Alhakamy, N. A., S. M. Badr-Eldin, O. A. A. Ahmed, H. M. Aldawsari, S. Z. Okbazghi, M. A. Alfaleh, W. H. Abdulaal, T. Neamatallah, O. D. Al-Hejaili, and U. A. Fahmy, "Green Nanoemulsion Stabilized by In Situ Self-Assembled Natural Oil/Native Cyclodextrin Complexes: An Eco-Friendly Approach for Enhancing Anticancer Activity of Costunolide against Lung Cancer Cells.", Pharmaceutics, vol. 14, issue 2, 2022. Abstract

Lung cancer is the second-most deadly malignancy worldwide, of which smoking is considered a major risk factor and causes 75-80% of lung cancer-related deaths. Costunolide (CTD) extracted from plant species exhibits potent anticancer properties, specifically in lung cancer and leukemia. Several nanoemulsions were prepared and optimized using a three-factor Box-Behnken experimental design. The optimized green nanoemulsion (GNE) showed a vesicle size of 199.56 nm. The IC values revealed that A549 cells were significantly more sensitive to the optimized CTD formula than the plain formula and raw CTD. A cell cycle analysis revealed that the optimized CTD formula treatment resulted in significant cell cycle arrest at the S phase. The results also indicated that treatment with the CTD formula significantly increased caspase-3, Bax, Bcl-2, and p53 mRNA expression compared to the plain formula and CTD raw. In terms of the inflammatory markers, the optimized formula significantly reduced the activity of TNF-α and NF-κB in comparison with the plain formula and raw drug only. Overall, the findings from the study proved that a CTD GNE formulation could be a promising therapeutic approach for the treatment of lung cancer.

Alhakamy, N. A., U. A. Fahmy, S. B. M. Eldin, O. A. A. Ahmed, H. M. Aldawsari, S. Z. Okbazghi, M. A. Alfaleh, W. H. Abdulaal, A. J. Alamoudi, and F. M. Mady, "Scorpion Venom-Functionalized Quercetin Phytosomes for Breast Cancer Management: In Vitro Response Surface Optimization and Anticancer Activity against MCF-7 Cells.", Polymers, vol. 14, issue 1, 2021. Abstract

Breast cancer is a dangerous type of cancer in women. Quercetin (QRT), a naturally occurring flavonoid, has wide biological effects including antioxidant, anticarcinogenic, anti-inflammatory, antiallergic, and antiviral activities. The anticancer activity is considered the most valuable effect of QRT against several types of cancer, including prostate, liver, lung, colon, and breast cancer. Scorpion venom peptides (SV) has been found to induce apoptosis and aggravate cancer cells, making it a promising anticancer agent. QRT, SV, and Phospholipon 90H (PL) were incorporated in a nano-based delivery platform to assess QRT's cellular uptake and antiproliferative efficacy against a lung cancer cell line derived from human breast cancer cells MCF-7. Several nanovesicles were prepared and optimized, using four-factor Box-Behnken, in an experimental design. The optimized phytosomes showed vesicle size and zeta potential values of 116.9 nm and 31.5 mV, respectively. The IC50 values revealed that MCF-7 cells were significantly more sensitive to the optimized QRT formula than the plain formula and raw QRT. Cell cycle analysis revealed that optimized QRT formula treatment resulted in significant cell cycle arrest at the S phase. The results also indicated that treatment with QRT formula significantly increased caspase-9, Bax, Bcl-2, and p53 mRNA expression, compared with the plain formula and QRT. In terms of the inflammatory markers, the QRT formula significantly reduced the activity of TNF-α and NF-κB, in comparison with the plain formula and QRT only. Overall, the findings from the study proved that a QRT formulation could be a promising therapeutic approach for the treatment of breast cancer.

Alhakamy, N. A., S. Z. Okbazghi, M. A. Alfaleh, W. H. Abdulaal, R. B. Bakhaidar, M. O. Alselami, M. A. Zahrani, H. M. Alqarni, A. F. Alghaith, S. Alshehri, et al., "Wasp venom peptide improves the proapoptotic activity of alendronate sodium in A549 lung cancer cells.", PloS one, vol. 17, issue 2, pp. e0264093, 2022. Abstract

BACKGROUND: Lung cancer in men and women is considered the leading cause for cancer-related mortality worldwide. Anti-cancer peptides represent a potential untapped reservoir of effective cancer therapy.

METHODOLOGY: Box-Behnken response surface design was applied for formulating Alendronate sodium (ALS)-mastoparan peptide (MP) nanoconjugates using Design-Expert software. The optimization process aimed at minimizing the size of the prepared ALS-MP nanoconjugates. ALS-MP nanoconjugates' particle size, encapsulation efficiency and the release profile were determined. Cytotoxicity, cell cycle, annexin V staining and caspase 3 analyses on A549 cells were carried out for the optimized formula.

RESULTS: The results revealed that the optimized formula was of 134.91±5.1 nm particle size. The novel ALS-MP demonstrated the lowest IC50 (1.3 ± 0.34 μM) in comparison to ALS-Raw (37.6 ± 1.79 μM). Thus, the results indicated that when optimized ALS-MP nanoconjugate was used, the IC50 of ALS was also reduced by half. Cell cycle analysis demonstrated a significantly higher percentage of cells in the G2-M phase following the treatment with optimized ALS-MP nanoconjugates.

CONCLUSION: The optimized ALS-MP formula had significantly improved the parameters related to the cytotoxic activity towards A549 cells, compared to control, MP and ALS-Raw.

Awan, Z. A., S. A. Alghamdi, N. A. Alhakamy, S. Z. Okbazghi, M. A. Alfaleh, S. M. Badr-Eldin, H. M. Aldawsari, M. A. S. Abourehab, H. Z. Asfour, S. A. Zakai, et al., "Optimized 2-methoxyestradiol invasomes fortified with apamin: a promising approach for suppression of A549 lung cancer cells.", Drug delivery, vol. 29, issue 1, pp. 1536-1548, 2022. Abstract

Certain anticancer agents selectively target the nucleus of cancer cells. One such drug is 2-methoxyestradiol (2ME), which is used for treating lung cancer. To improve the therapeutic effectiveness of these agents, many new methods have been devised. 2ME was entrapped into the core of hydrophobic invasomes (INVA) covered with Phospholipon 90G and apamin (APA). The Box-Behnken statistical design was implemented to enhance the composition. Using Design-Expert software (Stat-Ease Inc., Minneapolis, MN), the INVA component quantities were optimized to obtain spherical particles with the smallest size, that is, a diameter of 167.8 nm. 2ME-INVA-APA significantly inhibited A549 cells and exhibited IC of 1.15 ± 0.04 µg/mL, which is lower than raw 2ME (IC 5.6 ± 0.2 µg/mL). Post 2ME-INVA-APA administration, a significant rise in cell death and necrosis was seen among the A549 cells compared to those treated with plain formula or 2ME alone. This effect was indicated by increased Bax expression and reduced Bcl-2 expression, as well as mitochondrial membrane potential loss. Moreover, the cell cycle analysis showed that 2ME-INVA-APA arrests the G2-M phase of the A549 cells. Additionally, it was observed that the micellar formulation of the drug increased the cell count in pre-G1, thereby exhibiting phenomenal apoptotic potential. Furthermore, it up-regulates caspase-9 and p53 and downregulates TNF-α and NF-κβ. Collectively, these findings showed that our optimized 2ME-INVA-APA could easily seep through the cell membrane and induce apoptosis in relatively low doses.

Fahmy, U. A., S. M. Badr-Eldin, H. M. Aldawsari, N. A. Alhakamy, O. A. A. Ahmed, M. F. Radwan, B. G. Eid, S. R. M. Sayed, G. A. Elsherbiny, and W. Abualsunun, "Potentiality of raloxifene loaded melittin functionalized lipidic nanovesicles against pancreatic cancer cells.", Drug delivery, vol. 29, issue 1, pp. 1863-1877, 2022. Abstract

Pancreatic cancer (PC) frequency and incidence have grown rapidly in recent years. One of the most serious problems with PC is the existence of asymptotic manifestations, which frequently delays early detection, and until the diagnosis is established, tumor cells progress to the metastatic stage. Another significant concern with PC is the scarcity of well-defined pharmacotherapeutic drugs. The aim of this study was to develop an efficient nanocarrier system to augment the efficacy of raloxifene (RLX) against PC cells. As a result, the current investigation was carried out in order to give an effective treatment method, in which an optimum RLX loaded phospholipid-based vesicles with melittin (PL-MEL) was chosen using experimental design software, with particle size, zeta potential and entrapment efficiency % as dependent variables. Furthermore, anticancer activity against PANC1 cells was assessed. The optimized nanovesicle parameters were 172.5 nm for the measured size, zeta potential of -0.69 mV, and entrapment efficiency of 76.91% that were in good agreement with the expected ones. RLX-raw, plain formula, and optimized RLX-PL-MEL showed IC concentrations of 26.07 ± 0.98, 9.166 ± 0.34, and 1.24 ± 0.05 µg/mL, respectively. Furthermore, cell cycle analysis revealed that the nanovesicle was most effective in the G2-M phase, whereas Bax, and Bcl-2 estimates revealed that optimized RLX formula had the highest apoptotic activity among treatments investigated. However, as compared to RLX alone or plain formula alone, the optimized formula demonstrated higher expression of TNFα and Bax while a significant reduction of Bcl-2 and NF-κB expression was observed. mitochondrial membrane potential (MMP) analysis confirmed the apoptosis as well as the anticancer effect of the optimized formula. Thus, the present study results showed an improvement in the anti-PC effects of the RLX with phospholipid conjugated melittin, making it a novel treatment approach against PC.

Alfaleh, M. A., O. Fahmy, M. W. Al-Rabia, M. A. S. Abourehab, O. A. A. Ahmed, U. A. Fahmy, H. H. Alsulimani, S. M. Badr-Eldin, H. M. Aldawsari, B. M. Aldhabi, et al., "Hybrid nanoparticulate system of Fluvastatin loaded phospholipid, alpha lipoic acid and melittin for the management of colon cancer.", Scientific reports, vol. 12, issue 1, pp. 19446, 2022. Abstract

As a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, Fluvastatin (FLV) is used for reducing low-density lipoprotein (LDL) cholesterol as well as to prevent cardiovascular problems. FLV showed cell line cytotoxicity and antitumor effect. Melittin (MEL) exhibits antineoplastic activity and is known to be promising as a therapeutic option for cancer patients. The aim of this work was to investigate the combination of FLV with MEL loaded hybrid formula of phospholipid (PL) with alpha lipoic acid (ALA) nanoparticles to maximize anticancer tendencies. This study examines the optimization of the prepared formulation in order to minimize nanoparticles size and maximize zeta potential to potentiate cytotoxic potentialities in colon cancer cells (Caco2), cell viability, cell cycle analysis and annexin V were tested. In addition to biological markers as P53, Bax, bcl2 and Caspase 3 evaluation The combination involving FLV PL ALA MEL showed enhanced cytotoxic potentiality (IC50 = 9.242 ± 0.35 µg/mL), about twofold lower, compared to the raw FLV (IC50 = 21.74 ± 0.82 µg/mL). According to studies analyzing cell cycle, optimized FLV PL ALA MEL was found to inhibit Caco2 colon cancer cells more significantly than other therapeutic treatments, wherein a higher number of cells were found to accumulate over G2/M and pre-G1 phases, whereas G0/G1/S phases witnessed the accumulation of a lower number of cells. The optimized formulation may pave the way for a novel and more efficacious treatment for colon cancer.

Alharbi, W. S., R. H. Hareeri, M. Bazuhair, M. A. Alfaleh, N. A. Alhakamy, U. A. Fahmy, A. A. Alamoudi, S. M. Badr-Eldin, O. A. Ahmed, S. A. Alghamdi, et al., "Spanlastics as a Potential Platform for Enhancing the Brain Delivery of Flibanserin: In Vitro Response-Surface Optimization and In Vivo Pharmacokinetics Assessment.", Pharmaceutics, vol. 14, issue 12, 2022. Abstract

Flibanserin was licensed by the United States Food and Drug Administration (FDA) as an oral non-hormonal therapy for pre-menopausal women with inhibited sexual desire disorder. However, it suffers from susceptibility to first-pass metabolism in the liver, low aqueous solubility, and degradation in the acidic stomach environment. Such hurdles result in a limited oral bioavailability of 33%. Thus, the aim of the study was to utilize the principles of nanotechnology and the benefits of an intranasal route of administration to develop a formulation that could bypass these drawbacks. A response-surface randomized D-optimal strategy was used for the formulation of flibanserin spanlastics (SPLs) with reduced size and increased absolute zeta potential. Two numerical factors were studied, namely the Span 60: edge activator ratio (/) and sonication time (min), in addition to one categorical factor that deals with the type of edge activator. Particle size (nm) and zeta potential (mV) were studied as responses. A mathematical optimization method was implemented for predicting the optimized levels of the variables. The optimized formulation was prepared using a Span: sodium deoxycholate ratio of 8:2 /; a sonication time of 5 min showed particle sizes of 129.70 nm and a zeta potential of -33.17 mV. Further in vivo assessment following intranasal administration in rats showed boosted plasma and brain levels, with 2.11- and 2.23-fold increases (respectively) compared to raw FLB. The aforementioned results imply that the proposed spanlastics could be regarded as efficient drug carriers for the trans-nasal delivery of drugs to the brain.

Asfour, H. Z., N. A. Alhakamy, U. A. Fahmy, O. A. A. Ahmed, W. Y. Rizg, R. I. Felimban, A. B. Abdel-Naim, M. A. S. Abourehab, R. A. Mansouri, U. M. Omar, et al., "Zein-Stabilized Nanospheres as Nanocarriers for Boosting the Aphrodisiac Activity of Icariin: Response Surface Optimization and In Vivo Assessment.", Pharmaceutics, vol. 14, issue 6, 2022. Abstract

Icariin (ICA), a main active compound of the Epimedium genus, is used as an aphrodisiac in traditional Chinese herbal medicine. Despite its therapeutic efficacy, ICA displays reduced oral absorption, and therefore, low bioavailability hindered its clinical application. Implementing nanotechnology in the field of formulation has been a focus to improve the efficacy of ICA. In this regard, polymeric nanoparticles find a potential application as drug delivery systems. A nanosphere formula was designed, aiming to improve the drug's efficacy. The proposed ICA nanosphere formula (tocozeinolate) was optimized using D-optimal response surface design. The concentrations of ICA (X), D-α-tocopherol polyethylene glycol 1000 succinate (TPGS, X), zein (X), and sodium deoxycholate (SDC, X) expressed as percentages were investigated as quantitative independent variables. As per the experimental design, 23 formulations were developed, which were investigated for particle size (PS, nm), zeta potential (ZP, mV), and entrapment efficiency (EE, %) as response parameters. Numerical optimization and desirability approach were employed to predict the optimized variable levels that, upon combination, could result in minimized size and maximized zeta potential and ICA entrapment. The optimized ICA-tocozeinolate nanospheres showed a particle size of 224.45 nm, zeta potential of 0.961 mV, and drug entrapment of 65.29% that coincide well with the predicted values. The optimized ICA-tocozeinolate nanospheres were evaluated for sexual behavior in Wistar male rats compared to raw ICA at equivalent doses (20 mg/kg). In vivo assessment results showed significant sexual behavior enhancement by the optimized formulation, as evidenced by decreased average time of both mount latency (ML) and ejaculation latency (EL) to almost half those of raw ICA. Additionally, intromission latency (IL) time was reduced by 41% compared to the raw ICA. These results highlighted the potential of the proposed ICA-tocozeinolate nanospheres as a promising platform for improving the delivery and efficacy of therapeutic agents.

Alamoudi, A. A., A. S. Alharbi, A. B. Abdel-Naim, S. M. Badr-Eldin, Z. A. Awan, S. Z. Okbazghi, O. A. A. Ahmed, N. A. Alhakamy, U. A. Fahmy, and A. Esmat, "Novel Nanoconjugate of Apamin and Ceftriaxone for Management of Diabetic Wounds.", Life (Basel, Switzerland), vol. 12, issue 7, 2022. Abstract

Diabetic hyperglycemia delays wound healing, leading to serious consequences. Topical antibiotics can reduce the risk of a wound infection during healing; nevertheless, the microbial fight against antibiotics brings about public health challenges. Anti-microbial peptides (AMPs) belong to a novel class of drug that is used to prevent and treat systemic and topical infections. The aim of the current work was to achieve better wound healing in diabetic rats by conjugating the anti-microbial peptide "apamin" (APA) with the broad-spectrum antibiotic "ceftriaxone" (CTX) to form a nanocomplex. The CTX-APA nanoconjugate formulation was optimized using a Box-Behnken design. The optimized CTX-APA nanoconjugate formulation was evaluated for its size and zeta potential, and was then examined using transmission electron microscopy (TEM). The CTX-APA nanoconjugate was loaded onto a hydroxypropyl methylcellulose (2% /)-based hydrogel. It was observed that the application of the CTX-APA nanocomplex on the wounded skin of diabetic rats accelerated the regeneration of the epithelium, granulation tissue formation, epidermal proliferation, and keratinization. The nanocomplex was capable of significantly reducing the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), while increasing the expression of transforming growth factor beta-1 (TGF-β1) as well as the angiogenic markers: hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Conclusively, the application of an ion-paired CTX-APA nanocomplex enhances wound healing in diabetic rats.

Ahmed, O. A. A., S. M. Badr-Eldin, G. Caruso, U. A. Fahmy, W. S. Alharbi, A. M. Almehmady, S. A. Alghamdi, N. A. Alhakamy, A. I. Mohamed, H. M. Aldawsari, et al., "Colon Targeted Eudragit Coated Beads Loaded with Optimized Fluvastatin-Scorpion Venom Conjugate as a Potential Approach for Colon Cancer Therapy: In Vitro Anticancer Activity and In Vivo Colon Imaging.", Journal of pharmaceutical sciences, vol. 111, issue 12, pp. 3304-3317, 2022. Abstract

Preclinical studies suggest that most of statins or 3‑hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors possess pleiotropic anticancer activity. The aim of the present work was to investigate the conjugation of the statin fluvastatin (FLV) with scorpion venom (SV), a natural peptide with proven anticancer properties, to enhance FLV cytotoxic activity and prepare colon targeted FLV-SV nanoconjugate beads for management of colon cancer. Response surface design was applied for the optimization of FLV-SV nanoconjugates. FLV-SV particle size and zeta potential were selected as responses. Cytotoxicity of optimized FLV-SV nanoconjugates was carried out on Caco2 cell line. Colon targeted alginate coated Eudragit S100 (ES100) beads for the optimized formula were prepared with the utilization of barium sulfate (BaSO4) as radiopaque contrast substance. Results revealed that optimized FLV-SV nanoconjugates showed a size of 71.21 nm, while the zeta potential was equal to 29.13 mV. Caco2 cells were considerably more sensitive to the FLV-SV formula (half-maximal inhibitory concentration (IC50) = 11.91 µg/mL) compared to SV and FLV used individually, as shown by values of IC50 equal to 30.23 µg/mL and 47.68 µg/mL, respectively. In vivo imaging of colon targeted beads, carried out by employing real-time X-ray radiography, confirmed the efficiency of colon targeted beads. Overall our results indicate that the optimized FLV-SV nanoconjugate loaded alginate coated ES100 beads could represent a promising approach for colon cancer with efficient colon targeting ability.