gives by themselves a complete description of the mechanism of mucoadhesion. In general, the global phenomenon of mucoadhesion results from a combination of the four theories with a balance between them which depends on the nature of the mucoadhesive polymer and on the nature and composition of the mucus. In an actual scenario, it can be expected that the polymer gets wet and swells upon contact with the mucosal surface (wetting theory). Thereafter, noncovalent bonds including electrostatic and hydrophobic interactions are created at the mucus-polymer interface (electronic and adsorption theory). The nature and strength of these noncovalent bonds are greatly influenced by the chemical nature of the mucoadhesive polymer. Then, the polymer and mucin chains interpenetrate and entangle together (diffusion theory), to further reinforce the mucus-polymer interactions [START_REF] Smart | The basics and underlying mechanisms of mucoadhesion[END_REF].

Polymers should have some general characteristics which will enable them to function as mucoadhesive material (Table 1). The most widely investigated groups of mucoadhesives are hydrophilic macromolecules containing large number of hydrogen bond forming 7 functional groups. The presence of hydroxyl, carboxyl or amine groups on the molecules are favorable to promote adhesion to mucosal surfaces. They are mostly called wet adhesives because they are activated by moistening and adhere to mucosal surfaces through non-specific interactions. Typical example includes polyacrylic acid, chitosan, sodium alginate and cellulose derivatives (Lowman and Peppas 1999). In general they are used as part of the components of a drug formulation designed to be administered by a mucosal route. In general, mucoadhesive polymers are included in conventional drug delivery formulations dedicated for mucosal routes of administration. At present, they are also widely considered for the development of technologies to be applied in nanomedicine including approaches for oral delivery of insulin.

Mucoadhesive Nanoparticles for Drug Delivery Applications

Polymeric nanoparticles are particles of less than 1 μm in diameter that are prepared from natural or synthetic polymers [START_REF] Hans | Biodegradable nanoparticles for drug delivery and targeting[END_REF]. Nanoparticles offer numerous advantages over conventional drug delivery systems as smaller surface to volume ratio is their salient feature. The high specific surface area promotes a better interaction of a nanoparticle with the mucosal surface compared to conventional drug delivery systems such as gels, patches, tablets or capsules. However, from an experimental point of view, the current methods for mucoadhesion measurements do not provide direct evidence of the nature of polymer-mucin interactions, when applied to a nano-particulate system. The methods currently reported mostly exploit custom-made or modified equipments, and there is no universally accepted test method available yet. Although same hold true for any mucoadhesive material the situation appears more complex in the case of nanoparticles [START_REF] Peppas | Nanoscale technology of mucoadhesive interactions[END_REF]. This is the reason that so few comparisons of inter-group data appeared in the literature and that the definition of "good" mucoadhesives still remains a debatable issue. 1999). Despite these shortcomings, nanoparticles prepared from known mucoadhesive polymers are now widely investigated for developing novel drug delivery systems, especially for proteins and other large molecular weight drug molecules. A classification in four groups can be suggested based on the nature and properties of the material used to prepare them as explained below.

-Nanoparticles from conventional mucoadhesive polymers -Mucus penetrating and diffusing nanoparticles -Nanoparticles obtained by the functionalization of conventional mucoadhesive polymers.

-Nanoparticles becoming mucoadhesive by surface erosion.

Nanoparticles from conventional mucoadhesive polymers

Conventional or so-called first generation mucoadhesive nanoparticles are largely based on poly(acrylic acid) (PAA) including their crosslinked derivatives such as Carbopol, and

Polycarbophil, chitosan (CS), alginate and cellulose derivatives . Mostly used as pharmaceutical excipients, these mucoadhesive polymers have also gained significance to formulate mucoadhesive nanoparticles.

Polyanionic polymers such as PAA operate mainly via non-covalent interactions with mucus including hydrogen bonding and Van der Waals forces (Table 1, Figure 2).

Hydrogen bonding is expected to take place between carboxylic groups of the polymer chain and the mucin glycoproteins (Figure 1).

10 1999). Other interesting features of mucoadhesive polymers composed of PAA are noteworthy regarding applications for oral delivery of protein and peptide drugs. PAA can act as calcium chelators resulting in calcium concentration depletion. This has two major benefits for oral delivery of protein and peptide drugs. First, depletion of calcium concentration in the gut inhibits proteolytic activity of the major proteases of the GIT, trypsine and chymotrypsine (Madsen and Peppas1999). Protein and peptide drugs may be protected against degradation and their therapeutic activity may be protected. Secondly, it disturbs cell-cell adhesion by loosening the epithelial tight junctions hence drug permeation is improved (Staddon et.al.1995, Lowman andPeppas 1999). Regarding oral delivery of insulin, several types of mucoadhesive formulations were prepared considering polyanionic polymer-based nano and microparticles. Results for in vivo investigations showed a clear dose dependent effect occurring 2 h after oral administration of the insulin-containing formulation in both healthy and diabetic rats while the effect lasted at least 6 h (Lowman et al. 1999, Lowman and[START_REF] Peppas | Nanoscale technology of mucoadhesive interactions[END_REF].

Other conventional mucoadhesive polymers used to formulate mucoadhesive nanoparticles are polysaccharides including chitosan (CS) and alginate.

12 Chitosan is a polycation prepared from chitin isolated from shrimps. Its strong mucoadhesive properties are explained by hydrogen and ionic bonds occurring between the negatively charged sialic acid residues of mucins and the positive charges of the amino groups of CS (Figure 1). Apart from the mucoadhesive properties, the polycationic form of chitosan is also able to open epithelial tight junctions enhancing the paracellular permeation of hydrophilic macromolecular drugs including insulin. It is noteworthy that the effect on the integrity of the epithelium and on the cell membranes of CS is much lower compared to that of known absorption enhancers [START_REF] Illum | Chitosan and its use as a pharmaceutical excipient[END_REF]. CS then appears as a much safer permeation enhancing agent in addition to its mucoadhesive properties. Mucoadhesive nanoparticles can be formulated with chitosan by ionotropic gelation with tripolyphosphates which are negatively charge oligomers of phosphate ions.

The formulations were proposed as drug delivery systems for oral administration of insulin. Efficacy of CS-nanoparticles to enhance oral absorption of pharmacologically active insulin was investigated by monitoring the glycemia of alloxan-induced diabetic rats. Results indicated that the CS-nanoparticles enhanced the intestinal absorption of insulin to a greater extent in comparison with an aqueous solution of CS. CSnanoparticles increased remarkably the relative bioavailability of insulin compared to a subcutaneous injection of an insulin solution. While the glycemia was reduced over a period of 15 hrs after administration of 21 IU/kg insulin in the CS-NPs, the average relative pharmacological bioavailability was up to 14.9 % [START_REF] Pan | Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo[END_REF]. By increasing the dose in insulin in the CS-nanoparticles up to 100 IU/kg, the glycemia was markedly reduced at 10 hours post-administration. Then, an interesting prolonged pharmacological effect lasted for up to 24 hours (Pan et.al. 2002). In another work, CS nanoparticles were prepared with hydroxypropyl methyl cellulose phthalate (HPMCP) which is a pH sensitive and gastroresistant polymer. The mucoadhesion of the nanoparticles was 2 to 4 times superior to that of the corresponding CS-tripolyphosphate nanoparticles.

Penetration of the nanoparticles in the mucosa was improved compared to that of the 13 reference CS-tripolyphosphate nanoparticles. The hypoglycemic effect of insulin obtained after peroral administration of the CS/HPMCP nanoparticles was also markedly increased. It was more than 9.8 and 2.8-folds compared to that produced by oral administration of a solution of insulin and of insulin-loaded CS-tripolyphosphate nanoparticles, respectively [START_REF] Makhlof | Design and evaluation of novel pHsensitive chitosan nanoparticles for oral insulin delivery[END_REF]. In an earlier work, HPMCP was considered as a coating material for CS capsules to design a delivery system for insulin to the colon (Tozaki et.al. 1997). A significant improvement of the oral bioavailability of insulin in rats was reported after oral administration of a dose of 20 IU. While the oral bioavailability compared to the intravenous route was 5.3%, the glycemia dropped down 6 hours after oral administration and the effect lasted for 24 hrs thereafter. Improvement of the performance of CS-nanoparticles can be obtained by modifying the chemical structure of the polysaccharide on its amino groups. For instance, trimethylation was aimed to improve the solubility of CS in aqueous solutions at pH approaching neutral values. This has dual advantages as the trimethyl chitosan (TMC) is protonated at pH above 6.5. The polymer chains are fully soluble in aqueous media of pH above 6.5 which is in contrast with that of CS which tend to aggregate in these media (Thanou et.al. 2001).

The TMC mucoadhesion is improved at the intestinal pH compared to that of CS. The second advantage concerns the effect of CS on the opening of the tight junctions which is very important to facilitate the paracellular transport of hydrophilic compounds including insulin. As only the protonated CS occurring at pH below 6.5 can trigger the opening of the tight junctions, TMC providing with a protonated form at pH above 6.5 found in the gut is suitable to improve paracellular permeability of the intestinal epithelium [START_REF] Thanou | Chitosan and its derivatives as intestinal absorption enhancers[END_REF] [START_REF] Tønnesen | Alginate in drug delivery systems[END_REF]Karlsen 2002, Sarmento et al. 2007).

After oral administration to rats, the superiority of alginate nanoparticles to improve bioactivity and absorption of insulin was attributed to the mucoadhesive properties of the formulation and to an improvement of the internalization of the peptide within the intestinal mucosa [START_REF] Sarmento | Alginate/chitosan nanoparticles are effective for oral insulin delivery[END_REF]).

Mucus Penetrating and Diffusing Nanoparticles

Efficacy of conventional mucoadhesive nanoparticles can be compromised by the interaction of the drug delivery device with soluble mucins found in the lumen of the GIT [START_REF] Peppas | Nanoscale technology of mucoadhesive interactions[END_REF]. Therefore, they are removed quickly from the intestine before they can reach the epithelium where drug absorption takes place. In order to counterbalance this effect, it was suggested to develop mucoadhesive formulations that bind specifically to the mucus layer. The rational behind this approach was to improve the inter-diffusion and inter-penetration of polymeric system in the mucosal barrier by reducing the interfacial energy and glass transition temperature of the parent polymer composing the nanoparticles. To this purpose, adhesion promoters were introduced in conventional mucoadhesive polymers. A typical example of adhesion promoter is composed of a poly(ethylene glycol) (PEG) containing macromer which can be easily incorporated in the mucoadhesive PAA/PMAA copolymer by copolymerization.

Resultant material possesses good chain mobility/flexibility in order to diffuse across the mucosal layer and avoid soluble mucin binding through a steric effect. PEG was also used to coat poly(lactide-co-glycolide) (PLGA) nanoparticles and poly(sebacic acid) nanoparticles [START_REF] Lai | Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues[END_REF]) (Figure 3). The diffusivity of the PEG-PLGA nanoparticle across the mucus was only reduced by 4

and 12 times respectively compared to the diffusivity evaluated for the same nanoparticles in water. In comparison, the corresponding non-coated nanoparticles were 3300 folds lower in mucus than in water. Results of diffusivity measurements suggest that surface modification of the nanoparticles can greatly improve diffusivity of the nanoparticles in the mucus layer overcoming the soluble mucus barrier. A new generation of mucoadhesive nanoparticles is about to emerge while it is now necessary to combine these findings with developments of nanoparticles showing tailored drug release profile [START_REF] Lai | Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues[END_REF].

As alternative polymers to PEG, polysaccharides are other interesting material to be used to decorate polymeric nanoparticles aiming to improve their mucoadhesion properties.

This approach was mainly developed in a series of core-corona nanoparticles composed of a poly(isobutyl cyanoacrylate)hydrophobic core surrounded by an hydrophilic corona composed of polysaccharides such as CS and dextran [START_REF] Bertholon | Bioadhesive properties of poly(alkylcyanoacrylate) nanoparticles coated with polysaccharide[END_REF][START_REF] Bravo-Osuna | Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles[END_REF]. Large amounts of the CS-coated nanoparticles were entrapped in the 16 mucus layer even at low nanoparticles concentration. With dextran-coated nanoparticles, the mucoadhesive phenomena was non saturable in contrast to what was observed with CS decorated nanoparticles. This finding agreed with the fact that CS may adhere to mucus components through electrostatic interactions where the number of sites is finite giving saturable phenomenon. It can be concluded that that bioadhesive properties of nanoparticles can be finely tuned by modifying surface properties of the nanoparticles.

Functionalization of nanoparticles made of conventional mucoadhesive polymers

Highly specific mucoadhesive properties can be given to the nanoparticles by decorating them with defined motives (Figure 4). The rational behind this is to promote adhesion of nanoparticle drug delivery system on a define portion of the intestine where absorption sites of the drug are located. Improvements of mucoadhesive properties are obtained by modifying surface properties of nanoparticles. This can be achieved by chemical or physical coupling methods of ligands interacting with mucus on already prepared nanoparticles or on the polymer prior to the synthesis of the nanoparticles [START_REF] Lehr | Lectin-mediated drug delivery: The second generation of bioadhesives[END_REF][START_REF] Wood | Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery[END_REF].

Lectins are typical examples of relevant ligands. They are non-immunologic glycoproteins recognizing very define sugar molecules [START_REF] Lehr | Lectin-mediated drug delivery: The second generation of bioadhesives[END_REF]. They can bind to glycosylated membrane components overlaying the GIT mucosa surface. For instance, the wheat germ agglutinin (WGA) extracted from plants can bind to N-acetylglucosamine and sialic acid residues found in the GIT. In vitro, it shows the highest binding rate to intestinal cells of human origin compared to lectins from other plant sources. Interesting studies were reported in the literature considering the use of lectins to improve specificity of mucoadhesion of nanoparticles in the GIT (Ponchel andIrache 1998, Gao 2006). Only a limited number of works have considered application of the approach on biodegradable nanoparticles and were aimed to improve oral delivery of peptides. Nanoparticles can be functionalized with lectins (A) or thiol (B) groups promoting specific interactions with define sugar residues of mucins or thiol groups of the cysteinrich region of mucin respectively.

A lectin modified PLGA nanoparticles loaded with a synthetic pentapeptide used in immunomodulation therapy, the thymopentin, showed an enhanced interactions with pig mucus by 1.8 to 4.2 folds compared with interactions measured in same conditions with the unmodified PLGA nanoparticles (Yin et.al. 2006). In another example considering oral insulin delivery, WGA was conjugated to alginate microparticles (Kim et.al. 2005).

The hypoglycemic effect of the WGA-decorated particles was the most pronounced.

However, it was not demonstrated that the effect was actually due to an effective 18 prolonged residence time in the GIT tract thanks to an improvement of the mucoadhesiveness of the particles. In another work, the mucoadhesive nature of PMAAgrafted-ethylene glycol hydrogel particles was enhanced with WGA thanks to specific interactions with carbohydrates found on mucins [START_REF] Wood | Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery[END_REF]) (Figure 1 and4A).

Improved mucoadhesive properties was accompanied by enhanced absorption of insulin as evaluated in vitro suggesting that the WGA decorated PMAA grafted ethylene glycol holds great promise as an oral formulation for the delivery of insulin improving diabetes treatments [START_REF] Wood | Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery[END_REF].

Another approach improving mucoadhesion with mucus is based on the use of thiol (sulphide) groups of the cystein rich region of mucins to promote strong and specific interactions with nanoparticles (Figure 1 and4B). In this aim, the nanoparticle surface is decorated with thiol groups. The rational is to enhance adhesion of nanoparticles on the mucus by formation of covalent disulfide bonds between thiol groups available on the surfaces of the two partners. The strategy was initially suggested by the group of Bernkop-Schnurch. (Bernkop-Schnürch et.al. 2003). It was widely used for the development of drug delivery platforms for oral administration of hydrophilic macromolecules such as insulin, heparin and salmon calcitonin (Bernkop-Schnürch et.al. 2003). Addition of thiol groups can induce transient and reversible increase of the permeability of the intestinal epithelium thanks to specific interactions with proteins of the tight junctions. This interesting effect was recognized to increase transport of drugs by the paracellular pathway and could be applied to improve oral bioavailability of protein and peptide drugs.

Thiol groups can be introduced on the surface of nanoparticles by coupling molecules such as cysteine, thioglycolic acid, cysteamine on mucoadhesive polymers like PAA, CS, alginate, carboxymethyl cellulose. Standard conjugation methods are suitable and the approach was recently applied on nanoparticles designed for oral delivery of insulin. In 19 general, results showed improvement of both the efficacy of the thiol modified drug delivery system and of the mucoadhesion. For instance, nanoparticles containing PAAcysteine increased 2.3 folds the area under the curve (AUC) of insulin administered orally to diabetic rats compared with the efficacy given by the corresponding non modified PAA formulation [START_REF] Deutel | Novel insulin thiomer nanoparticles: in vivo evaluation of an oral drug delivery system[END_REF]. Although the incorporation of cystein in one of the nanoparticle formulations improved the efficacy of insulin delivery, the relative bioavailability of the peptide remained extremely low (0.2 and 0.1 respectively for PAA-Cysteine and PAA systems) compared to the subcutaneous injection of insulin. In another example, thiol modification achieved by grafting of cystein on surface of poly(methacrylic acid)-chitosan-poly(ethylene glycol) (PCP) particles (diameter around 1 µm) significantly improved transport of insulin across Caco 2 cells and mucoadhesion on excised rat intestinal tissue compared with the unmodified PCP particles (Sajeesh et.al. 2010). In vivo, the thiol modified PCP particles were more effective in reducing blood glucose level in diabetic rats. The benefit brought by the introduction of thiol groups on the drug delivery systems to promote mucoadhesion and efficacy of oral administration of insulin was further confirmed in a recent work considering TMC nanoparticles. The introduction of cysteine residues in the structure of the TMC nanoparticles improved mucoadhesive properties by 4.7 folds compared with the non-modified TMC nanoparticles. This improvement correlated well with the increase of insulin transport through the rat intestine which ranged from 1.7 to 2.6 for the TMC nanoparticles and from 3.3 to 11.7 folds with the TMC-cysteine nanoparticles [START_REF] Yin | Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery[END_REF]). Further work is required to elucidate whether the improvement of the mucoadhesion was the only mechanism responsible for the increase of the oral efficacy of insulin delivered by the thiol modified nanoparticles or whether the permeation of the epithelium was increased because of the presence of the thiol groups. It is probable that both mechanisms have contributed to the enhanced delivery of insulin observed with the thiol modified particles but the part of each mechanism in this contribution remained to be determined. 20

Nanoparticles becoming mucoadhesive by surface erosion.

In general, hard bioerodible thermoplastic polymers such as polyesters and polyanhydrides are not mucoadhesives. However, they were found interesting potential mucoadhesive polymers to formulate nanoparticles for oral drug delivery applications.

Actually, polymers like poly(fumaric-co-sebacic) (PFASA) anhydride can rapidly degrade in contact with aqueous media relealing mucoadhesive properties thanks to the appearance of carboxylic acid groups resulted from the hydrolytic cleavage of the anhydride bond (Figure 5). The increase of the number of carboxylic acid groups appearing at the nanoparticle surface during degradation enhances the ability of the polymer to form hydrogen bonds with mucus and epithelial cell glycoproteins promoting the adherence of the nanoparticles on the mucosa. Mucoadhesive properties shown by such polyanhydride nanoparticles on small intestinal tissue of rats appeared stronger than those of all other nanoparticles made of other polymers [START_REF] Chickering | Poly(fumaric-co-sebacic) microspheres as oral drug delivery systems[END_REF]. In vivo, the transit of the 21 nanoparticles is delayed while the relative bioavailability of a drug can be improved. It was proposed that formulations including such hydrophobic polymers can be retained in the GIT and possibly increase intestinal absorption of drugs [START_REF] Chickering | Poly(fumaric-co-sebacic) microspheres as oral drug delivery systems[END_REF]. The prolonged residence time of particles in the GIT may increase the chance of particles to be taken up by enterocytes which were observed to cross the mucosal epithelium and the follicle-associated epithelium covering the lymphatic elements of Peyer's patches (Mathiowitz et.al. 1997). Particles of p(FASA) with a diameter around 5 µm were applied to the oral delivery of insulin on type 1 diabetic rats and dogs. Different bioavailabilities of insulin were observed depending on the type of animals and on the administration schedule In diabetic rats, the fed double dose experiment produced a relative bioavailability of 23.3% while the fed single dose experiment produced a bioavailability of only 5.5 ± 1.7% [START_REF] Furtado | Oral delivery of insulin loaded poly(fumaric-co-sebacic) anhydride microspheres[END_REF]. In fasted diabetic dogs, the relative bioavailability was 5.5 ± 3.4%. Oral delivery of active zinc insulin was also achieved with another type of particles formulated with a combination of PLGA and oligomers of fumaric acid as polyanhydrides. The formulation was able to control plasma glucose levels when faced with a simultaneously administered glucose challenge. The efficacy was 11.4% of that of an intraperitoneal delivery of zinc insulin. The rational behind the use of these polymers revealing mucoadhesive properties upon degradation is a tricky approach allowing to immobilize the drug delivery device in the mucus releasing high local concentration of drugs close to absorption sites of the intestinal epithelium.

Conclusion

Bioadhesive polymers are promising materials in designing drug delivery systems for mucosal barriers. However, oral delivery of proteins and peptides still remains a bottleneck. Attempts to improve oral delivery of proteins and peptides have been quite

unsuccessful so far in obtaining satisfactory and consistent results. For instance, a therapeutic protein like insulin used in the treatment of diabetes needs to be administered 22 on a daily basis with a very precise and robust delivery method able to regulate the pharmacological response at an optimum level. In this review, it was shown that technologies from nanomedicines can improve oral insulin delivery just by improving mucoadhesion of the nanoparticles. Although still insufficient, further improvements can be expected from a better optimization of the mucoadhesive properties and adding to the delivery systems other functionalities including permeation enhancing properties. A lot of work remained to be done but technologies from nanomedicine bring new hopes to succeed in obtaining an oral treatment of insulin available to diabetic patients.

Summary Points

 Penetration of drugs in mucus is required to reach absorption sites stranded on the gut epithelium  Mucoadhesion is a complex phenomenon that results from the combination of 4 main mechanisms.  Certain polymers exhibit mucoadhesion phenomenon.  Mucoadhesive nanoparticles promote oral delivery efficacy of active insulin  Mucoadhesion should take place in the mucus layer and not with soluble mucins of the lumen  Mucus penetrating/diffusing nanoparticles showed better mucoadhesive performance  Mucoadhesion of polymeric nanoparticles can be improved by specific modification approach  Thiol modification improves mucoadhesion of nanomedicine by formation of disulfide bonds with cystein rich region of mucins and confers enhancing permeation properties promoting drug absorption through the paracellular pathway.  Mucoadhesive technologies from nanomedicine bring true hope for the development of a treatment of diabetes based on oral insulino-therapy.

Key Facts

 Key facts on oral delivery of insulin: Oral delivery of insulin is facing many challenges due to the physiology of the gastrointestinal tract.  Key facts on nanomedicine technologies: Technologies from nanomedicines provide opportunities to improve drug delivery efficacy and targeting. It can make oral insulino-therapy a reality to patients in near future.  Key facts on mucoadhesion: Mucoadhesion promotes interactions of exogenous particles with mucus overlaying the epithelial cells in the gastrointestinal tract. This can help mucoadhesive nanoparticles to reach absorption sites found on the gut epithelium.  Key facts on designing mucoadhesive nanoparticles: Mucoadhesiveness of nanoparticles depends on nanoparticle surface properties which in turn govern the different types and strength of interactions promoted between particles and mucus components. Major difficulty here is to find a proper balance between strength of interactions allowing adequate adherence of nanoparticles onto the mucus and at the same time allowing particles to penetrate and diffuse through the mucus layer to access enterocytes. Modulating surface properties of nanoparticle delivery system seems to be the key in improving their efficacy towards oral insulin delivery.

Figure 1 :

 1 Figure 1: General structure of the gut mucosa and of mucins, main component of mucus.Cells in the epithelium are enterocytes (E), M cells (M) and goblet cells (G). Inserted frame:The different relevant chemical groups involved in mucoadhesion are indicated as well as the different possible specific interactions that can be promoted with mucoadhesive drug delivery systems.

Figure 2 :

 2 Figure 2: Interactions of conventional mucoadhesive nanoparticles with the gut mucosa. Nature of interactions is mainly nonspecific including non-ionic interactions between the nanoparticles and the mucus layer.However, drug delivery systems formulated with PAA often failed to demonstrate strong mucoadhesive efficacy in vivo. This is attributed to the ionization of carboxylic groups of PAA at neutral/alkaline pH of the Gut. Indeed, PAA which has a pKa close to 5.0 gets easily ionized at the intestinal pH hence reducing hydrogen bonding interactions with the mucus layer. Another minor effect is attributed to the rigid structure of the polymers. In some examples, PAA was used in combination with poly(methacrylic acid) (PMAA) to obtain hydrogel nanoparticles adding several other values to the mucoadhesive formulation(Lowman and Peppas 1999).

Figure 3 :

 3 Figure 3: Interactions of mucus penetrating and diffusing nanoparticles with gut mucosa. The nanoparticles are coated with polymer chains enabling the nanoparticles to diffuse through the mucus layer avoiding strong interactions with mucins.

Figure 4 :

 4 Figure 4: Interactions of functionalized nanoparticles with the gut mucosa.Nanoparticles can be functionalized with lectins (A) or thiol (B) groups promoting specific interactions with define sugar residues of mucins or thiol groups of the cysteinrich region of mucin respectively.

Figure 5 :

 5 Figure 5: Interactions of surface eroding nanoparticles with the gut mucosa. Typical example are poly(anhydride)-coated nanoparticles that reveal carboxylic group during hydrolysis.

8 Table 1 :

 81 Mucoadhesive polymersIn general, in vitro techniques currently used to evaluate mucoadhesion process involve destruction of the adhesive bond between the polymer and the mucus or tissue predominantly via application of mechanical force. It is obvious that many of these techniques are not applicable with nanoparticles. Therefore, it remains extremely difficult to investigate exact mechanism of nanoparticle mucoadhesion(Lowman and Peppas

	Polymers and	Type of	Mode of interaction	Advantage	Disadvantage
	functionalities	interactions
	Polyacrylic acid and	Non-specific	Carboxyl groups in these	Safe for oral delivery,	Low in vivo efficacy
	derivatives,		polymers make H-bonding with	non-specific binding with
	Carboxymethyl		the hydroxyl groups in the	the intestinal epithelium,
	Cellulose,		mucus layer	inexpensive
	Alginate
	Chitosan and	Non-specific	Amino groups in the polymer	Safe for oral delivery,	Poor solubility at physiological
	derivatives,		interact with the carboxyl group	electrostatic interaction with	conditions, strong interactions at
	amino dextran,		residues in the mucus layer	mucus layer	mucus layer, poor diffusion
	polydiaminomethyl				property
	methacrylates
	PEG and their	Non-specific	Non-adhesive in nature, works	Non-toxic,	Excellent in vitro results, poor in
	derivatives		as adhesion promoter when	no interaction with the	vivo performance
			used along with a polymeric	intestinal mucosal layer
			system such as hydrogel
	Lectins	Highly specific	Specifically binds to define	Specific binding,	Safety issues related to the
			sugar residues of glycoproteins	non-toxic and non-	repeated use, cost factor etc.
			found on the epithelial surfaces	immunogenic
			and in mucus
	Thiols	Specific	Make di-sulphide bonds with	Specific binding mechanism,	Poor in vivo results, stability
			mucus glycoproteins	ability of open epithelial	issue
				tight junctions
	Specific functions enhancing mucoadhesive properties and principal characteristics of polymer interactions with mucus.
				9

  . Promising results were obtained from TMC nanoparticles in experiments based on in vitro cell cultures of the cell monolayer model of Caco 2 (Thanou et al.

	Alginate is another polysaccharide use to formulate mucoadhesive nanoparticles for oral
	delivery of insulin. Most methods of preparation include an ionotropic pre-gelation of the
	alginate with divalent calcium followed by complexation with a polyelectrolyte. Calcium
	crosslinked alginate particles can protect peptides from gastric degradation by virtue of
	pH dependent release mechanism
	2001). Apart from nanoparticles obtained from TMC, other derivatives of CS were used
	to develop nano and microparticles for oral delivery of proteins. Typical examples
	included phthalate, succinate, PEGylated derivatives of CS.
	14

Abbreviations

Definitions

Copolymer: polymer made of at least two monomers Copolymerization: Method of synthesis of copolymer based on the polymerization of at least two types of monomer together.

Homopolymer: Macromolecule formed from one type of repeated unit Hydrogel: polymeric material formed by a tridimensional network of polymer chains swelled with water.

Macromer: monomer of high molecular weight.

Monomer: generally small chemical molecule which can polymerize meaning that they can be react together to form long polymer chains

Mucin: glycoproteins found in mucus

Mucoadhesion: adhesion to mucosa.

Mucus: hydrogel material composed of mucins found on the top of the epithelium in mucosa.

Nanoparticles: small polymer nanoparticles characterized by a diameter ranging from 1 to 1000 nm. Most nanoparticles designed as drug delivery systems have size ranging from 70 to 300 nm or around 1 µm for the largest.

Polymer: general term to design macromolecule resulted from the self addition of the same monomer unit or by the addition of several types of monomer units.