| 1. |
- Ahlgren, Sara, 1979-, et al.
(författare)
-
EGF-targeting lipodisks for specific delivery of poorly water-soluble anticancer agents to tumour cells
- 2017
-
Ingår i: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 7:36, s. 22178-22186
-
Tidskriftsartikel (refereegranskat)abstract
- Concerns regarding poor aqueous solubility, high toxicity and lack of specificity impede the translation of many hydrophobic anticancer agents into safe and effective anticancer drugs. The application of colloidal drug delivery systems, and in particular the use of lipid-based nanocarriers, has been identified as a promising means to overcome these issues. PEG-stabilized lipid nanodisks (lipodisks) have lately emerged as a novel type of biocompatible, nontoxic and adaptable drug nanocarrier. In this study we have explored the potential of lipodisks as a platform for formulation and tumour targeted delivery of hydrophobic anticancer agents. Using curcumin as a model compound, we show that lipodisks can be loaded with substantial amounts of hydrophobic drugs (curcumin/lipid molar ratio 0.15). We demonstrate moreover that by deliberate choice of preparation protocols the lipodisks can be provided with relevant amounts of targeting proteins, such as epidermal growth factor (EGF). Data from in vitro cell studies verify that such EGF-decorated curcumin-loaded lipodisks are capable of EGF-receptor specific targeting of human A-431 tumour cells, and strongly suggest that the interaction between the lipodisks and the tumour cells results in receptor-mediated internalization of the disks and their cargo.
|
|
| 2. |
- Ali, Sajid, et al.
(författare)
-
Dual centrifugation as a novel and efficient method for the preparation of lipodisks
- 2024
-
Ingår i: International Journal of Pharmaceutics. - : Elsevier. - 0378-5173 .- 1873-3476. ; 653
-
Tidskriftsartikel (refereegranskat)abstract
- Polyethylene glycol (PEG)-stabilized lipodisks have emerged as innovatiive, promising nanocarriers for several classes of drugs. Prior research underscores the important role of lipid composition and preparation method in determining the lipodisk size, uniformity, and drug loading capacity. In this study, we investigate dual centrifugation (DC) as a novel technique for the production of PEG-stabilized lipodisks. Moreover, we explore the potential use of DC for the encapsulation of two model drugs, curcumin and doxorubicin, within the disks. Our results show that by a considerate choice of experimental conditions, DC can be used as a fast and straightforward means to produce small and homogenous lipodisks with a hydrodynamic diameter of 20-30 nm. Noteworthy, the technique works well for the production of both cholesterol-free and cholesterol-containing disks and does not require pre-mixing of the lipids in organic solvent. Furthermore, our investigations confirm the efficacy of DC in formulating curcumin and doxorubicin within these lipodisks. For doxorubicin, careful control and optimization of the experimental conditions resulted in formulations displaying an encouraging encapsulation efficiency of 84 % and a favourable drug-to-lipid ratio of 0.13 in the disks.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Björkelund, Hanna, et al.
(författare)
-
Avoiding false negative results in specificity analysis of protein-protein interactions
- 2011
-
Ingår i: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 24:1, s. 81-89
-
Tidskriftsartikel (refereegranskat)abstract
- The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × K(D) is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed.
|
|
| 6. |
- Björkelund, Hanna, et al.
(författare)
-
Comparing the epidermal growth factor interaction with four different cell lines : intriguing effects imply strong dependency of cellular context
- 2011
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:1, s. e16536-
-
Tidskriftsartikel (refereegranskat)abstract
- The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of (125)I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the (125)I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K(D)≈200 pM on SKBR3 cells to K(D)≈8 nM on A431 cells. The (125)I-EGF - EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the (125)I-EGF - EGFR affinity, in particular when the cells are starved. The (125)I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.
|
|
| 7. |
- Björkelund, Hanna, et al.
(författare)
-
Gefitinib Induces Epidermal Growth Factor Receptor Dimers Which Alters the Interaction Characteristics with (125)I-EGF
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:9, s. e24739-
-
Tidskriftsartikel (refereegranskat)abstract
- The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer (R) Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of (125)I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0-3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8-2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF - EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with (125)I-EGF.
|
|
| 8. |
- Björkelund, Hanna, 1983-
(författare)
-
Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions : Resolving How the Epidermal Growth Factor Binds to Its Receptor
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.
|
|
| 9. |
- Björkelund, Hanna, 1983-, et al.
(författare)
-
Resolving the EGF-EGFR interaction characteristics through a multiple-temperature, multiple-inhibitor, real-time interaction analysis approach
- 2013
-
Ingår i: Molecular and Clinical Oncology. - : Spandidos Publications. - 2049-9469 .- 2049-9450. ; 1:2, s. 343-352
-
Tidskriftsartikel (refereegranskat)abstract
- Overexpression and aberrant activity of the epidermal growth factor (EGF) have been observed in various cancer types, rendering it an important target in oncology research. The interaction between EGF and its receptor (EGFR), as well as subsequent internalization, is complex and may be affected by various factors including tyrosine kinase inhibitors (TKIs). By combining real‑time binding curves produced in LigandTracer® with internalization assays conducted at different temperatures and with different TKIs, the processes of ligand binding, internalization and excretion was visualized. SKOV3 cells had a slower excretion rate compared to A431 and U343 cells, and the tested TKIs (gefitinib, lapatinib, AG1478 and erlotinib) reduced the degree of internalization. The kinetic analysis of the binding curves further demonstrated TKI‑dependent balances of EGFR monomer and dimer populations, where lapatinib promoted the monomeric form, while the other TKIs induced dimers. The dimer levels were found to be associated with the apparent affinity of the EGF‑EGFR interaction, with EGF binding stronger to EGFR dimers compared to monomers. This study analyzed how real‑time molecular interaction analysis may be utilized in combination with perturbations in order to understand the kinetics of a ligand‑receptor interaction, as well as some of its associated intracellular processes. Our multiple‑temperature and ‑inhibitor assay setup renders it possible to follow the EGFR monomer, dimer and internalized populations in a detailed manner, allowing for a new perspective of the EGFR biology.
|
|
| 10. |
- Bohl Kullberg, Erika, et al.
(författare)
-
Development of EGF-conjugated liposomes for targeted delivery of boronated DNA-binding agents
- 2002
-
Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 13:4, s. 737-743
-
Tidskriftsartikel (refereegranskat)abstract
- Liposomes are of interest as drug delivery tools for therapy of cancer and infectious diseases. We investigated conjugation of epidermal growth factor, EGF, to liposomes using the micelle-transfer method. EGF was conjugated to the distal end of PEG−DSPE lipid molecules in a micellar solution and the EGF−PEG−DSPE lipids were then transferred to preformed liposomes, either empty or containing the DNA-binding compound, water soluble acridine, WSA. We found that the optimal transfer conditions were a 1-h incubation at 60 °C. The final conjugate, 125I-EGF−liposome−WSA, contained approximately 5 mol % PEG, 10−15 EGF molecules at the liposome surface, and 104 to 105 encapsulated WSA molecules could be loaded. The conjugate was shown to have EGF-receptor-specific cellular binding in cultured human glioma cells.
|
|
