The formation of functionalized polyester nanoparticles containing amine, keto and allyl groups enabled the adaptation of the particles for the conjugation of bioactive building blocks, such as a dendritic molecular transporter and peptides. The versatile nature of the nanoparticle platform made it possible to tailor the particles to meet the needs of specific drug delivery applications. The versatile nature of the nanoparticle platform allowed for tailoring the particles to meet the needs of specific drug delivery applications (Chapter V).
The diamine cross-linking reagent, 2,2-(ethylenedioxy)bis(ethylamine), was specifically chosen to improve the hydrophilic and amorphous properties of the resulting particle. It was observed that during particle formation, the linear precursor and the formed particles were both soluble in dichloromethane and the reaction mixture remained clear for the duration of the cross-linking reaction. The chemical structure of the cross-linked particle and the correct proton resonance assignments are shown.
Parallel to the formation of the AB1 nanoparticles, a polynomial increase in the size of the ABC, ABD, and ABCD nanoparticles was observed as the amounts of diamine increased (Figure II-7). The allyl functionality was converted into epoxide units as one of the critical cross-linking entities to facilitate nanoparticle formation. The reaction of the epoxide units with diamine 2,2'-(ethylenedioxy)bis(ethylamine) led to the controlled preparation of nanoparticles, the size of which depended on the amount of diamine present during the cross-linking process.
An important change is the decrease of the epoxide protons at and 2.47 ppm and the appearance of signals at 3.64 and 2.97 ppm, corresponding to protons adjacent to the secondary amine of the PEG linker after cross-linking.
Nanoparticle formation using alkyne-azide click cross-linking
Both DLS and TEM emphasize the versatility of the alkyne-azide click approach to produce well-defined nanoparticles in narrow nanoscopic size dimensions, controlled by equivalents of bisazide. Using 1H NMR, the nanoparticle formation was further confirmed, as evidenced by a reduction of the signal at 2.03 ppm due to the alkyne proton and the appearance of the peak at 7.49 ppm due to the proton from triazole formation as a result of cross-linking (Figure III-2). The ability to control the size of the nanoparticles was investigated in more detail by performing a second set of experiments where the effect of increasing the amount of alkyne incorporated into the linear polymer from 5% to 12% was investigated.
For these reactions, the equivalences of the bisazide were varied from 2 to 8 azides per alkyne group in the polymer AC2, which contained 12% of the alkyne crosslinking unit, and it was found that this resulted in well-defined larger particles compared to those formed from AC1. It can be concluded that with the alkyne-azide cross-linking chemistry, the size of nanoparticles can be controlled both by the equivalence of the azide cross-linker and the percentage of incorporated alkyne entities in the linear polymer, in the same way as was observed. for the epoxide-amine crosslinking reaction.24 This has found another suitable reaction to carry out the intermolecular crosslinking in a controlled manner. By increasing the percentage of incorporated alkyne groups in the linear polymer, the size of the particles can be systematically increased, as was seen in the case of polymer AC2, which incorporated 12% alkyne groups.
As the crystallinity decreased with increasing nanoparticle diameter, a shift in melting temperature occurred from 42.0 °C to 41.8 °C. Therefore, by varying the alkyne-azide cross-linking reaction temperature, not only can the size of the nanoparticle be adjusted, but also the thermal properties of the particles can be changed. In addition to alkyne-azide chemistry, there has been significant development in click reactions that do not require a metal catalyst while exhibiting all the beneficial properties of the copper-catalyzed alkyne-azide click reaction, such as the thiol-ene click reaction.
The assembly of the nanoparticles using thiolene click coupling begins in a very similar way to that of the particles formed by the alkyne-azide reaction, with the synthesis of a low molecular weight linear copolymer, however with pendant allyl groups instead of alkyne units. Integration of the allyl moieties, the critical functionality for cross-linking, was accomplished by copolymerizing α-allyl-δ-valerolactone (b) with δ-valerolactone (A) via ROP as previously reported24 to give poly(valerolactone-allylvalerolactone) (poly) (vl -avl)), Ab1, with 5% allyl groups incorporated (Table III-1).
Nanoparticle formation using thiol-ene click cross-linking
As a result of varying the thiol equivalents, the particle size can be precisely controlled, as shown by DLS analysis (Figure III-4). By relating the particle size to the equivalents of dithiol used, it is apparent that there is a polynomial increase in the nanoscopic diameter. Both resonance shifts together with the reduction of allylic protons confirm the successful cross-linking of the dithiol with the linear polymer.
Important information about the morphology of the thiol-linked nanoparticles was inferred from their thermal properties collected through DSC. Increasing the duration of the cross-linking reaction from 12 to 24 h not only increased the particle size but also decreased the crystallinity of the particle. As a further evaluation of thiol–ene cross-linking efficiency, a series of reactions was performed at 45 °C for only 12 h, which remains a sufficient time to form discrete particles, with cross-linker equivalence other than 1.
The biocompatibility of the particles formed from thiol-ene cross-linking was evaluated using an MTT assay (Figure III-7B). By reducing the duration of the thiol-ene cross-linking reaction from 24 h to 12 h, the ability to form separated nanoparticles efficiently in half the time was demonstrated in contrast to the alkyne-azide cross-linking reaction at 45°C. °C. The degree of crystallinity was determined by quantifying the enthalpy associated with the melting temperature of the nanoparticles.
After first capping the amine groups of the nanoparticles with N-acetoxysuccinimide, the modified particle and the peptide were solubilized in. incorporate a fluorophore for imaging purposes.
Nanoparticle formation from linear polyester precursor AbBD
The allyl resonance peaks were still present in the 1H NMR spectra of the particles and were found to be analogous to the resonances of the allyl functionalities in the linear precursor. The focus of the dendritic molecular transporter can be functionalized with 3-(2-pyridinyldithio)propanoic acid which is recognized as a valuable moiety in thiol exchange reactions and can be easily cleaved by reducing reagents, such as dithiothreitol (DTT). The conjugation strategy began by linking NHS Alexa Fluor® 594 to the free amine groups on the particles, which are present as a result of the diamine cross-linking.
These results underline the versatility of the thiol-ene reaction, which was found to be independent of the solvent chosen. Therefore, the free amines of the AbBD nanoparticle were first modified with the NHS Alexa Fluor® and. As a next step, the complementary biological functions of the targeting unit and the molecular transporter unit were combined in the same nanoparticle scaffold.
As a result, two strategies for the attachment of both the biological units, the dendritic transporter and the targeting unit have been developed. For the first approach, the free amines of the linear peptide GCGGGNHVGGSSV were capped with N-acetoxysuccinimide. After the reductive amination reaction was completed in the same manner as described for compound 3 and analyzed by 1H NMR (Figure IV-4 seen at the end of this chapter), a thiolene reaction between the allyl groups of the nanoparticle and the thiol group of the molecular carrier (Scheme.
1H NMR (300MHz, CDCl3\Me4Si): δ An important change is the appearance of the peak at 1.43 ppm due to the Boc protecting group. The solution remaining in the upper part of the concentrator tube was blue in color. After removal of the solvent under reduced pressure, the crude product was purified by RP-HPLC.
For the further development and optimization of the nanoparticles for clinical use, it has been shown that the kinetics of drug release can be controlled.
4 days
Untreated GIRLRG
3 Gy XRT GIRLRG
7 days4 days
7 days
After the injections were completed, the eyes were removed and dissected to examine the distribution. However, most of the nanoparticles in the retina were localized in the nerve fiber layer and the RGC layer (Figure V-15). The surface of the retina represented by the DiO marker was compared with the entire surface of the retina.
Therefore, it can be concluded that the particles provide a controlled release of brimonidine to lower intraocular pressure for the duration of the in vivo study. By achieving higher drug loading due to the increased hydrophobicity of the therapeutic, it is hypothesized that AB-NP-BIM will have a longer-lasting IOP reduction. Using 0.25 equivalents of amine per epoxide in the linear polymer, we obtained 240.8 nm particles with wider cross-links due to long-pegylated Jeffamine ® specifically for siRNA encapsulation.
As the reaction time progressed from 0 to 12 h, there was a significant decrease in the intensity of the allylic protons of the polymer (Figure V-19). Previous studies have demonstrated the success of brimonidine-loaded nanoparticles in lowering and maintaining IOP. Nanoparticle degradation was monitored by the change in molecular weight, as determined by SLS, with incubation time.
The degradation of the nanoparticles was monitored by the change in molecular weight, as determined by static light scattering, with incubation time. The solutions were given to the collaborator, who then performed the injections and the rest of the studies.
