Mahmoud A . Younis | Assiut University (original) (raw)

Papers by Mahmoud A . Younis

3 Biotech, 2024

Silver nanoparticles (AgNPs) have attracted increasing attention in nanomedicine, with versatile ... more Silver nanoparticles (AgNPs) have attracted increasing attention in nanomedicine, with versatile applications in drug delivery, antimicrobial treatments, and cancer therapies. While chemical synthesis remains a common approach for AgNP production, ensuring environmental sustainability requires a shift toward eco-friendly, “green” synthesis techniques. This article underscores the promising role of plant extracts in the green synthesis of AgNPs, highlighting the importance of their natural sources and diverse bioactive compounds. Various characterization methods for these nanomaterials are also reviewed. Furthermore, the anticancer potential of green AgNPs (Gr-AgNPs) is examined, focusing on their mechanisms of action and the challenges to their clinical implementation. Finally, future directions in the field are discussed.

Pharmacogenomics and Personalized Medicine, 2024

Hepatocellular carcinoma (HCC) is the dominant type of liver cancers and is one of the deadliest ... more Hepatocellular carcinoma (HCC) is the dominant type of liver cancers and is one of the deadliest health threats globally. The conventional therapeutic options for HCC are hampered by low efficiency and intolerable side effects. Gene therapy, however, now offers hope for the treatment of many disorders previously considered incurable, and gene therapy is beginning to address many of the shortcomings of conventional therapies. Herein, we summarize the involvement of genes in the pathogenesis and prognosis of HCC, with a special focus on dysregulated signaling pathways, genes involved in immune evasion, and non-coding RNAs as novel two-edged players, which collectively offer potential targets for the gene therapy of HCC. Herein, the opportunities and challenges of HCC gene therapy are discussed. These include innovative therapies such as genome editing and cell therapies. Moreover, advanced gene delivery technologies that recruit nanomedicines for use in gene therapy for HCC are highlighted. Finally, suggestions are offered for improved clinical translation and future directions in this area of endeavor.

Colloids and Surfaces B: Biointerfaces, 2024

Breast cancer is a wide-spread threat to the women’s health. The drawbacks of conventional treatm... more Breast cancer is a wide-spread threat to the women’s health. The drawbacks of conventional treatments necessitate the development of alternative strategies, where gene therapy has regained hope in achieving an efficient eradication of aggressive tumors. Monocarboxylate transporter 4 (MCT4) plays pivotal roles in the growth and survival of various tumors, which offers a promising target for treatment. In the present study, pH-responsive lipid nanoparticles (LNPs) based on the ionizable lipid,1,2-dioleoyl-3-dimethylammonium propane (DODAP), were designed for the delivery of siRNA targeting MCT4 gene to the breast cancer cells. Following multiple steps of characterization and optimization, the anticancer activities of the LNPs were assessed against an aggressive breast cancer cell line, 4T1, in comparison with a normal cell line, LX-2. The selection of the helper phospholipid to be incorporated into the LNPs had a dramatic impact on their gene delivery performance. The optimized LNPs enabled a powerful MCT4 silencing by ∼90 % at low siRNA concentrations, with a subsequent ∼80 % cytotoxicity to 4T1 cells. Meanwhile, the LNPs demonstrated a 5-fold higher affinity to the breast cancer cells versus the normal cells, in which they had a minimum effect. Moreover, the MCT4 knockdown by the treatment remodeled the cytokine profile in 4T1 cells, as evidenced by 90 % and ∼64 % reduction in the levels of TNF-α and IL-6; respectively. The findings of this study are promising for potential clinical applications. Furthermore, the simple and scalable delivery vector developed herein can serve as a breast cancer-targeting platform for the delivery of other RNA therapeutics.

AAPS PharmSciTech, 2024

Colorectal cancer (CRC) is a widespread cancer that starts in the digestive tract. It is the thir... more Colorectal cancer (CRC) is a widespread cancer that starts in the digestive tract. It is the third most common cause of cancer deaths around the world. The World Health Organization (WHO) estimates an expected death toll of over 1 million cases annually. The limited therapeutic options as well as the drawbacks of the existing therapies necessitate the development of non-classic treatment approaches. Nanotechnology has led the evolution of valuable drug delivery systems thanks to their ability to control drug release and precisely target a wide variety of cancers. This has also been extended to the treatment of CRC. Herein, we shed light on the pertinent research that has been performed on the potential applications of nanoparticles in the treatment of CRC. The various types of nanoparticles in addition to their properties, applications, targeting approaches, merits, and demerits are discussed. Furthermore, innovative therapies for CRC, including gene therapies and immunotherapies, are also highlighted. Eventually, the research gaps, the clinical potential of such delivery systems, and a future outlook on their development are inspired.

Journal of Controlled Release, 2023

We report on a novel strategy for treating liver fibrosis through reprogramming activated Hepatic... more We report on a novel strategy for treating liver fibrosis through reprogramming activated Hepatic Stellate Cells (aHSCs) into quiescent Hepatic Stellate Cells (qHSCs) using siRNA-loaded lipid nanoparticles (LNPs). The in vivo screening of an array of molecularly-diverse ionizable lipids identified two candidates, CL15A6 and CL15H6, with a high siRNA delivery efficiency to aHSCs. Optimization of the composition and physico-chemical properties of the LNPs enabled the ligand-free, selective, and potent siRNA delivery to aHSCs post intravenous administration, with a median effective siRNA dose (ED50) as low as 0.08 mg/Kg. The biosafety of the LNPs was confirmed by escalating the dose to 50-fold higher than the ED50 or by chronic administration. The recruitment of the novel LNPs for the simultaneous knockdown of Hedgehog (Hh) and Transforming Growth Factor Beta 1 (TGFβ1) signaling pathways using an siRNA cocktail enabled the reversal of liver fibrosis and the restoration of the normal liver function in mice. Analysis of the key transcription factors in aHSCs suggested that the reprogramming of aHSCs into qHSCs mediated the therapeutic outcomes. The scalable ligand-free platform developed in this study as well as the novel therapeutic strategy reported herein are promising for clinical translation.

Colloids and Surfaces B: Biointerfaces, 2023

Lung cancer is one of the most aggressive and deadliest health threats. There has been an increas... more Lung cancer is one of the most aggressive and deadliest health threats. There has been an increasing interest in non-coding RNA (ncRNA) recently, especially in the areas of carcinogenesis and tumour progression. However, ncRNA-directed therapies are still encountering obstacles on their way to the clinic. In the present article, we provide an overview on the potential of targeting ncRNA in the treatment of lung cancer. Then, we discuss the delivery challenges and recent approaches enabling the delivery of ncRNA-directed therapies to the lung cancer cells, where we illuminate some advanced technologies including chemically-modified oligonucleotides, nuclear targeting, and three-dimensional in vitro models. Furthermore, advanced non-viral delivery systems recruiting nanoparticles, biomimetic delivery systems, and extracellular vesicles are also highlighted. Lastly, the challenges limiting the clinical trials on the therapeutic targeting of ncRNAs in lung cancer and future directions to tackle them are explored.

Drug Development and Industrial Pharmacy, 2023

Objective and significance: Silver nanoparticles (AgNPs) have become an interesting therapeutic m... more Objective and significance: Silver nanoparticles (AgNPs) have become an interesting therapeutic modality and drug delivery platform. Herein, we aimed to investigate the impact of functional coating on the in vivo performance of AgNPs as an economic and scalable method to modulate their behavior.

Methods: AgNPs were coated with chitosan (CHI) as a model biopolymer using a one-pot reduction-based method, where CHI of two molecular weight ranges were investigated. The resultant CHI-coated AgNPs (AgNPs-CHI) were characterized using UV-VIS spectroscopy, DLS, and TEM. AgNPs were administered intravenously to rats and their biodistribution and serum levels of hepato-renal function markers were monitored 24 h later compared to plain AgNO3 as a positive control.

Results: UV-VIS spectroscopy confirmed the successful coating of AgNPs with CHI. DLS revealed the superiority of medium molecular weight CHI over its low molecular weight counterpart. AgNPs-CHI demonstrated a semi-complete clearance from the systemic circulation, a liver-dominated tissue tropism, and limited renal exposure. On the other hand, AgNO3 was poorly cleared from the circulation, with relatively high renal exposure and a non-specific tissue tropism. AgNPs-CHI were well-tolerated by the liver and kidney without signs of toxicity or inflammation, in contrary with AgNO3 which resulted in a significant elevation of Creatinine (CRE), Urea, and Total Protein (TP), suggesting a significant nephrotoxicity and inflammation.

Conclusions: Functional coating of AgNPs with CHI substantially modulated their in vivo behavior, promoting their hepatic selectivity and biotolerability, which can be invested in the development of drug delivery systems for the treatment of liver diseases.

Nanotechnology Reviews, 2023

Herein, we report on the development of chitosan-capped silver nanoparticles (AgNPs-CHI) with an ... more Herein, we report on the development of chitosan-capped silver nanoparticles (AgNPs-CHI) with an intrinsic activity against breast cancer cells. Following chemical synthesis via a simple, one-pot reaction, the chitosan coating of AgNPs was verified using Fouriertransform infrared and ultraviolet-visible spectroscopies. The physicochemical properties and morphology were characterized using dynamic light scattering, scanning electron microscopy, and transmission electron microscopy. The shelf stability of the optimized platform was tracked for 3 months upon storage at either room temperature or 4°C. Then, the anticancer activities of AgNPs-CHI on human breast cancer cells, MCF-7, versus normal human cells, human skin fibroblasts (HSF), were evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay and tumor-associated biomarkers determination by enzyme-linked immunosorbent assay, in comparison with plain silver nitrate (AgNO 3) solution. AgNPs were successfully coated with chitosan and demonstrated acceptable physicochemical properties, with a spherical morphology and high stability upon long-term storage. Although AgNPs-CHI and AgNO 3 demonstrated comparable cytotoxicity to MCF-7 cells, AgNPs-CHI resulted in 10-fold lower toxicity to HSF cells, suggesting a higher selectivity. In addition, AgNPs-CHI lowered IL-6 and tumor necrosis factor-alpha levels in MCF-7 cells by 90 and 30%, respectively, compared to 60 and 10% in the case of plain AgNO 3. The interesting therapeutic modality presented in this study is promising for potential clinical applications.

Colloids and Surfaces B: Biointerfaces, 2023

Throughout decades, the intrinsic power of the immune system to fight pathogens has inspired rese... more Throughout decades, the intrinsic power of the immune system to fight pathogens has inspired researchers to develop techniques that enable the prevention or treatment of infections via boosting the immune response against the target pathogens, which has led to the evolution of vaccines. The recruitment of Lipid nanoparticles (LNPs) as either vaccine delivery platforms or immunogenic modalities has witnessed a breakthrough recently, which has been crowned with the development of effective LNPs-based vaccines against COVID-19. In the current article, we discuss some principles of such a technology, with a special focus on the technical aspects from a translational perspective. Representative examples of LNPs-based vaccines against cancer, COVID-19, as well as other infectious diseases, autoimmune diseases, and allergies are highlighted, considering the challenges and promises. Lastly, the key features that can improve the clinical translation of this area of endeavor are inspired.

Journal of Controlled Release, 2023

Herein, we report on the development of a platform for the selective delivery of mRNA to the hard... more Herein, we report on the development of a platform for the selective delivery of mRNA to the hard-to-transfect Activated Hepatic Stellate Cells (aHSCs), the fundamental player in the progression of liver fibrosis. Using a microfluidic device (iLiNP), we prepared a series of lipid nanoparticles (LNPs) based on a diverse library of pH-sensitive lipids. After an in-depth in vivo optimization of the LNPs, their mRNA delivery efficiency, selectivity, potency, robustness, and biosafety were confirmed. Furthermore, some mechanistic aspects of their selective delivery to aHSCs were investigated. We identified a promising lipid candidate, CL15A6, that has a high affinity to aHSCs. Tweaking the composition and physico-chemical properties of the LNPs enabled the robust and ligand-free mRNA delivery to aHSCs in vivo post intravenous administration, with a high biosafety at mRNA doses of up to 2 mg/Kg, upon either acute or chronic administrations. The mechanistic investigation suggested that CL15A6 LNPs were taken up by aHSCs via Clathrin-mediated endocytosis through the Platelet-derived growth factor receptor beta (PDGFRβ) and showed a pKa-dependent cellular uptake. The novel and scalable platform reported in this study is highly promising for clinical applications.

Advanced Drug Delivery Reviews, 2022

A new era of nanomedicines that involve nucleic acids/gene therapy has been opened after two deca... more A new era of nanomedicines that involve nucleic acids/gene therapy has been opened after two decades in 21st century and new types of more efficient drug delivery systems (DDS) are highly expected and will include extrahepatic delivery. In this review, we summarize the possibility and expectations for the extrahepatic delivery of small interfering RNA/messenger RNA/plasmid DNA/genome editing to the spleen, lung,
tumor, lymph nodes as well as the liver based on our studies as well as reported information. Passive targeting and active targeting are discussed in in vivo delivery and the importance of controlled intracellular trafficking for successful therapeutic results are also discussed. In addition, mitochondrial delivery as a novel strategy for nucleic acids/gene therapy is introduced to expand the therapeutic dimension of nucleic acids/gene therapy in the liver as well as the heart, kidney and brain.

Nature Communications, 2022

There is currently no therapy available for periprosthetic osteolysis, the most common cause of a... more There is currently no therapy available for periprosthetic osteolysis, the most common cause of arthroplasty failure. Here, the role of AnxA1 in periprosthetic osteolysis and potential therapeutics were investigated. Reducing the expression of AnxA1 in calvarial tissue was found to be associated with increased osteolytic lesions and the osteolytic lesions induced by debris implantation were more severe in AnxA1-defecient mice than in wild-type mice. AnxA1 inhibits the differentiation of osteoclasts through suppressing NFκB signaling and promoting the PPAR-γ pathway. Administration of N-terminal-AnxA1 (Ac2-26 peptide) onto calvariae significantly reduced osteolytic lesions triggered by wear debris. These therapeutic effects were abrogated in mice that had received the PPAR-γ antagonist, suggesting that the AnxA1/ PPAR-γ axis has an inhibitory role in osteolysis. The administration of Ac2-26 suppressed osteolysis induced by TNF-α and RANKL injections in mice. These findings indicate that AnxA1 is a potential therapeutic agent for the treatment of periprosthetic osteolysis.

International Journal of Nanomedicine, 2022

Despite the massive advancements in the nanomedicines and their associated research, their transl... more Despite the massive advancements in the nanomedicines and their associated research, their translation into clinicallyapplicable products is still below promises. The latter fact necessitates an in-depth evaluation of the current nanomedicines from a clinical perspective to cope with the challenges hampering their clinical potential. Quantum dots (QDs) are semiconductors-based nanomaterials with numerous biomedical applications such as drug delivery, live imaging, and medical diagnosis, in addition to other applications beyond medicine such as in solar cells. Nevertheless, the power of QDs is still underestimated in clinics. In the current article, we review the status of QDs in literature, their preparation, characterization, and biomedical applications. In addition, the market status and the ongoing clinical trials recruiting QDs are highlighted, with a special focus on the challenges limiting the clinical translation of QDs. Moreover, QDs are technically compared to other commercially-available substitutes. Eventually, we inspire the technical aspects that should be considered to improve the clinical fate of QDs.

Advanced Drug Delivery Reviews, 2022

Despite the massive interest and recent developments in the field of nanomedicine, only a limited... more Despite the massive interest and recent developments in the field of nanomedicine, only a limited number of formulations have found their way to the clinics. This shortcoming reveals the challenges facing the clinical translation of this technology. In the current article, we summarize and evaluate the status, market situation, and clinical profiles of the reported nanomedicines, the shortcomings limiting their clinical translation, as well as some approaches designed to break through this barrier. Moreover, some emerging technologies that have the potential to compete with nanomedicines are highlighted. Lastly, we identify the key factors that should be considered in nanomedicine-related research to be clinically-translatable. These can be classified into five areas: rational design during the research and development stage, the recruitment of representative preclinical models, careful design of clinical trials, development of specific and uniform regulatory protocols, and calls for non-classic sponsorship. This new field of endeavor was firmly established during the last two decades and more in-depth progress is expected in the coming years.

Journal of Controlled Release , 2021

Hepatocellular carcinoma (HCC) is a fatal disease with limited therapeutic choices. The stroma-ri... more Hepatocellular carcinoma (HCC) is a fatal disease with limited therapeutic choices. The stroma-rich tumor microenvironment hinders the in vivo delivery of most nanomedicines. Ultra-small lipid nanoparticles (usLNPs) were designed for the selective co-delivery of the cytotoxic drug, sorafenib (SOR), and siRNA against the Midkine gene (MK-siRNA) to HCC in mice. The usLNPs composed of a novel pH-sensitive lipid, a diversity of phospholipids and a highly-selective targeting peptide. A microfluidic device, iLiNP, was used and a variety of factors were controlled to tune particle size aiming at maximizing tumor penetration efficiency. Optimizing the composition and physico-chemical properties of the usLNPs resulted in an enhanced tumor accumulation, selectivity and in vivo gene silencing. The optimized usLNPs exerted potent gene silencing in the tumor (median effective dose, ED50~0.1 mg/Kg) with limited effect on the healthy liver. The novel combination synergistically-eradicated HCC in mice (~85%) at a surprisingly-low dose of SOR (2.5 mg/Kg) which could not be achieved via individual monotherapy. Toxicity studies revealed the biosafety of the usLNPs upon either acute or chronic treatment. Furthermore, the SOR-resistant HCC established in mice was eradicated by 70% using this approach. We conclude that our strategy is promising for potential clinical applications in HCC treatment.

Biological and Pharmaceutical Bulletin, 2020

The last few years have witnessed a great advance in the development of nonviral systems for in v... more The last few years have witnessed a great advance in the development of nonviral systems for in vivo targeted delivery of nucleic acids. Lipid nanoparticles (LNPs) are the most promising carriers for producing clinically approved products in the future. Compared with other systems used for nonviral gene delivery, LNPs provide several advantages including higher stability, low toxicity, and greater efficiency. Additionally, systems based on LNPs can be modified with ligands and devices for controlled biodistribution and inter-nalization into specific cells. Efforts are ongoing to improve the efficiency of lipid-based gene vectors. These efforts depend on the appropriate design of nanocarriers as well as the development of new lipids with improved gene delivery ability. Several ionizable lipids have recently been developed and have shown dramatically improved efficiency. However, enhancing the ability of nanocarriers to target specific cells in the body remains the most difficult challenge. Systemically administered LNPs can access organs in which the capil-laries are characterized by the presence of fenestrations, such as the liver and spleen. The liver has received the most attention to date, although targeted delivery to the spleen has recently emerged as a promising tool for modulating the immune system. In this review, we discuss recent advances in the use of LNPs for cell-specific targeted delivery of nucleic acids. We focus mainly on targeting liver hepatocytes and spleen immune cells as excellent targets for gene therapy. We also discuss the potential of endothelial cells as an alternate approach for targeting organs with a continuous endothelium.

Journal of Drug Delivery Science and Technology, 2020

Rofecoxib (ROF), a potent selective COX-II inhibitor NSAID, has been withdrawn from the market du... more Rofecoxib (ROF), a potent selective COX-II inhibitor NSAID, has been withdrawn from the market due to its high risk on patients with cardiovascular disorders (CVD). In the current study, an attempted to re-introduce ROF into the market via co-formulation with the antiplatelet drug, Aspirin, to neutralize ROF effect on the platelet function has been investigated. Solubility and dissolution profiles of ROF were studied and improved through formulation into solid dispersions with Soluplus® before being mixed with a low dose of Aspirin. The novel drug combination was incorporated into oral tablets with various compositions to optimize the tablets physico-chemical performance. In addition, a new validated HPLC method was developed for simultaneous assessment of both drugs in the tablets. The optimized tablets demonstrated satisfactory physico-chemical performance. The developed HPLC method was accurate, precise, robust and sensitive with limits of detection (LODs) of 2.96 and 0.25 μg/mL and limits of quantitation (LOQs) of 8.77 and 0.77 μg/mL for Aspirin and ROF, respectively. The novel co-therapy showed normal platelet function in rabbits in opposite to ROF monothrapy. The data presented in this study can be promising to re-utilize ROF as an effective and selective NSAID, to satisfy the market's demand, while ensuring its safety in CVD patients.

Advanced Therapeutics, 2020

Treatment for hepatocellular carcinoma (HCC) is currently limited to early stages where surgical ... more Treatment for hepatocellular carcinoma (HCC) is currently limited to early stages where surgical intervention is applicable. Meanwhile, the response for most chemotherapies is still low, leaving patients in advanced stages without an effective therapeutic approach. Other therapeutic strategies based on immunotherapies or radiotherapy have a narrow spectrum with multiple limitations. These collective drawbacks necessitate the development of alternative strategies. Gene therapy has achieved a breakthrough in the treatment of several diseases, especially tumors. In the current review, a discussion is provided on the various strategies that have been developed for HCC gene therapy, the functional and genetic materials used, and their diverse delivery systems, with a special focus on novel targeting strategies based on biomaterials, peptide libraries, and aptamers. In addition, animal models that have been used in preclinical evaluation of HCC gene therapies are highlighted and compared. Lastly, ongoing clinical trials and future perspectives for these strategies are discussed.

Molecular Pharmaceutics, 2019

Hepatocellular carcinoma (HCC), a common deadly malignancy, requires novel therapeutic strategies... more Hepatocellular carcinoma (HCC), a common deadly malignancy, requires novel therapeutic strategies to improve the survival rate. Combining chemotherapy and gene therapy is a promising approach for increasing efficiency and reducing side effects. We report on the design of highly-specific lipid nanoparticles (LNPs) encapsulating both the chemotherapeutic drug, sorafenib (SOR), and siRNA against the Midkine gene (MK), thereby conferring a novel highly-efficient anticancer effect on HCC. The LNPs were modified with a targeting peptide, SP94, which is selective for hepatic cancer cells (HCCs), thus permitting the specific delivery of the payload. MK-siRNA increased the sensitivity of HCCs, HepG2, to SOR (IC50 for SOR+MK-siRNA: 5±1.50 μM compared to 9±2.20 μM and 17±2.60 μM for SOR+control siRNA and MK-siRNA, respectively). The selectivity was confirmed by cellular uptake, cytotoxicity and gene silencing studies in HCCs, HepG2 and Hepa 1-6, compared to other cancerous cells, HeLa, and normal cells, FL83B. The use of a novel pH-sensitive lipid, YSK05, increased the cytotoxic and gene knockdown efficiencies and limited extracellular drug release. The nanoparticles were also compatible with serum and showed no aggregation after long storage. The efficient and specific co-delivery system reported here is a highly promising strategy for the treatment of HCC.

Journal of Drug Targeting, 2020

Sulpiride (SUL), anti-dopaminergic drug, has a specific site for absorption located in the upper ... more Sulpiride (SUL), anti-dopaminergic drug, has a specific site for absorption located in the upper portion of the gastrointestinal tract hence, its oral delivery represents a challenge regarding SUL absorption and bioavailability. So, a gastro-retentive oral platform of SUL was developed to increase its gastric residence time, release SUL at a controlled rate in the stomach and consequently, enable it to reach its specific absorption site. Floating microsponges were prepared via quasi-emulsion solvent diffusion method and characterized for their physico-chemical properties. In addition, Taguchi design of experiment was utilized to optimize some independent variables affecting microsponges performance. The optimized SUL microsponges showed a yield of 79.82 ± 2.37%, an encapsulation efficiency of 89.11 ± 2.28% and in vitro time for floatation of 8.0 hr. Additionally, pharmacokinetics were investigated in rabbits and compared with the commercial SUL formulation, Dogmatil® capsules. Optimized SUL microsponges showed a significantly (p < 0.05) higher Cmax, AUC and 2-fold increase in oral bioavailability compared with the commercial product. Moreover, the optimized SUL microsponges remained present in the stomach up to 8.0 hr post administration when viewed via X-ray radiographs in rabbits. It could be concluded that the floating microsponges could be useful as an oral platform to enhance the sulpiride absorption and bioavailability.

3 Biotech, 2024

Pharmacogenomics and Personalized Medicine, 2024

Colloids and Surfaces B: Biointerfaces, 2024

AAPS PharmSciTech, 2024

Journal of Controlled Release, 2023

Colloids and Surfaces B: Biointerfaces, 2023

Drug Development and Industrial Pharmacy, 2023

Nanotechnology Reviews, 2023

Colloids and Surfaces B: Biointerfaces, 2023

Journal of Controlled Release, 2023

Advanced Drug Delivery Reviews, 2022

Nature Communications, 2022

International Journal of Nanomedicine, 2022

Advanced Drug Delivery Reviews, 2022

Journal of Controlled Release , 2021

Biological and Pharmaceutical Bulletin, 2020

Journal of Drug Delivery Science and Technology, 2020

Advanced Therapeutics, 2020

Molecular Pharmaceutics, 2019

Journal of Drug Targeting, 2020

9th Annual meeting of the Nucleic Acids Therapeutics Society of Japan (NATSJ), Sendai International Center, Sendai, Miyagi, Japan, 2024

Background: The recent advancements in gene therapy has regained the hope in the treatment of inc... more Background: The recent advancements in gene therapy has regained the hope in the treatment of incurable diseases. Lipid nanoparticles (LNPs) have demonstrated a promising performance as non-viral delivery vectors for RNA therapeutics. Nevertheless, targeting hard-to-transfect cellular populations in complex disease models is still challenging. Ligand-based active targeting is a successful strategy in basic research, but it encounters several obstacles during scale-up, which subtracts from the clinical translatability of LNPs. Liver fibrosis is a fatal chronic disorder that is progressed through the activation of hepatic stellate cells from the quiescent phenotype (qHSCs) into the activated phenotype (aHSCs), with a subsequent deposition of extracellular matrix that hampers the delivery of nanocarriers to their target cells in the liver.
Research Contents: In the present study, we designed self-homing LNPs for selective mRNA/siRNA delivery to aHSCs in mice undergoing liver fibrosis. A library of ionizable lipids was synthesized and screened, while the composition and physico-chemical properties of the LNPs were tweaked to manipulate the nature of their relevant protein corona that can enable ligand-free targeting. A microfluidic device was recruited for scalable preparation of LNPs and precise control of their physico-chemical properties. Then, the optimized LNPs were loaded with a novel siRNA cocktail targeting Smoothened (SMO) and Transforming growth factor beta 1 (TGFβ1) in aHSCs with the purpose of treating liver fibrosis. Two ionizable lipids, CL15A6 and CL15H6, were identified with high selectivity to aHSCs. The composition and physico-chemical properties of LNPs dramatically affected their in vivo performance. The application of microfluidics technology enabled precise particle size control for efficient penetration through the stroma-rich microenvironment in the fibrotic liver. The novel approach adopted herein enabled efficient and robust ligand-free delivery of either mRNA or siRNA to aHSCs, with a high biosafety. Mechanistic elucidation suggested the involvement of Platelet-derived growth factor (PDGF) pathway in the delivery of our LNPs. Delivery of siRNA cocktail to aHSCs in vivo succeeded in the reprogramming of aHSCs into qHSCs and reversed liver fibrosis with restoration of the normal liver function in mice.
In conclusion, a scalable ligand-free strategy was developed for robust delivery of RNA therapeutics to aHSCs and reversal of liver fibrosis, which is promising for clinical translation.
Younis MA et al. J Control Release. 361: 592–603 (2023)
Younis MA et al. J Control Release. 353: 685-698 (2023)

18th Liposome Research Days (LRD 2024), Technology and Innovation Center, University of Strathclyde, Glasgow, Scotland, UK, 2024

Liver fibrosis is a chronic liver disease that can proceed into life-terminating complications. A... more Liver fibrosis is a chronic liver disease that can proceed into life-terminating complications. Activated Hepatic Stellate Cells (aHSCs) play a pivotal role in the initiation and progression of liver fibrosis. Small molecules targeting aHSCs are limited by their low selectivity and high toxicity. Therefore, gene therapy can provide a promising alternative in this area of endeavor. Yet, the challenging fibrotic liver microenvironment, the hard-to-transfect nature of aHSCs, and the poor scalability of most nanocarriers reduce the opportunities for clinical translation of these innovative therapies. We developed self-homing lipid nanoparticles (LNPs) that harness the endogenous serum proteins post intravenous administration to generate a targeting protein corona that enables the ligand-free targeting of aHSCs. A library of ionizable lipids as well as a variety of helper phospholipids were screened. In addition, microfluidics technology was applied for scalable and precise preparation of the LNPs. The LNPs were loaded with a novel siRNA cocktail for the simultaneous knockdown of Hedgehog (Hh) and Transforming growth factor beta 1 (TGFβ1) pathways in the aHSCs, and the therapeutic outcomes were evaluated in a mouse model of liver fibrosis. The composition and physico-chemical properties of the LNPs significantly affected their selectivity and gene silencing efficiency. The novel LNPs demonstrated a potent and selective silencing of the target genes in aHSCs and reprogrammed them into the quiescent status (qHSCs) with a subsequent reversal of liver fibrosis and restoration of liver functions in mice. Furthermore, the LNPs were biosafe upon either dose escalation or chronic administration. This novel, economic, and scalable platform as well as the novel therapeutic strategy introduced herein are promising for clinical translation.

15th HOPE meeting with Nobel Laureates, Japan Society for the Promotion of Science (JSPS), Kyoto International Conference Hall, Kyoto, Japan, 2024

There has been a massive breakthrough in the development of nanomedicines, especially after the U... more There has been a massive breakthrough in the development of nanomedicines, especially after the US-FDA approval of anti-COVID-19 messenger RNA (mRNA) vaccines in 2020. Nevertheless, the extension of such a technology to other applications faces formidable obstacles due to the poor clinical translatability of most nanomedicines. Ligand-based targeting is a successful strategy in basic research. However, the attachment of ligands to nanoparticles decreases their in vivo stability and complicates their scale-up process, which collectively decrease their possibility to reach the clinics. In the present study, we tackled the above challenges and developed clinically-translatable RNA delivery systems for the gene therapy of liver fibrosis, as a model of chronic liver diseases. Tweaking the composition and physico-chemical properties of the nanocarriers facilitated harnessing the endogenous transport pathways for highly-selective ligand-free targeting. A library of lipid nanoparticles (LNPs) based on diverse ionizable lipids was screened for the delivery of either mRNA or small interfering RNA (siRNA) to the activated Hepatic Stellate Cells (aHSCs), the key player in the pathogenesis of liver fibrosis. The application of the optimized LNPs for the delivery of a novel gene therapy enabled reprogramming the aHSCs into the quiescent status, with a subsequent reversal of liver fibrosis in mice. The novel, simple, and scalable platform introduced in this study is highly promising for clinical applications.

50th Annual Meeting of Controlled Release Society (CRS), Paris Hotel Las Vegas, Las Vegas, NV, USA, 2023

Introduction: Liver fibrosis is a chronic hepatic disorder that can proceed to cirrhosis, ascites... more Introduction: Liver fibrosis is a chronic hepatic disorder that can proceed to cirrhosis, ascites, cancer, and death. The activation of quiescent hepatic stellate cells (qHSCs) into myofibroblasts (aHSCs) is a key process in hepatofibrogenesis (1). Despite breakthrough in RNA delivery technologies, the extension of such progress to the hard-to-transfect cellular populations is still a formidable process (2). In addition, most classic ligand-based targeting approaches are impractical in terms of scalability and clinical translatability (3). Herein, we report on a novel targeting approach that is based on the recruitment of endogenous serum proteins to achieve selective mRNA delivery to the hard-to-transfect aHSCs in vivo post intravenous administration.

Methods: A scalable microfluidic-based approach was adopted in the preparation of mRNA-loaded lipid nanoparticles (LNPs). A library of diverse ionizable lipids was synthesized and screened in vitro. The top-performing candidates were further optimized and evaluated in vivo in a murine model of thioacetamide (TAA)-induced liver fibrosis. The mRNA delivery efficiency, selectivity, robustness, and biosafety were investigated. Furthermore, the mechanism for in vivo delivery of LNPs to aHSCs was explored.

Results: Five ionizable lipids were recognized to have high affinity to aHSCs in vitro. A model candidate, CL15A6, was selected for in vivo experiments. Tweaking the lipid composition and physico-chemical properties of LNPs enabled the manipulation of the endogenous protein corona that encapsulates the LNPs in the systemic circulation, with a subsequent robust and efficient mRNA delivery to aHSCs, in comparison with the other cellular populations in the liver. The optimized LNPs demonstrated a high biosafety upon either acute or chronic administrations. Moreover, mechanistic studies revealed that the novel LNPs recruit the endogenous platelet-derived growth factor (PDGF) pathway to target aHSCs in vivo.

Conclusion: We report on the first ligand-free delivery platform for the robust and selective RNA delivery to aHSCs in vivo. This simple, efficient, and scalable delivery system is highly promising for clinical applications in the gene therapy of liver fibrosis.

References: (1) Higashi T, et al. Adv Drug Deliv Rev. 2017: 27-42. (2) Munk M, et al. Biomed Phys Eng Exp. 2017: 045002. (3) Younis MA, et al. Adv Drug Deliv Rev. 2022: 114083.

The Now and Future of RNA Therapeutics 2023, Awaji Yumebutai Conference Center, Awaji, Japan, 2023

We have been developing our own library of ionizable cationic lipids (iCL). From in vivo screenin... more We have been developing our own library of ionizable cationic lipids (iCL). From in vivo screening, we have identified a few ionizable cationic lipids such as CL4H6 which can exert silencing activity of siRNA more efficient than MC3 in hepatocytes after intravenous administration. CL4H6-LNP can also be applied for genome editing successfully. We established to formulate CRISPR/Cas9 ribonucleoprotein (RNP) into LNP. Intravenous administration of RNP-LNP can induce more than 70 % of genome editing in liver to achieve 80% reduction of serum TTR protein level. Then we challenged to find a new system which can target activated stellate cells (aHSC) which play an important role in the progression of liver fibrosis based on our library of iCL. After screening both in vivo as well as in vitro based on liver fibroblast model, we have identified CL15A6 (pKa=7.3, one double bond in lipid tail), which has different hydrophilic head group and hydrophobic lipid tail from CL4H6 (pKa=6.25, two double bonds in lipid tail). It was found that CL15A6-LNP can be taken up by aHSC via ApoE independent manner, possibly PDGFRb-mediated endocytosis, and could not be taken up by liver efficiently in healthy condition. Transfection efficiency of mRNA in aHSC is deeply dependent of helper lipid. Another example is an optimized formulation to target splenic B-cells for pDNA, which can induce efficient antitumor effects via enhanced CTL activity. It was found that enhanced transfection activity in splenic B-cells should be taken-up via a complement receptor-mediated endocytosis, which increased translational efficiency 3-order of magnitude. These results indicate that a tailored formulation with ionizable cationic lipids library can provide a cell selective targeting of nucleic acids/RNP without ligands.

The 7th Japan-Taiwan Joint Symposium For Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan, virtual platform, 2022

The last few years have witnessed a breakthrough in the development of RNA therapeutics, especial... more The last few years have witnessed a breakthrough in the development of RNA therapeutics, especially those based on the lipid nanoparticles (LNPs) technology. Nevertheless, the clinical translation of the vast majority of the reported delivery systems has encountered several technical obstacles in terms of scalability and stability. In an attempt to improve the clinical potential of RNA-loaded LNPs, we applied a novel microfluidic device for one-step, scalable, and controlled preparation of LNPs. In addition, our results revealed that the manipulation of the composition and physico-chemical properties of LNPs dramatically affected their in vivo performance and subsequently enabled the recruitment of such a strategy as an alternative to the classic active targeting that solely depends on the attachment of targeting ligands.

7th Annual meeting of the Nucleic Acids Therapeutics Society of Japan (NATSJ), Sola City Conference Center, Tokyo, Japan, 2022

The recent evolution of RNA therapeutics such as COVID-19 vaccines holds promise for treatment of... more The recent evolution of RNA therapeutics such as COVID-19 vaccines holds promise for treatment of various life-threatening diseases. Although active targeting was recruited in most DDS, a limited focus was given to the impact of the composition and properties of the nanocarriers on their in vivo performance. We designed lipid nanoparticles based on novel pH-sensitive lipids for the targeted delivery of diverse RNA therapeutics to various liver diseases models, including hepatocellular carcinoma and liver fibrosis. The composition and physico-chemical characteristics of the nanocarriers were manipulated to understand their impact in vivo following intravenous administration to mice. Moreover, the selectivity, biosafety, robustness of gene delivery, and therapeutic applications were investigated. Based on the composition of the nanocarrier, we could target different cell populations involved in hepatopathogenesis and the optimum conditions to target each cell type and disease model were identified. The designed nanocarriers demonstrated a high biosafety while maintaining a robust and selective gene delivery efficiency and outstanding therapeutic potential. Interestingly, our data revealed that the composition and physico-chemical properties of DDS are the key determinants of its in vivo behavior, enabling efficient targeting with a higher clinical translatability.

48th Annual Meeting of Controlled Release Society (CRS), Virtual Platform, 2021

Introduction: Hepatocellular Carcinoma (HCC) is one of the deadliest malignancies with high liabi... more Introduction: Hepatocellular Carcinoma (HCC) is one of the deadliest malignancies with high liability for chemoresistance (1). Sorafenib (SOR) is a multiple kinase inhibitor designated by the FDA as the drug of choice for resistant HCC. Nevertheless, there are no available therapies if the tumor develops resistance to SOR (2). While gene therapy holds promise for the treatment of multiple incurable diseases, gene delivery to HCC is challenged by the stroma-rich tumor microenvironment preventing most nanomedicines from efficient access to the tumor cells. Midkine (MK) is a growth cytokine overexpressed in HCC and associated with tumor growth and survival (3). In the current study, the mechanism of resistance to SOR via MK signaling pathway was explored and reversed by highly-efficient gene delivery nanovectors.

Methods: SOR-resistant HCC was established in vitro and in vivo. The recruitment of MK signaling pathway was investigated in presence/absence of MK knockdown by small interfering RNA (MK-siRNA). Combo lipid nanoparticles (cLNPs) were loaded with SOR and MK-siRNA and their composition and physico-chemical characteristics were tailored for efficient and selective MK knockdown in vivo. Then, the optimum cLNPs were used to eradicate SOR-resistant HCC in mice.

Results: The overexpression of MK gene correlated with the acquired resistance to SOR. Knocking down MK reversed the resistance to SOR by downregulating Signal transducer and activator of transcription 3 (STAT3)and Nuclear factor kabba (NF-kB) while upregulating Caspase-3. Tailoring the lipid composition and physico-chemical characteristics of cLNPs dramatically-affected their in vivo knockdown activity. The optimum cLNPs demonstrated highly-efficient and selective MK knockdown in the tumor with a median effective dose(ED~0.1 mg/Kg), and a minimal activity in the healthy liver tissues. Moreover, the cLNPs re-sensitized the resistant HCC to SOR at an interestingly-low SOR dose (2.5 mg/Kg) and demonstrated ~70% eradication of the tumor in mice.

Conclusion: We revealed a potential mechanism for the acquired resistance to SOR in HCC via MK signaling pathway. Manipulating the composition and physico-chemical properties of cLNPs significantly-affected their in vivo performance, with a highly-efficient and selective MK knockdown activity and reversal of the resistance to SOR. The novel findings offer a new therapeutic approach for incurable chemoresistant HCC with potential clinical applications.

6th Annual meeting of the Nucleic Acids Therapeutics Society of Japan (NATSJ), Tokushima University, Japan, virtual platform, 2021

Gene therapy has achieved a breakthrough in the treatment of numerous challenging disorders. Neve... more Gene therapy has achieved a breakthrough in the treatment of numerous challenging disorders. Nevertheless, delivery of nucleic acids to stroma-rich tumors like hepatocellular carcinoma (HCC) and pancreatic cancer faces solid obstacles due to the stroma barrier that hampers nanomedicines from accessing the tumor cells. In the current study, we designed lipid nanovectors based on a pH-sensitive lipid combined with various phospholipids. The particle size of the nanovectors was controlled in the range of 50-200 nm using a novel microfluidic device (iLiNP) to screen the optimum particle diameter capable of penetration through the stroma barrier following intravenous administration to an aggressive mouse model of HCC. The nanovectors were loaded with siRNA targeting the Midkine gene associated with tumorigenesis (MK-siRNA). Tweaking the lipid composition and particle size of the nanovectors created a powerful DDS (average particle size ~ 60 nm) that achieved a potent MK knockdown activity, with a median effective dose (ED50) ~ 0.1 mg / Kg. Furthermore, the optimized vectors delivered a cocktail of MK-siRNA and the cytotoxic drug, sorafenib (SOR), efficiently to HCC resulting in a synergistic eradication of the tumor with high selectivity and biosafety. It can be concluded that our DDS holds promise for potential clinical applications in the gene therapy of stroma-rich tumors.

The 1st ITB International Conference on Pharmaceutical Sciences and Pharmacy (ICPSP), Institut Teknologi Bandung, virtual platform, 2020

Delivery of nucleic acids to their target is challenging resulting from their delicate nature and... more Delivery of nucleic acids to their target is challenging resulting from their delicate nature and multiple biological obstacles. Despite the progress and achievements in nucleic acid delivery to in vitro tumor models as a proof-of-concept, the in vivo small interfering RNA (siRNA) delivery to tumors remains disappointing. In the current study, we designed multifunctional lipid nanocarriers for selective delivery of Midkine siRNA (MK-siRNA) to hepatocellular carcinoma (HCC) in vitro. After confirming their efficacy, an in vivo stroma-rich mouse model of HCC was challenged. Step-by-step optimization facilitated the elucidation of numerous challenges and their overcome through tweaking the composition and physico-chemical properties of these nanocarriers. Eventually, the optimized nanocarriers were loaded with a cocktail of MK-siRNA and the cytotoxic drug, sorafenib (SOR), for integrative eradication of HCC in mice. The designed nanocarriers showed high selectivity and efficiency in vitro with a median effective siRNA dose (ED50) as low as 7.5 nM with diminished effects on the other cell lines. Nevertheless, no significant tumor accumulation or gene silencing were achieved following intravenous administration to HCC-bearing mice. Extensive optimization of the pharmacokinetic performance, particle size and composition of the aforementioned nanocarriers resulted in a dramatic impact on their in vivo performance with a final ED50 of 0.1 mg/Kg in the tumor, the lowest reported so far, while minimal activity was shown in the healthy liver tissues. Furthermore, our nanoparticles facilitated highly-selective and efficient chemo-gene therapy of HCC in mice with a synergistic effect between SOR and MK-siRNA that lead to the eradication of the tumor at the lowest SOR dose that has ever been investigated, 2.5 mg/Kg. In a conclusion, understanding the successive obstacles encountered upon shifting from the in vitro to the in vivo microenvironments is a key factor that will determine the clinical potential of anticancer nucleic acid therapeutics.

The 6th Vancouver Nanomedicine Day (VND), University of British Columbia, virtual platform, 2020

Hepatocellular carcinoma (HCC) is a global challenge with limited efficient therapies. The stroma... more Hepatocellular carcinoma (HCC) is a global challenge with limited efficient therapies. The stroma-rich tumor microenvironment restricts most nanomedicines from accessing the tumor cells following systemic administration. Combinational therapy based on chemotherapy and gene therapy is a promising strategy for synergistic eradication of the tumor at lower doses. The rational design of nanocarriers dramatically-affect their in vivo performance and the fate of treatment. Combo lipid nanoparticles (cLNPs) were designed based on a novel pH-sensitive lipid, a diversity of phospholipids and a Highly-selective targeting peptide. cLNPs were loaded with the cytotoxic drug, sorafenib (SOR), and a small interfering RNA targeting the Midkine gene (MK-siRNA). The lipid composition of cLNPs was tweaked and the physico-chemical properties were manipulated using a novel microfluidic device, iLiNP. The lipid composition and physico-chemical properties of cLNPs significantly-controlled their pharmacokinetics, tumor penetration and gene knockdown efficiency. The optimized cLNPs showed highly-potent gene silencing in the tumor with an siRNA median effective dose of 0.1 mg/Kg following intravenous administration to HCC-bearing mice, compared to minimal effect on the healthy liver. Moreover, the novel combination recruited in this study synergistically-eradicated HCC in mice at surprisingly-low doses of SOR and MK-siRNA. We believe that our strategy has a promising potential for clinical application in HCC therapy.

47th Annual Meeting of Controlled Release Society (CRS), virtual platform, 2020

Introduction: Hepatocellular carcinoma (HCC) is the fifth most common cancer type worldwide and t... more Introduction: Hepatocellular carcinoma (HCC) is the fifth most common cancer type worldwide and the second cause of cancer-related deaths (1). The first line therapy by surgical intervention fails in advanced cases, while conventional chemotherapy has limited value due to the challenges facing HCC delivery including the presence of stroma barrier hindering most nanomedicines, the low sensitivity of HCC cells to cytotoxic drugs and the emergence of chemoresistance (2). The design of innovative nanocarriers capable of facing these challenges has become critical. The recruitment of combinational therapy in cancer treatment holds promise to maximize the benefits and reduce the side effects.

Methods: We introduced a novel combination of Sorafenib (SOR), the FDA’s drug of choice for resistant HCC, and a small interfering RNA against the Midkine gene (MK-siRNA), an overexpressed gene with multiple malignant roles in HCC. Co-delivery lipid nanoparticles (LNPs) were prepared based on the pH-sensitive lipid, YSK05, developed in our laboratory and were modified with a highly-selective ligand for HCC, SP94 peptide. The LNPs were evaluated in vitro for their efficiency and selectivity to various cell lines. To extend their applicability in vivo, we engineered the composition and physico-chemical properties of our LNPs to overcome the physiological barriers and the challenging tumor microenvironment. The optimized LNPs were applied to eradicate HCC tumor in mice after intravenous administration.

Results: Our LNPs showed high selectivity and cytotoxicity to human and murine HCC cells, HepG2 and Hepa 1-6, respectively compared to limited effect on other cancerous cells, HeLa, and negligible effect on normal hepatocytes, FL83B. We showed the first evidence that MK-siRNA increases the sensitivity of HCC cells to SOR. In vivo tweaking of LNPs composition and characteristics resulted in ultra-small nanoparticles with high tumor accumulation and powerful highly-selective gene knockdown in the tumor (half-maximal effective dose, ED50= 0.1 mg/Kg) compared to the healthy liver after intravenous administration in mice. Furthermore, the LNPs loaded with SOR and MK-siRNA at low doses could dramatically eradicate HCC in mice (~85%) compared to mice received monotherapy suggesting the synergism between the two agents used. No toxicity was observed during the treatment.

Conclusion: The novel combination of SOR and MK-siRNA showed synergistic anticancer effect against HCC both in vitro and in vivo. Optimization of the nanocarrier’s composition and characteristics could dramatically affect their in vivo fate and performance. The promising results shown by our novel nanocarrier are encouraging for future translation into clinical HCC therapy.

The 7th International Life Science Symposium for Young Scientists (7th ILSS), Hokkaido University , 2019

Hepatocellular carcinoma (HCC) is the 5 th most common cancer type and the worldwide's third caus... more Hepatocellular carcinoma (HCC) is the 5 th most common cancer type and the worldwide's third cause of cancer-related deaths. Conventional chemotherapy is the only available therapeutic option for non-resectable or metastatic tumors, but it is challenged by high off-target cytotoxicity and the emergence of multidrug resistance by cancer cells. In the current study, we used RNA interference gene therapy (RNAi) by small interfering RNA (siRNA) to knockdown Midkine gene (MK), an overexpressed gene in HCC, which is responsible for anti-apoptotic and chemoresistant functions. We proposed that using such approach in combination with the cytotoxic drug, sorafenib (SOR), would act synergistically to maximize the anticancer activity and reduce the therapeutic dose of the drug. We designed a novel lipid-based nanocarrier for the co-delivery of SOR and MK-siRNA to HCC cells based on YSK05, a novel pH-responsive lipid synthesized in our laboratory. Nanoparticles were modified with a novel targeting peptide to ensure highly-specific delivery of the payload. The performance of the delivery system and the cargo was evaluated by cytotoxicity and gene knockdown studies. Furthermore, optimization of the different formulation variables maximized the efficiency of the combination and the biotolerability of the nanocarrier. We showed the first proof that MK-siRNA increases the sensitivity of HCC cells to SOR. In addition, our system showed amazing selectivity to HCC cells compared to other cancerous and normal cells. Our selective and efficient co-delivery system, encapsulating the novel anticancer combination, holds promise as a novel strategy for HCC treatment. We are currently optimizing it for the in vivo application.

34th Annual Meeting of The Academy of Pharmaceutical Science and Technology, Japan (APSTJ), Toyama International Conference Hall, Toyama, Japan, 2019

Hepatocellular carcinoma (HCC) is the worldwide's fifth cause of death. In the present study, we ... more Hepatocellular carcinoma (HCC) is the worldwide's fifth cause of death. In the present study, we designed a novel lipid-based nanoplatform for highly-specific and efficient co-delivery of the cytotoxic drug, sorafenib (SOR), and small interfering RNA against Midkine gene (MK-siRNA) to HCC cells. SOR is designated by the FDA as the drug of choice for resistant HCC. Midkine (MK) is a growth cytokine highly-expressed in HCC with multiple malignant functions. The system was modified with a specific peptide ligand for selective uptake into HCC cells. We showed the first evidence that the combination of SOR and MK-siRNA had a synergistic cytotoxic effect on HCC cells. Our laboratory's novel pH-sensitive cationic lipid, YSK05, showed the highest efficiency among the investigated lipids. Moreover, optimization of formulation variables improved the system's outcome and resulted in a highly-specific internalization in both human and mouse HCC cells, (HepG2 and Hepa 1-6, respectively) compared to a significantly-lower internalization in other cancerous cells from non-HCC origin (HeLa), and no internalization in normal hepatocytes (FL83B). Cell viability studies confirmed the same highly-selective cytotoxicity pattern. The nanoplatform developed in this study has high potential as a promising therapy for HCC with minimum effect on normal cells.

16th Liposome Research Days (LRD), Hokkaido University Conference Hall, Sapporo, Japan, 2019

Hepatocellular carcinoma (HCC) is the fifth most common malignancy type around the world. The cur... more Hepatocellular carcinoma (HCC) is the fifth most common malignancy type around the world. The current study aimed at the design of a novel smart liposome for efficient and highly-specific co-delivery of the cytotoxic drug, Sorafenib (SOR), and a small interfering RNA against Midkine gene (MK-siRNA) to HCC cells. SOR is a multiple kinase inhibitor approved as the drug of choice for resistant HCC, but it is limited by its non-selective cytotoxicity and liability for the emergence of chemoresistance. Midkine is a growth cytokine overexpressed in HCC cells with several malignant functions including resistance to apoptosis. YSK05, a novel smart pH-sensitive cationic lipid synthesized in our laboratory, was selected as the main lipid for the formulation of the co-delivery liposome and showed superior efficiency over other investigated lipids in terms of both chemotherapy and gene therapy. Optimization of various formulation variables improved the efficiency and biosafety of the liposome. Furthermore, the introduction of a novel targeting peptide, SP94, imparted high selectivity to hepatic cancer cells with a minimal effect on normal hepatocytes. Selectivity was confirmed in terms of cellular uptake, cytotoxicity and gene knockdown activities. Moreover, we showed the first evidence for synergism between SOR and MK-siRNA via the increase of HCC cell sensitivity to SOR by MK-siRNA. In addition, our liposome showed a pH-sensitive controlled release pattern of SOR which secures prolonged action and minimizes off-target cytotoxicity. It can be concluded that the SP94-modified smart liposome is a promising co-delivery system for the targeted combinational treatment of HCC.

The 6th International Life Science Symposium for Young Scientists (6th ILSS), Hokkaido University, 2018

Hepatocellular carcinoma (HCC) is the 5 th most common cancer type and one of the most aggressive... more Hepatocellular carcinoma (HCC) is the 5 th most common cancer type and one of the most aggressive tumors with poor recovery rates. Sorafenib (SOR) is a potent multiple kinase inhibitor drug assigned by FDA as the drug of choice for resistant HCC. Midkine (MK) is a heparin-binding cytokine known to be overexpressed in several malignant tumors with mitogenic, angiogenic, anti-apoptotic and chemo-resistant functions. The aim of the current study was to design a nano-sized lipid-based co-delivery system encapsulating both SOR and a small interfering RNA (siRNA) against MK gene to create a synergistic formulation for maximal efficacy. Various lipids were screened and the highest efficiency was obtained with YSK05, a novel pH-sensitive lipid synthesized in our laboratory. Cell viability studies confirmed synergism between SOR and MK-siRNA with efficient gene silencing as demonstrated by quantitative real time polymerase chain reaction (qRT-PCR) studies. Optimization of different formulation variables created an efficient system with minimum carrier's toxicity. To improve system's specificity towards HCC, the system was decorated with a novel targeting ligand exclusive for HCC, namely SP94 peptide, and further optimization has been carried out to balance efficiency and specificity. The system was tested on HCC cell line, HepG2, versus normal hepatocytes, FL83B. Cellular uptake studies by flow cytometry indicated a significant difference between HCC and normal hepatocytes in the uptake of the optimized system proving system's selectivity for HCC. Cell viability data confirmed the high efficiency and specificity of the optimized system in HCC cells with a minimum effect on normal hepatocytes (Fig.1). The current data are promising and will be extrapolated to the in vivo level in the future.

Glibenclamide (GLC) is a potent oral hypoglycemic drug widely-used in the treatment of type II di... more Glibenclamide (GLC) is a potent oral hypoglycemic drug widely-used in the treatment of type II diabetes mellitus. It is practically-insoluble in water which hindering its dissolution at the gastric pH hence, lowering its absorption and oral bioavailability (45% only). The aim of this work was to improve such dissolution via formulation of adsorbates and co-adsorbates to enhance the oral bioavailability of GLC. This will enable its use at a lower dose to minimize the cost and possible side effects. Adsorption of drug onto different adsorbents including Laponite RD, Laponite FP, Neusilin US2, Aerosil 200 and Florite R was studied and Langmuir adsorption isotherms were constructed. Moreover, the effect of surfactant addition on the adsorption process was studied also using different concentrations of different surfactants namely; Tween 80, Pluronic F-127 and Pluronic F-68. Laponite RD as an adsorbent and Pluronic F-68 as a surfactant showed the best results and therefore, they were used in the formulation of adsorbates and co-adsorbates. Adsorbates of drug with Laponite RD were prepared in different weight ratios using two different techniques; physical mixing and solvent evaporation technique. Co-adsorbates of drug with pluronic F-68 and Laponite RD were prepared in different weight ratios by solvent evaporation technique. The prepared systems were tested for their drug content and in-vitro dissolution rate. The results showed marked enhancement of GLC in-vitro dissolution rate compared with untreated GLC which would be promising in further incorporation of the optimized formulation into several dosage forms with enhanced bioavailability.

The aim of this study was to improve the bioavailability of water-insoluble, anti-emetic drug; do... more The aim of this study was to improve the bioavailability of water-insoluble, anti-emetic drug; domperidone (DMP) which has a poor oral bioavailability (13-17%) due to extensive first pass metabolism. DMP was solubilized via formulation of solid dispersions with pluronic F-68 by fusion method in different weight ratios and the prepared solid dispersions were tested for their in-vitro dissolution rate to select the best ratio for final formulation. Then, the selected solid dispersions were incorporated into sublingual tablets together with different soluble excipients. Sublingual tablets were prepared by direct compression technique and they were evaluated for their physical properties and in-vitro dissolution rate. Sublingual tablets formulae S16 (containing fructose and 10% w/w Ac-Di-Sol) and S20 (containing fructose and 10% w/w Explotab) showed the best results and thus; they were selected for in-vivo studies in rabbits in comparison with the commercially-available oral tablets; Motinorm®. The selected formulae showed marked enhancement of DMP bioavailability compared with the marketed oral tablets, with relative bioavailability values of 432.49 ±10.13% and 409.32 ±11.59 % for S16 and S20, respectively. The results confirmed that sublingual tablets were promising tool for DMP delivery with marked enhancement of bioavailability.

Purpose: Domperidone (Dp) is a dopamine (D 2) receptor antagonist widely used as gastrointestinal... more Purpose: Domperidone (Dp) is a dopamine (D 2) receptor antagonist widely used as gastrointestinal prokinetic and antiemetic drug. It is practically insoluble in water and it is also a weak base having poor dissolution rates at relatively high pH values. The objective of this study is to enhance its dissolution via formulation in gastro-retentive floating tablets. While the system is floating on the gastric contents, the drug is released slowly at a desirable rate imparting a sustained-release effect. Methods: Several hydrophilic and hydrophobic polymers were used in the preparation of tablets matrices. Tablets were evaluated for their physical properties including weight uniformity, drug content, friability, hardness, thickness, floating time as well as the in vitro drug release. Results: The order of sustaining Dp release was exhibited by carbopol 934P > Eudragit RLPO > Eudragit RS100 > Eudragit RL100 > ethyl cellulose> hyroxypropylmethyl cellulose 15000>sodium alginate. Increasing the polymer/Dp ratio in the tablets increased the sustaining effect. Tablets containing 30% and 40 % (w/w) of either HPMC 15000 or sodium alginate showed the best floating properties and release profiles. Analysis of release data revealed that formulae containing cabopol 934P showed zero-order kinetics while the other formulae showed Higuchi model kinetics. All formulae showed non-fickian release pattern. Conclusion: formulation of floating tablets has successfully improved and sustained domperidone release over a time period of 12 hours. This may be beneficial in prolonging the prophylaxis against nausea and vomiting for a longer time eliminating the need for multiple dosing which also improves the patient compliance. Future work includes adding an immediate release component for instant prokinetic effect and in vivo comparative studies.

Domperidone is a dopamine antagonist antiemetic drug [1] , water-insoluble and weakly basic with ... more Domperidone is a dopamine antagonist antiemetic drug [1] , water-insoluble and weakly basic with poor dissolution rates at high pH values [2]. The current study aimed to improve such dissolution via adsorbates and co-adsorbates formulations. Adsorbates of drug with Avicel PH 101, Florite R and Aerosil 200 were prepared in different weight ratios by physical mixing, grinding and solvent deposition methods. Co-adsorbates of drug with Tween 80 and Aerosil 200 were prepared by solvent deposition method in different weight ratios. These systems were characterized using Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), powder X-ray diffractometry (P-XRD) and in-vitro dissolution. The results showed marked enhancement of domperidone dissolution at both pH1.2 and pH6.8 (7 fold and 5 fold, respectively) compared to drug alone.

Doctoral Dissertation, Graduate School of Life Science, Hokkaido University, Japan, 2020

Hepatocellular carcinoma (HCC), is the 5th most common cancer worldwide and the third most common... more Hepatocellular carcinoma (HCC), is the 5th most common cancer worldwide and the third most common cause of cancer-related deaths. Treating advanced stages of HCC is difficult and the response rate to most chemotherapeutics is still low (10-15%) mainly due to the emergence of multidrug resistance (MDR). These drawbacks necessitate looking for novel approaches such as gene therapy that can effectively cure the disease by targeting its primary cause at the molecular level. Sorafenib (SOR) is a multiple kinase inhibitor designated by FDA as the drug of choice for resistant HCC. However, it remains the last therapeutic approach and there are no effective solutions if the tumor develops resistance to it. In addition, its systemic administration is restricted by numerous toxicities and side effects. Meanwhile, Midkine (MK) is a growth cytokine overexpressed in HCC with anti-apoptotic, mitogenic, angiogenic and chemoresistant roles. In the current study, we propose that combining SOR with anti-Midkine nucleic acid therapy (MK-siRNA) would exert a synergistic therapeutic activity against HCC. Furthermore, we developed novel lipid nanoparticles (LNPs) for the co-delivery of such payload to HCC with high efficiency and selectivity. The selected main lipid was YSK05, a novel pH-sensitive lipid designed in our laboratory. LNPs were modified with a novel highly-selective HCC-targeting peptide, SP94. In the first part of this study, we tested our hypothesis in vitro. We optimized various aspects regarding the formulation and composition of these LNPs to control their efficiency and selectivity. Our results revealed significant uptake in human and murine HCC (HepG2 and Hepa 1-6, respectively) compared to other cancerous cells (HeLa) or normal hepatocytes (FL83B). Evaluation of the cytotoxicity and MK gene knockdown activity confirmed the same pattern of selectivity. In addition, we showed the first evidence that MK-siRNA increases the sensitivity of HCC cells to SOR as suggested from its effect on SOR dose-response curve which significantly differed when MK-siRNA was replaced by a control siRNA (siCntrl). The promising in vitro results triggered us to extend the applicability of our system to the in vivo level. Preliminary in vivo evaluation of the system after intravenous administration to HCC-bearing mice indicated that it could not accumulate in the tumor or exert MK knockdown activity there which reflected the challenges of the in vivo delivery to HCC. To overcome this, we designed a step-wise optimization process considering the possible challenges restricting the delivery of LNPs to the tumor; the pharmacokinetic performance, the stroma-rich tumor microenvironment hindering the penetration of LNPs to HCC cells, and the inadequate endosomal escape capability upon shifting from the in vitro to the in vivo situation. Controlling PEG-SP94 density in the LNPs dramatically affected their pharmacokinetic performance which lead to improvement in MK knockdown activity in the tumor, but the selectivity remained poor as indicated upon comparing the activity in the tumor versus the healthy liver. We applied a microfluidic device (iLiNP) and optimized diverse factors related to the operation or lipid composition to tune the particle size. Interestingly, reducing the particle size to 70 nm dramatically improved the gene knockdown activity and increased the selectivity to the tumor indicating successful penetration through the stroma barrier. However, further reduction in the particle size by increasing YSK05 ratio above 50 mol% sensitized Apolipoprotein E (ApoE) transporter in the serum to redirect the LNPs to the healthy liver, therefore LNPs with 70 nm size (50 mol% YSK05) were considered to be the optimum. To avoid significant effect on the healthy liver, we attempted masking ApoE recognition of our LNPs by filling the interspaces in their surface by additional DSPE-PEG2K-MA. Surprisingly, at 3 mol% ratio, the LNPs showed strong knockdown activity in the tumor compared to limited activity in the healthy liver. Moreover, the particle size of the new LNPs was reduced to 60 nm by the effect of the additional PEG. Eventually, we screened various helper lipids to maximize the endosomal escape performance which revealed the superiority of DOPE. The optimized system was referred to as ultra-small lipid nanoparticles (usLNPs). We evaluated the therapeutic activity of these usLNPs for the treatment of HCC-bearing mice. SOR and siRNA doses were adjusted to 2.5 mg/Kg and 0.5 mg/Kg, respectively. Treatment was given on days 7,10,13,16,19,22 and 25 post tumor inoculation and the mice were terminated on the 28th day. Surprisingly, SOR+MK-siRNA combination showed ~85% eradication of the tumor compared with SOR+siCntrl (~40%) or MK-siRNA (~18%) which suggested the synergism between SOR and MK-siRNA. The treatment did not result in significant body weight loss commonly seen with ordinary chemotherapeutics suggesting the biosafety of our usLNPs. Furthermore, treatment with SOR+MK-siRNA combination resulted in 3-fold down regulation of the characteristic HCC marker, Alpha fetoprotein (AFP), and 4-fold down regulation of Osteopontin (OSP) and vascular endothelial growth factor 1 (VEGF-1). We raised the challenge and attempted the treatment of SOR-resistant tumor. In vitro results suggested the ability of MK-siRNA to reverse the resistance to SOR in established SOR-resistant HepG2 cells. The upregulation of MK gene by 3-folds in the resistant cell line suggested the role of MK pathway in acquiring the resistance to SOR. Surprisingly, SOR+MK-siRNA combination could eradicate SOR-resistant tumor in mice by ~70% which was almost insensitive to SOR+siCntrl at the same dose. In conclusion, we designed LNPs for efficient and selective co-delivery of SOR and MK-siRNA to HCC both in vitro and in vivo. We reported the synergism between SOR and MK-siRNA for the first time. According to our knowledge, our system is the most efficient for siRNA delivery to HCC so far (ED50=0.1 mg/Kg). We could eradicate HCC in mice using the lowest dose of SOR so far (2.5 mg/Kg). We challenged SOR-resistant HCC for the first time. We believe that our system holds a great promise for potential clinical application in HCC treatment.

Master Thesis, Faculty of Pharmacy, Assiut University, Egypt, 2016

Domperidone (DMP) is widely used in the management of nausea and vomiting; especially those assoc... more Domperidone (DMP) is widely used in the management of nausea and vomiting; especially those associated with anticancer chemotherapy. Its oral bioavailability is only 13-17% due to its weakly-basic nature lowering the dissolution rate and absorption at high pH values. Moreover, the drug is subjected to extensive first-pass metabolism. This thesis has focused on the formulation of DMP into new dosage forms to enhance its bioavailability. Gastro-retentive floating tablets were formulated to retain the drug in the acidic medium favoring the drug release. Also, sublingual tablets were formulated to overcome first pass metabolism.

Silver Nanoparticles for Drug Delivery, 2024

Recently, there has been a growing interest in silver nanoparticles (AgNPs) and their biomedical ... more Recently, there has been a growing interest in silver nanoparticles (AgNPs) and their biomedical applications. Nevertheless, the clinical translation of such a promising technology into the global healthcare market encounters multiple obstacles. In the current chapter, we put some light on the market status and clinical profile of AgNPs as well as the technical, industrial, and regulatory challenges hampering their translation from the bench to the bedside. Then, some promising advancements that enable the clinical translation of AgNPs are highlighted, including the innovative high-throughput production technologies and the recruitment of AgNPs into the battle against the emerging infectious diseases and pandemics. Furthermore, AgNPs are compared versus other competitive nanotechnologies from a technical and clinical perspective. Lastly, we inspire the key aspects that should be considered for the successful translation of this area of endeavor into the clinics and the healthcare market.