Concurrent Tissue Engineering for Wound Healing in Diabetic Rats Utilizing Dual Actions of Green Synthesized CuO NPs Prepared from Two Plants Grown in Egypt.

Purpose: Diabetes mellitus is among the disrupting factors of orchestrated events in wound healing. This necessitates the urge for tailored medications, which are continually offered by nano-sized materials. Herein, we present greenly synthesized copper oxide nanoparticles (CuO NPs), obtained from either Punica granatum L. (PG) or Pisidium guajava L. (GV) extract, to function as potent bactericidal and fungicidal materials that promote regeneration and healing of the targeted diabetic wounded tissues. Methods: PG or GV plant extracts were compared as source of reducing agents for CuO NPs synthesis process. The yield and photocatalytic degradation potential were compared. NPs obtained from the superior extract, PG, were characterized using particles size, zeta potential, XRD, TEM, SEM, and EDX. The antimicrobial effects were evaluated on multidrug-resistant human pathogens and then the percentage biofilm inhibitory concentration was determined. The cytotoxicity and wound scratch study were conducted on a normal human skin cell line. In-vivo wound healing activity in diabetic rats was assessed along with histopathological and immunohistochemical examination of CD45 and α-SMA. Results: The greenly synthesized CuO NPs are spherical in shape having a diameter of 233nm. CuO NPs (250µg/mL) acted as promising biocontrol agent against a variety of multidrug-resistant human pathogens. They significantly exhibited 29.460±0.811% healing of the scratched wound compared to only 2.001±0.155% for the control. Wound healing experiments revealed the safety of a low CuO NPs concentration in a diabetic animal model as well as on human normal skin fibroblast cell line. The treated group with a dose of 2mg/cm2 showed superior results with a WC50 value of 7.2 days, and 92% wound contraction after 13-days. Immunohistochemical investigation of the same group demonstrated well-established fibrous tissue (5.7±3.7/HPF), and an amplified granulation tissue of recently developed blood vessels (70±1.5/HPF). Conclusion: Green synthesized CuO NPs could overcome drug resistance and promote wound healing process effectively.

Graphene oxide crosslinked-zein nanofibrous scaffolds for prominent Cu-adsorption as tissue regeneration promoters in diabetic rats: Nanofibers optimization and in vivo assessment.

Diabetic ulcers are prone to bacterial contamination and can severely affect patient's quality of life. This study is first report to explore copper-grafted graphene oxide-crosslinked zein scaffolds (Cu-GZS) for promoting cutaneous excision wounds healing as a promising therapeutic modality in diabetic male-rats. Cu-GZS scaffolds were fabricated using electrospinning technique, where GO was employed as an echo-friendly crosslinker to meliorate mechanical stability and swellability of scaffolds. To circumvent risk of infection, copper ions were grafted into GZS as bactericidal agents and angiogenesis promoters, through soaking GZS nanofibers into Cu-solution or direct loading during electrospinning process. SEM images showed GO encapsulation by wrapping around/or trapping within nanofibrous. Interestingly, formation of chemical amide bond between zein and GO was proven by FTIR spectra. Crosslinking of zein nanofibers with GO increased tensile strength of nanofibrous by 3-folds compared to uncrosslinked zein nanofibers. Optimized Cu-GZS exhibited constant release rate of copper over a period of 8 days (~53.42%). Cu-GZS immensely accelerated wound closure demonstrated by diminished infiltration of leukocytes, absence of α-SMA positive cells, presence of fully intact epithelium with normal keratinization and accelerated wound size reduction, compared to control. Cu-GZS scaffolds could serve as promising biomaterials for effective topical wound healing in diabetic rats.

α-Bisabolol-Loaded Cross-Linked Zein Nanofibrous 3D-Scaffolds For Accelerating Wound Healing And Tissue Regeneration In Rats.

OBJECTIVES: Novel α-bisabolol (BIS)-loaded citric acid cross-linked zein nanofibrous scaffolds (C-ZNFs) were proposed to serve as safe platforms for promoting wound repair in rats. METHODS: ZNFs were synthesized using electrospinning technique, then NFs, with adequate water resistance, were produced using citric acid as a safe cross-linker. RESULTS: Compared to the uncross-linked ZNFs, cross-linking with 7% w/w citric acid decreased swelling index by 3 folds, while the tensile strength and the contact angle were enhanced to 2.5 and 3.8 folds, respectively. SEM images showed beads-free homogeneous NFs with a fully inter-connected 3D-network, where the average diameter of optimized C-ZNFs was 181.7±50 nm. After 24 h, C-ZNFs exhibited a decreased BIS release rate (45.6%), compared to uncross-linked mats (84.9%). By increasing BIS concentration, the cell adhesion (WI38 fibroblasts) was improved which can be attributed mainly to BIS activation of transforming growth factor-beta (TGF-ß1). The MTT-OD obtained values indicated that all tested zein scaffolds significantly enhanced the viability of WI38 fibroblasts, compared to the control after 48h of incubation which can be referred to the proliferative potential of zein by provoking cell spreading process. The scratch wound assay demonstrated that BIS-loaded ZNF scaffolds showed accelerated migration and proliferation of fibroblasts expressed by significantly higher wound closure rates compared to the control sample. BIS-loaded-C-ZNFs prominently accelerated tissue regeneration for wound closure demonstrated by entirely grown epithelium with normal keratinization and rapid wound contraction, compared to the control. Immunohistochemical results confirmed the superiority of BIS-loaded-C-ZNFs, where the observed reduced NF-κB and the elevated cytokeratin expressions confirmed the anti-inflammatory and proliferative effects of the scaffolds, respectively. CONCLUSION: In-vitro, optimized C-ZNFs offered a satisfactory cytocompatibility, adhesion and healing which were consistent with the in-vivo results. BIS-loaded-C-ZNFs could be regarded as a promising and effective biomaterial for tissue regeneration and for accelerating the wound healing process.

Lactoferrin-decorated vs PEGylated zein nanospheres for combined aromatase inhibitor and herbal therapy of breast cancer.

OBJECTIVES: Herein, we propose combined aromatase inhibitor and herbal therapy of breast cancer as a synergistic therapeutic modality. METHODS: Zein nanospheres were prepared by phase separation for co-delivery of exemestane and luteolin. To enhance their tumor-targeting capability, the nanospheres were coated with PEGylated phospholipids and lactoferrin for passive and active targeting, respectively. RESULTS: The developed nanospheres demonstrated a small particle size and controlled drug release. In addition, the nanospheres revealed high serum stability, acceptable hemocompatibility, and good physical stability. Moreover, a 5-fold scale-up of zein nanospheres could be enabled followed by spray-drying using 2.5% mannitol as a drying adjuvant. PEGylated and lactoferrin-targeted nanospheres showed enhanced cytotoxicity against MCF-7 and 4T1 breast cancer cells with higher selectivity to cancer cells rather than normal fibroblasts. The in-vivo pharmacokinetics and anti-tumor efficacy confirmed the superiority of zein nanospheres particularly after PEGylation compared to free drug(s). The enhanced anti-cancer activity of nanocarriers was revealed as prolonged circulation half-life, lower % change in tumor volume, reduced expression of aromatase, Cyclin D1 and VEGF markers as well as amplified apoptosis and necrosis. CONCLUSION: Overall, combined delivery of aromatase inhibitors and herbal drugs via tumor-targeted zein nanospheres could serve as a promising strategy for breast cancer therapy.

Shell-crosslinked zein nanocapsules for oral codelivery of exemestane and resveratrol in breast cancer therapy.

AIM: Oral administration of exemestane (EXM) and resveratrol (RES) for breast cancer therapy has been limited by their poor solubility and low permeability. METHODS: In this study, these issues were tackled using zein nanocapsules (ZNCs) for oral EXM/RES codelivery combining drug solubilization within oily core and resistance to digestion via hydrophobic protein shell. Furthermore, higher oral stability and sustained release could be enabled by glutaraldehyde crosslinking of zein shell. RESULTS & CONCLUSION: EXM/RES-ZNCs showed enhanced cytotoxicity against MCF-7 and 4T1 breast cancer cells compared with free drug combination with higher selectivity to cancer cells rather than normal fibroblasts. In vivo, crosslinked EXM/RES-ZNCs markedly reduced the percentage increase of Ehrlich ascites mammary tumor volume in mice by 2.4-fold compared with free drug combination.

HPLC Methods for Quantitation of Exemestane-Luteolin and Exemestane-Resveratrol Mixtures in Nanoformulations.

Two HPLC-DAD assays for the simultaneous quantitation of exemestane (EXE) and resveratrol (RES)-Mix 1-and EXE and luteolin (LUT)-Mix 2-in novel breast cancer therapy nanoformulations were developed. Calibration curves 15-30 µg/mL and samples were injected through an Inertsil ODS-3 (250 × 4.6 mm, 5 µm) column. The gradient elution for Mix 1 was methanol : 0.05% (v/v) acetic acid in water (60 : 40 to 80 : 20, linear over 2 min), and for Mix 2, it was methanol : water (60 : 40 for 4 min, then ramped linearly to 90 : 10, over 12 min) pumped at 1.5 mL/min for 4 min, then 1 mL/min till the end of run. EXE, RES, LUT and flutamide (internal standard (IS)) were measured at 246, 307, 350 and 300 nm, respectively. For Mix 1, RES, EXE and IS eluted at 3.5, 6.8 and 7.4 min, respectively, while for Mix 2, LUT, EXE and IS eluted at 7.5, 11.4 and 12.7 min, respectively. The mean r(2) for the standard curves was ≥0.99, and percentage coefficient of variation and % error of the mean were <2. Both assays successfully quantitated Mix 1 and Mix 2 in their nanoformulations. The two developed assays were sensitive and selective for the analysis of EXE-LUT and EXE-RES mixtures in nanoformulations according to International Conference on Harmonization guidelines.