Effect of polymer architecture and tunable PEG content on micro-structuration of polymeric nanoparticles

Rabanel, Jean-Michel (Faculté de pharmacie, Université de Montréal)
Hildgen, Patrice (Faculté de pharmacie, Université de Montréal)
Banquy, Xavier (Faculté de pharmacie, Université de Montréal)

Introduction

Poly(ethylene glycol) grafted on polyesters polymers, such as PEG-PLGA diblock, have proved their usefulness in the preparation of nanosized drug delivery systems and other biomedical devices [1]. Pegylation have been shown to be of paramount importance to increase the in vivo efficacy of encapsulated drugs. Surface PEG coverage-density has been related to plasma protein repellency, increased circulation time and modified biodistribution [2]. The introduction of PEG chains in the polymer have been proposed through different architectures. The architecture could play a role in modifying not only the device surface but also the bulk properties. The polymer organization is hypothesized to have consequences on the structuration of particulate drug carriers. To test this hypothesis, we proposed to generate a library of polymers composed of diblock and comb copolymers of poly(lactic acid) and PEG chains. From these well-characterized materials, we prepared nanoparticles (NP) with different PEG content by nano-precipitation. NPs were characterized for their size while surface PEG coverage-densities were determined by NMR. Electronic microscopy techniques were used to characterize the internal structure of the particles.

Materials and Methods

Polymer synthesis. Bz-g-PLA (PLA grafted with pendant O-benzyl groups) were synthesized by random ring-opening copolymerization of D,L dilactide in presence of variable molar ratio of Benzyl glycidyl ether (BGE). After polymer purification, alcohol pendant group were uncovered by catalytic hydrogenation to yield OH-g-PLA. Methoxy-PEGs 2kD were attached at different grafting densities by acylation (0.5-2 % mole of lactic acid monomer) on OH groups to yield PEG-g-PLA. PEG-PLA diblocks were synthesized by ring-opening polymerisation using Methoxy-PEG2000 as a macro-initiator of the polyesters chains. The molecular weights of the polymers were characterized by GPC, 1H NMR, 13C NMR and 2-D 1H/1H (Bruker, 400 MHz) experiments were performed to asses polymer structure and calculate mass fraction of PEG as well as y (number of grafted chains of PEG per polymer backbone chain), as described earlier [3]. Nanoparticle preparation. NPs were prepared by nanoprecipitation. Briefly, polymers were dissolved in acetone (20 mg/ml) followed by a transfer into an aqueous phase (8 mg/ml final concentration) under stirring. After solvent removal by evaporation and dialysis, NPs were kept in suspension at 4oC until use. The NP size was determined by photo correlation spectroscopy on a Malvern Zetasizer (Malvern, UK). Surface PEG quantification. NPs prepared by nanoprecipitation in D2O were analyzed by 1H NMR, and surface PEG chains quantified using an internal standard. PEG coverage density was calculated based on the NP weight in sample measured by gravimetry, the DLS radius and on NP density assimilated to PLA polymer density (1.253 g.cm-3) determined by pycnometry [3]. Electronic microscopy. NP deposited on copper carbon grid were examined by TEM on a JEOL, JEM 2100 F. Dual beam FIB/SEM was performed on NP sample deposited on double side copper tape with a FEI Strata (DB-235) equipped with a Gallium ion source.

Results

Different analyses confirmed the structure of the polymers (fig. 1). BGE insertion ratio were found close to the feed ratio in the range tested. Different polymer batches were synthesized with different Mn and PEG content to generate a library of PEG-g-PLA polymers. Characterizations of some representative batches are shown in Table 1. PEG high coverage-densities, above brush regimen threshold, were found by 1H NMR analysis of NPs suspended in D2O (Table 2). The percentage of total PEG theoretically present in the particle found on the surface was between 65 and 85%. TEM at low dose allows the imaging of NP without staining. Characteristic core-shell structures were observed for NP prepared from PEG-g-PLA polymers. This was not observed for pure PLA or diblock polymers. Shell structure appeared with a slightly lower density to electrons compared to the particle core. Shell thickness was found to be around 10-20 nm. FIB/SEM image allow observation of the inside of the particle upon Gallium sputtering of surface polymeric material. Observations are showing the presence of region of lower electronic densities at the outer layer of the particle as well as on the internal face of cavities present inside the matrix.

Discussion and Conclusion

The obtained polymer architecture is characterized by a higher ratio of grafted chains number to backbone chains number in comparison with the most used diblock polymer architecture. NMR confirms non-ambiguously the presence of dense PEG coating on the NP surface. The observed structuration seen in TEM or FIB/SEM, seems to correspond to a PEG enriched region rather than to the PEG corona itself. The PEG corona is not expected to be dense enough to electrons to be directly visualized. The observed structuration due to self-organization upon nanoprecipitation may have implications on drug encapsulation, release and fate of particle. To our knowledge it is the first time FIB/SEM is used to elucidate the internal structure of polymeric drug delivery NP.

Figure 1. (A) Copolymer of BGE with dilactide (Bz-g-PLA); (B) after catalytic hydrogenation (OH-g-PLA); (C) after pegylation (PEG-g-PLA); (D) PEG-PLA diblock.

Table 1. Polymer characteristics

Table 2. Nanoparticle batch characteristics

Acknowledgements

JMR wish to thank the FRQNT (QC, Canada) and the Faculte de pharmacie for doctoral grants and research funding (Dean's fund, Faculte de pharmacie). XB thanks the financial support of the Canadian Research Chair program. Assistance of J.P. Masse and M.H. Bernier (Ecole Polytechnique, Montreal, QC) for TEM and FIB/SEM respectively is acknowledged.

References

1. Hrkach J, Von Hoff D, Ali MM, Andrianova E, Auer J, Campbell T, et al. Preclinical Development and Clinical Translation of a PSMA-Targeted Docetaxel Nanoparticle with a Differentiated Pharmacological Profile. Sci Transl Med. 2012;4(128):128-39. 2. Vonarbourg A, Passirani C, Saulnier P, Benoit JP. Parameters influencing the stealthiness of colloidal drug delivery systems. Biomaterials. 2006;27(24):4356-73. 3. Rabanel J.M., Aoun V. Hildgen P. Impact of Polymer Physico-chemical Properties on PEG-grafted-PLA Nanoparticles Structure, CRS Annual Meeting 2012, Québec, Canada 12-15 july 2012, Abst. 100649

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