Enzyme mimic to develop antioxidant nanoreactors : from synthesis to application

Reactive oxygen species have been implicated in various diseases, but attempts to find efficient antioxidant treatments for such conditions have met with only limited success. We have demonstrated the first successful global structure–activity relationship based on statistical analysis of all classes of copper complexes proposed to have a SOD like activity. We took into account all copper-based SODm with electronic and EPR parameters that have been published, and established the specificity of the geometry around the metal as an essential criterion for a highly active SODm. The model we obtained for highly active SODm is that a slightly distorted square planar geometry seems to favour high catalytic activity for 4-coordination sphere in agreement with the geometry of native SOD and for 5-coordinated SODm is square pyramidal, with a slightly distorted basal plane. We have successfully synthesized a highly active enzyme mimics (CuIIENZm). The detailed structure of CuIIENZm in solid state as well as in solution was studied by X-ray crystallography and Electron paramagnetic resonance. CuIIENZm is shown to a change from a di-copper complex in the solid state to a mono-copper complex in solution and obtained a square-planar conformation. Upon a weak ligation of the solvent molecule, it preserves a pyramidal structure, as established by the combined pulsed EPR and DFT analysis. In this respect, the flexible solvent coordination site of CuIIENZm can be related to its high SOD activity. The presence of a solvent molecule in an equatorial position may be related to the labile bridge present in native SOD between Cu and Zn centers, which breaks to allow the geometric change during the catalytic reaction. Therefore, the structural geometry of CuIIENZm can be related to the obtained structural model for highly active SODm based on SAR analysis. We developed a robust antioxidant nanoreactor based on the encapsulation of highly active CuIIENZm that permits higher encapsulation efficiency as compared to a nanoreactor containing the enzymes themselves. This is attributable to intrinsic properties of the CuIIENZm, such as improved solubility and elimination of the severe constraints required to avoid enzyme denaturation. CuIIENZm was encapsulated inside the aqueous cavity of polymeric nanovesicles generated by the self-assembly of PMOXA-PDMS-PMOXA amphiphilic copolymers. The mild, structure-preserving procedure used to encapsulate CuIIENZm had no affect on the size/shape of the vesicles, as indicated by light scattering and TEM, or on the metal-coordination sphere that served as a center for catalytic activity, as demonstrated by EPR and UV-Vis spectroscopy. The cellular interaction of CuIIENZm containing nanoreactors was studied with THP-1 cells such as internalization, cytotoxicity, and in vitro activity. Approximately 11 % of cells contained internalized nanoreactors after 24 h incubation even without a functionalized vesicle surface to support a targeted approach. The antioxidant nanoreactors showed minimal cytotoxicity after 24 h incubation compared to free CuIIENZm. The population of THP-1 cells containing the antioxidant nanoreactors was significantly increased after 48 h incubation, and a protective antioxidant effect (23 %) against oxidative stress by paraquat was established. As CuIIENZm is both a mimic of SOD and of catalase, it serves to detoxify superoxide radicals and related H2O2 inside vesicles, preventing the generation of ROS as a side effect. The well pronounced antioxidant activity of nanoreactors in cells clearly points to a unique method to provide enzyme- mimics based antioxidant therapy, whereby encapsulating mimics in polymeric nanoreactors avoids mimic degradation within biological compartments and simultaneously allows CuIIENZm to act in situ when concentrated inside nanometer-range polymer cavities. Compared with conventional drug nanocarriers made of liposomes or polymers, our system is more stable and combines the advantages of a polymer shield with an in situ active antioxidant compound. This type of nanoreactors is proposed to function as versatile antioxidants, able to escape the immune system, for medical applications. Development of this simple, robust antioxidant nanoreactor represents a new direction in fighting oxidative stress efficiently

Evaluation of the effects of nanoprecipitation process parameters on the size and morphology of poly(ethylene oxide)-block-polycaprolactone nanostructures

Nanoprecipitation is a straightforward method for the production of block copolymer nanoparticles for drug delivery applications. However, the effects of process parameters need to be understood to optimize and control the particle size distribution (PSD). To this end, we investigated the effects of material and process factors on PSD and morphology of nanoparticles prepared from an amphiphilic diblock copolymer, poly(ethylene oxide)-block-polycaprolactone. Using a Design of Experiments approach, we explored the joint effects of molecular weight, block length ratios, water volume fraction, stirring rate, polymer concentration and organic phase addition rate on hydrodynamic size and polydispersity index of the nanostructures and created statistical models explaining up to 94 % of the variance in hydrodynamic diameter. In addition, we performed morphological characterization by cryogenic transmission electron microscopy and showed that increasing the process temperature may favor the formation of vesicles from these polymers. We showed that the effects of process parameters are dependent on the polymer configuration and we found that the most useful parameters to fine-tune the PSD are the initial polymer concentration and the stirring rate. Overall, this study provides evidence on the joint effects of material and process parameters on PSD and morphology, which will be useful for rational design of formulation-specific optimization studies, scale-up and process controls.Peer reviewe

Systematic in vitro biocompatibility studies of multimodal cellulose nanocrystal and lignin nanoparticles

Natural biopolymer nanoparticles (NPs), including nanocrystalline cellulose (CNC) and lignin, have shown potential as scaffolds for targeted drug delivery systems due to their wide availability, cost‐efficient preparation, and anticipated biocompatibility. Since both CNC and lignin can potentially cause complications in cell viability assays due to their ability to scatter the emitted light and absorb the assay reagents, we investigated the response of bioluminescent (CellTiter‐Glo®), colorimetric (MTT® and AlamarBlue®) and fluorometric (LIVE/DEAD®) assays for the determination of the biocompatibility of the multimodal CNC and lignin constructs in murine RAW 264.7 macrophages and 4T1 breast adenocarcinoma cell lines. Here, we have developed multimodal CNC and lignin NPs harboring the radiometal chelator DOTA (1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) and the fluorescent dye Cyanine 5 for the investigation of nanomaterial biodistribution in vivo with nuclear and optical imaging, which were then used as the model CNC and lignin nanosystems in the cell viability assay comparison. CellTiter‐Glo® based on the detection of ATP‐dependent luminescence in viable cells revealed to be the best assay for both nanoconstructs for its robust linear response to increasing NP concentration and lack of interference from either of the NP types. Both multimodal CNC and lignin NPs displayed low cytotoxicity and favorable interactions with the cell lines, suggesting that they are good candidates for nanosystem development for targeted drug delivery in breast cancer and for theranostic applications. Our results provide useful guidance for cell viability assay compatibility for CNC and lignin NPs and facilitate the future translation of the materials for in vivo applications.Peer reviewe

Microfluidic preparation and optimization of sorafenib-loaded poly(ethylene glycol-block-caprolactone) nanoparticles for cancer therapy applications

The use of amphiphilic block copolymers to generate colloidal delivery systems for hydrophobic drugs has been the subject of extensive research, with several formulations reaching the clinical development stages. However, to generate particles of uniform size and morphology, with high encapsulation efficiency, yield and batch-to-batch reproducibility remains a challenge, and various microfluidic technologies have been explored to tackle these issues. Herein, we report the development and optimization of poly(ethylene glycol)-block-(ε-caprolactone) (PEG-b-PCL) nanoparticles for intravenous delivery of a model drug, sorafenib. We developed and optimized a glass capillary microfluidic nanoprecipitation process and studied systematically the effects of formulation and process parameters, including different purification techniques, on product quality and batch-to-batch variation. The optimized formulation delivered particles with a spherical morphology, small particle size (dH < 80 nm), uniform size distribution (PDI < 0.2), and high drug loading degree (16 %) at 54 % encapsulation efficiency. Furthermore, the stability and in vitro drug release were evaluated, showing that sorafenib was released from the NPs in a sustained manner over several days. Overall, the study demonstrates a microfluidic approach to produce sorafenib-loaded PEG-b-PCL NPs and provides important insight into the effects of nanoprecipitation parameters and downstream processing on product quality.Peer reviewe

Lipid-Polymer Hybrid Nanoparticles for Controlled Delivery of Hydrophilic and Lipophilic Doxorubicin for Breast Cancer Therapy

Background: Lipid polymer hybrid nanoparticles (LPHNPs) for the controlled delivery of hydrophilic doxorubicin hydrochloride (DOX.HCl) and lipophilic DOX base have been fabricated by the single step modified nanoprecipitation method. Materials and methods: Poly (D, L-lactide-co-glicolide) (PLGA), lecithin, and 1,2-distearoyl-Sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000 (DSPE-PEG 2000) were selected as structural components. Results: The mean particle size was 173–208 nm, with an encapsulation efficiency of 17.8±1.9 to 43.8±4.4% and 40.3±0.6 to 59. 8±1.4% for DOX.HCl and DOX base, respectively. The drug release profile was in the range 33–57% in 24 hours and followed the Higuchi model (R2,=0.9867–0.9450) and Fickian diffusion (n<0.5). However, the release of DOX base was slower than DOX.HCl. The in vitro cytotoxicity studies and confocal imaging showed safety, good biocompatibility, and a higher degree of particle internalization. The higher internalization of DOX base was attributed to higher permeability of lipophilic component and better hydrophobic interaction of particles with cell membranes. Compared to the free DOX, the DOX.HCl and DOX base loaded LPHNPs showed higher antiproliferation effects in MDA-MB231 and PC3 cells. Conclusion: Therefore, LPHNPs have provided a potential drug delivery strategy for safe, controlled delivery of both hydrophilic and lipophilic form of DOX in cancer cellsPeer reviewe

In vitro Evaluation of the Therapeutic Effects of Dual-Drug Loaded Spermine-Acetalated Dextran Nanoparticles Coated with Tannic Acid for Cardiac Applications

Myocardial infarction results in a massive loss of cardiomyocytes (CMs). Unfortunately, current therapies are unsuccessful in replacing lost CMs, and thus, there is an urgent need for innovative approaches. Here, a nanosystem based on spermine-acetalated dextran (AcDXSp) and encapsulating two drug compounds able to stimulate in vitro CMs proliferation is developed. The nanosystem is coated by deposition of a film constituted by tannic acid (TA) and Fe3+ ions. The coating with TA increases the retention of the nanocarrier in cell co-cultures of CMs and fibroblasts stimulated with transforming growth factor (TGF)-β, due to the high affinity of TA for components of the cardiac extracellular matrix. The system exhibits biocompatibility toward primary CMs and induces their proliferation, as indicated by the two-fold increase of CMs in the active cell cycle. At the same time, the presence of TA synergistically helps contrasting fibrosis by reducing profibrotic genes expression, such as collagen 1 and osteopontin, by approximately 80% compared to the control. Overall, the developed nanosystem demonstrates the capability to stimulate CMs proliferation and reduce fibrosis, showing potential benefits for future in vivo applications.Peer reviewe

Dual-Peptide Functionalized Acetalated Dextran-Based Nanoparticles for Sequential Targeting of Macrophages during Myocardial Infarction

The advent of nanomedicine has recently started to innovate the treatment of cardiovascular diseases, in particular myocardial infarction. Although current approaches are very promising, there is still an urgent need for advanced targeting strategies. In this work, the exploitation of macrophage recruitment is proposed as a novel and synergistic approach to improve the addressability of the infarcted myocardium achieved by current peptide-based heart targeting strategies. For this purpose, an acetalated dextran-based nanosystem is designed and successfully functionalized with two different peptides, atrial natriuretic peptide (ANP) and linTT1, which target, respectively, cardiac cells and macrophages associated with atherosclerotic plaques. The biocompatibility of the nanocarrier is screened on both macrophage cell lines and primary macrophages, showing high safety, in particular after functionalization of the nanoparticles' surface. Furthermore, the system shows higher association versus uptake ratio towards M2-like macrophages (approximately 2-fold and 6-fold increase in murine and human primary M2-like macrophages, respectively, compared to M1-like). Overall, the results demonstrate that the nanosystem has potential to exploit the "hitchhike" effect on M2-like macrophages and potentially improve, in a dual targeting strategy, the ability of the ANP peptide to target infarcted heart.Peer reviewe