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  • 1. Biswas, Abhijit
    et al.
    Maloverjan, Maria
    Padari, Kärt
    Abroi, Aare
    Rätsep, Margus
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. CellPept Sweden AB, Sweden.
    Jarvet, Jüri
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. CellPept Sweden AB, Sweden.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. CellPept Sweden AB, Sweden.
    Kisand, Vambola
    Löhmus, Rünno
    Pooga, Margus
    Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery2023Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 15, nr 2, artikel-id 396Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP–NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide–cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

  • 2. Carissimi, Guzmán
    et al.
    Montalbán, Mercedes G.
    Villora, Gloria
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Direct Quantification of Drug Loading Content in Polymeric Nanoparticles by Infrared Spectroscopy2020Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 12, nr 10, artikel-id 912Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Nanotechnology has enabled the development of novel therapeutic strategies such as targeted nanodrug delivery systems, control and stimulus-responsive release mechanisms, and the production of theranostic agents. As a prerequisite for the use of nanoparticles as drug delivery systems, the amount of loaded drug must be precisely quantified, a task for which two approaches are currently used. However, both approaches suffer from the inefficiencies of drug extraction and of the solid-liquid separation process, as well as from dilution errors. This work describes a new, reliable, and simple method for direct drug quantification in polymeric nanoparticles using attenuated total reflection Fourier transform infrared spectroscopy, which can be adapted for a wide variety of drug delivery systems. Silk fibroin nanoparticles and naringenin were used as model polymeric nanoparticle carrier and drug, respectively. The specificity, linearity, detection limit, precision, and accuracy of the spectroscopic approach were determined in order to validate the method. A good linear relation was observed within 0.00 to 7.89% of naringenin relative mass with an R-2 of 0.973. The accuracy was determined by the spike and recovery method. The results showed an average 104% recovery. The limit of detection and limit of quantification of the drug loading content were determined to be 0.3 and 1.0%, respectively. The method's robustness is demonstrated by the notable similarities between the calibrations carried out using two different equipment setups at two different institutions.

  • 3. El-Garawani, Islam
    et al.
    El-Seedi, Hesham
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Menoufia University, Egypt; Jiangsu University, China.
    Khalifa, Shaden
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    El Azab, Islam H.
    Abouhendia, Marwa
    Mahmoud, Shaymaa
    Enhanced Antioxidant and Cytotoxic Potentials of Lipopolysaccharides-Injected Musca domestica Larvae2020Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 12, nr 11, artikel-id 1111Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The usage of insects as a sustainable and functional natural products resource is a new promise in complementary and alternative medicine. The present study aimed to investigate the ability of Musca domestica (housefly) larval hemolymph (insect blood) to display the enhanced in vitro antioxidant and cytotoxic effects. The oxidative stress (OS) was elicited by inducing lipopolysaccharides (LPS) treatment as an exogenous stressor. Determination of superoxide dismutase 1 (SOD1), glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity (TAC), and mRNA and protein expressions of SOD1, was investigated as confirmatory markers of oxidative stress induction. Cytotoxicity on cancerous MCF-7 and normal Vero cells were also evaluated using an MTT assay at 24 h post-injection. The injection of LPS induced a significant (p < 0.05) increase in SOD, GSH and TAC, whereas, the MDA was diminished. Hemolymph was collected from normal and treated larvae after 6, 12 and 24 h. The M. domestica superoxide dismutase (MdSOD1) transcripts were significantly (p < 0.05) upregulated 6 and 12 h post-treatment, while a significant downregulation was observed after 24 h. Western blot analysis showed that MdSOD1 was expressed in the hemolymph of the treated larvae with an increase of 1.2 folds at 6 and 12 h and 1.6 folds at 24 h relative to the control group. LPS-treated larval hemolymphs exhibited significant cytotoxicity with respect to the untreated ones against MCF-7 while Vero cells showed no cytotoxicity for both hemolymphs. The DPPH free radical scavenging activity was examined and a significant antioxidant potential potency was observed at 6 h (50% maximal inhibitory concentration (IC50): 63.3 +/- 3.51 mu g/mL) when compared to the control M. domestica larval hemolymph (IC50: 611.7 +/- 10.41 mu g/mL). Taken together, M. domestica larval hemolymph exhibited enhanced antioxidant and consequently increased cytotoxic capacities under stressed conditions.

  • 4. El-Garawani, Islam
    et al.
    Emam, Mahmoud
    Elkhateeb, Waill
    El-Seedi, Hesham
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Jiangsu University, China; Al-Rayan Colleges, Saudi Arabia; Uppsala University, Sweden.
    Khalifa, Shaden
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Oshiba, Salwa
    Abou-Ghanima, Shaimaa
    Daba, Ghoson
    In Vitro Antigenotoxic, Antihelminthic and Antioxidant Potentials Based on the Extracted Metabolites from Lichen,Candelariella vitellina2020Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 12, nr 5, artikel-id 477Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lichens have recently received great attention due to their pharmacological potentials. The antigenotoxic potential ofC. vitellinaextract (25 and 50 mu g/mL) was assessed in normal human peripheral blood lymphocytes (HPBL) against Mitomycin C (MMC) co-treatments. Flow cytometric analyses of cell cycle distribution, as well as apoptosis (Annexin V/PI), revealed that the extract had significantly (p <= 0.05) ameliorated the MMC toxicity by reducing the apoptotic cells and normalized the cell cycle phases.C. vitellinaexhibited antigenotoxicity by ameliorating the diminished mitotic index and DNA single-strand breaks caused by MMC. Herein, the hydromethanolic extract (80%) ofCandelariella vitellina(Japan) lichen, exhibited very low cytotoxicity towards normal human peripheral lymphocytes (HPBL) with IC50>1000 mu g/mL. In order to explore the antihelminthic effect,Echinococcus granulosusprotoscoleces were used in vitro. Eosin staining revealed significant (p <= 0.05) dose and time-dependent scolicidal effects of the extract confirmed by degenerative alterations as observed by electron scan microscopy. Furthermore, primary and secondary metabolites were investigated using GC-MS and qualitative HPLC, revealing the presence of sugars, alcohols, different phenolic acids and light flavonoids. Significant antioxidant capacities were also demonstrated by DPPH radical-scavenging assay. In conclusion, the promising antigenotoxic, antihelminthic and antioxidant potentials ofC. vitellinaextract encourage further studies to evaluate its possible therapeutic potency.

  • 5.
    Holmbäck, Jan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). Lipidor AB, Sweden.
    Rinwa, Vibhu
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK). Lipidor AB, Sweden.
    Halthur, Tobias
    Rinwa, Puneet
    Carlsson, Anders
    Herslöf, Bengt
    AKVANO®: A Novel Lipid Formulation System for Topical Drug Delivery—In Vitro Studies2022Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 14, nr 4, artikel-id 794Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel formulation technology called AKVANO® has been developed with the aim to provide a tuneable and versatile drug delivery system for topical administration. The vehicle is based on a water-free lipid formulation where selected lipids, mainly phospholipids rich in phosphatidylcholine, are dissolved in a volatile solvent, such as ethanol. With the aim of describing the basic properties of the system, the following physicochemical methods were used: viscometry, dynamic light scattering, NMR diffusometry, and atomic force microscopy. AKVANO formulations are non-viscous, with virtually no or very minute aggregates formed, and when applied to the skin, e.g., by spraying, a thin film consisting of lipid bilayer structures is formed. Standardized in vitro microbiological and irritation tests show that AKVANO formulations meet criteria for antibacterial, antifungal, and antiviral activities and, at the same time, are being investigated as a non-irritant to the skin and eye. The ethanol content in AKVANO facilitates incorporation of many active pharmaceutical ingredients (>80 successfully tested) and the phospholipids seem to act as a solubilizer in the formulation. In vitro skin permeation experiments using Strat-M® membranes have shown that AKVANO formulations can be designed to alter the penetration of active ingredients by changing the lipid composition.

  • 6. Kiisholts, Kristina
    et al.
    Kurrikoff, Kaido
    Arukuusk, Piret
    Porosk, Ly
    Peters, Maire
    Salumets, Andres
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Tartu, Estonia.
    Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models2021Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 13, nr 10, artikel-id 1618Artikel i tidskrift (Refereegranskat)
    Abstract [en]

     Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.

  • 7.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Tartu, Estonia.
    Cell-Penetrating Peptides and Transportan2021Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 13, nr 7, artikel-id 987Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    In the most recent 25-30 years, multiple novel mechanisms and applications of cell-penetrating peptides (CPP) have been demonstrated, leading to novel drug delivery systems. In this review, I present a brief introduction to the CPP area with selected recent achievements. This is followed by a nostalgic journey into the research in my own laboratories, which lead to multiple CPPs, starting from transportan and paving a way to CPP-based therapeutic developments in the delivery of bio-functional materials, such as peptides, proteins, vaccines, oligonucleotides and small molecules, etc.

  • 8. Saldanha, Leonor
    et al.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Tartu, Estonia.
    Vale, Nuno
    In Silico Studies to Support Vaccine Development2023Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 15, nr 2, artikel-id 654Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The progress that has been made in computer science positioned in silico studies as an important and well-recognized methodology in the drug discovery and development process. It has numerous advantages in terms of costs and also plays a huge impact on the way the research is conducted since it can limit the use of animal models leading to more sustainable research. Currently, human trials are already being partly replaced by in silico trials. EMA and FDA are both endorsing these studies and have been providing webinars and guidance to support them. For instance, PBPK modeling studies are being used to gather data on drug interactions with other drugs and are also being used to support clinical and regulatory requirements for the pediatric population, pregnant women, and personalized medicine. This trend evokes the need to understand the role of in silico studies in vaccines, considering the importance that these products achieved during the pandemic and their promising hope in oncology. Vaccines are safer than other current oncology treatments. There is a huge variety of strategies for developing a cancer vaccine, and some of the points that should be considered when designing the vaccine technology are the following: delivery platforms (peptides, lipid-based carriers, polymers, dendritic cells, viral vectors, etc.), adjuvants (to boost and promote inflammation at the delivery site, facilitating immune cell recruitment and activation), choice of the targeted antigen, the timing of vaccination, the manipulation of the tumor environment, and the combination with other treatments that might cause additive or even synergistic anti-tumor effects. These and many other points should be put together to outline the best vaccine design. The aim of this article is to perform a review and comprehensive analysis of the role of in silico studies to support the development of and design of vaccines in the field of oncology and infectious diseases. The authors intend to perform a literature review of all the studies that have been conducted so far in preparing in silico models and methods to support the development of vaccines. From this point, it was possible to conclude that there are few in silico studies on vaccines. Despite this, an overview of how the existing work could support the design of vaccines is described.

  • 9.
    Österlund, Nicklas
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wärmländer, Sebastian K. T. S.
    Stockholms universitet, Humanistiska fakulteten, Institutionen för arkeologi och antikens kultur. CellPept Sweden AB, Sweden.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. CellPept Sweden AB, Sweden.
    Cell-Penetrating Peptides with Unexpected Anti-Amyloid Properties2022Ingår i: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 14, nr 4, artikel-id 823Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) with sequences derived originally from a prion protein (PrP) have been shown to exhibit both anti-prion and anti-amyloid properties particularly against prion proteins and the amyloid-β (Aβ) peptide active in Alzheimer’s disease. These disease-modifying properties are so far observed in cell cultures and in vitro. The CPP sequences are composed of a hydrophobic signal sequence followed by a highly positively charged hexapeptide segment. The original signal sequence of the prion protein can be changed to the signal sequence of the NCAM1 protein without losing the anti-prion activity. Although the detailed molecular mechanisms of these CPP peptides are not fully understood, they do form amyloid aggregates by themselves, and molecular interactions between the CPPs and PrP/Aβ can be observed in vitro using various spectroscopic techniques. These initial intermolecular interactions appear to re-direct the aggregation pathways for prion/amyloid formation to less cell-toxic molecular structures (i.e., co-aggregates), which likely is why the disease-inducing PrP/Aβ aggregation is counteracted in vivo.

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