HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies

Michels, Luana Roberta; Fachel, Flávia Nathiely Silveira; Azambuja, Juliana H.; Gelsleichter, Nicolly Espindola; Braganhol, Elizandra; Teixeira, Hélder Ferreira

doi:10.1002/bmc.4615

Cited by 12 publications

(5 citation statements)

References 20 publications

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“…Intracellular TMZ levels were quantified using high-performance liquid chromatography (HPLC) according to protocols from previous studies with modifications. 20 GPCs were treated with p38MAPK inhibitors for 24 h, followed by 500 µM TMZ. The cells were collected and lysed at 0, 5, 15, 30, 60, 120, and 180 min after TMZ exposure using modified RIPA lysis buffer (pH 3) containing 100 µM dexamethasone as the internal standard.…”

Section: Methodsmentioning

confidence: 99%

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide

Cheng,

Chong,

Lim

et al. 2024

Neuro-Oncology

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Background Precision treatment of glioblastoma is increasingly focused on molecular subtyping, with the mesenchymal subtype particularly resistant to temozolomide. Here, we aim to develop a targeted therapy for temozolomide resensitization in the mesenchymal subtype. Methods We integrated kinomic profiles and kinase inhibitor screens from patient-derived proneural and mesenchymal glioma-propagating cells public clinical datasets to identify key protein kinases implicated in temozolomide resistance. RNAseq, apoptosis assays and comet assays were used to examine the role of p38MAPK signaling and adaptive chemoresistance in mesenchymal cells. The efficacy of dual p38MAPK and MEK/ERK inhibition using ralimetinib (selective orally active p38MAPK inhibitor; phase I/II for glioblastoma) and binimetinib (approved MEK1/2 inhibitor for melanoma; phase II for high-grade glioma) in primary and recurrent mesenchymal tumors was evaluated using an intracranial patient-derived tumor xenograft model, focusing on survival analysis. Results Our transcriptomic-kinomic integrative analysis revealed p38MAPK as the prime target whose gene signature enables patient stratification based on their molecular subtypes and provides prognostic value. Repurposed p38MAPK inhibitors synergize favourably with temozolomide to promote intracellular retention of temozolomide and exacerbate DNA damage. Mesenchymal cells exhibit adaptive chemoresistance to p38MAPK inhibition through a pH-/calcium-mediated MEK/ERK pathway. Dual p38MAPK and MEK inhibition effectively maintains temozolomide sensitivity in primary and recurrent intracranial mesenchymal glioblastoma xenografts. Conclusion Temozolomide resistance in mesenchymal glioblastoma is associated with p38MAPK activation. Adaptive chemoresistance in p38MAPK-resistant cells is mediated by MEK/ERK signaling. Adjuvant therapy with dual p38MAPK and MEK inhibition prolongs temozolomide sensitivity, which can be developed into a precision therapy for the mesenchymal subtype.

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Section: Methodsmentioning

confidence: 99%

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide

Cheng,

Chong,

Lim

et al. 2024

Neuro-Oncology

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“…Blank NP was also produced for comparative purposes. The encapsulation efficiency analyses (EE%) of TMZ into PLGA nanoparticles were performed applying validated reverse-phase high-performance liquid chromatography (RP-HPLC) methodology adapted from Michels and co-workers ( Michels et al, 2019 ) with standard analytical curve built-in acetic acid 0.5% (y = 74177x + 1360,6; r 2 = 1,00).…”

Section: Methodsmentioning

confidence: 99%

Using design of experiments (DoE) to optimize performance and stability of biomimetic cell membrane-coated nanostructures for cancer therapy

Ferreira

Miranda

Moreno

et al. 2023

Front. Bioeng. Biotechnol.

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Introduction: Cell membrane-covered biomimetic nanosystems have allowed the development of homologous nanostructures to bestow nanoparticles with enhanced biointerfacing capabilities. The stability of these structures, however, still represents a challenge for the scientific community. This study is aimed at developing and optimizing cell derived membrane-coated nanostructures upon applying design of experiments (DoE) to improve the therapeutic index by homotypic targeting in cancer cells.Methods: Important physicochemical features of the extracted cell membrane from tumoral cells were assessed by mass spectrometry-based proteomics. PLGA-based nanoparticles encapsulating temozolomide (TMZ NPs) were successfully developed. The coating technology applying the isolated U251 cell membrane (MB) was optimized using a fractional two-level three-factor factorial design. All the formulation runs were systematically characterized regarding their diameter, polydispersity index (PDI), and zeta potential (ZP). Experimental conditions generated by DoE were also subjected to morphological studies using negative-staining transmission electron microscopy (TEM). Its short-time stability was also assessed. MicroRaman and Fourier-Transform Infrared (FTIR) spectroscopies and Confocal microscopy were used as characterization techniques for evaluating the NP-MB nanostructures. Internalization studies were carried out to evaluate the homotypic targeting ability.Results and Discussion: The results have shown that nearly 80% of plasma membrane proteins were retained in the cell membrane vesicles after the isolation process, including key proteins to the homotypic binding. DoE analysis considering acquired TEM images reveals that condition run five should be the best-optimized procedure to produce the biomimetic cell-derived membrane-coated nanostructure (NP-MB). Storage stability for at least two weeks of the biomimetic system is expected once the original characteristics of diameter, PDI, and ZP, were maintained. Raman, FTIR, and confocal characterization results have shown the successful encapsulation of TMZ drug and provided evidence of the effective coating applying the MB. Cell internalization studies corroborate the proteomic data indicating that the optimized NP-MB achieved specific targeting of homotypic tumor cells. The structure should retain the complex biological functions of U251 natural cell membranes while exhibiting physicochemical properties suitable for effective homotypic recognition.Conclusion: Together, these findings provide coverage and a deeper understanding regarding the dynamics around extracted cell membrane and polymeric nanostructures interactions and an in-depth insight into the cell membrane coating technology and the development of optimized biomimetic and bioinspired nanostructured systems.

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“…Standard solutions of TMZ have been prepared in the 10–180 µg/mL concentration range by appositely diluting in water a stock TMZ solution containing 147.1 mg of TMZ in 25 mL of a 1:1 mixture of MeOH and diluted aqueous HCl (pH = 4.5). After microfiltration of the prepared solutions, they have been injected in HPLC by adapting a method described previously 52 . In particular, HPLC analysis was performed on an Agilent LC1120 system equipped with a manual injector using a C-18 column (300 mm × 4.6 mm i.d., 5 μm particle size) and a methanol/acetic acid (0.03% in water) 35/65 as mobile phase.…”

Section: Methodsmentioning

confidence: 99%

Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models

Gherardini

Buratti

Maturi

et al. 2023

Sci Rep

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Glioblastoma multiforme (GBM) is the most aggressive primary tumor of the central nervous system and the diagnosis is often dismal. GBM pharmacological treatment is strongly limited by its intracranial location beyond the blood–brain barrier (BBB). While Temozolomide (TMZ) exhibits the best clinical performance, still less than 20% crosses the BBB, therefore requiring administration of very high doses with resulting unnecessary systemic side effects. Here, we aimed at designing new negative temperature-responsive gel formulations able to locally release TMZ beyond the BBB. The biocompatibility of a chitosan-β-glycerophosphate-based thermogel (THG)-containing mesoporous SiO2 nanoparticles (THG@SiO2) or polycaprolactone microparticles (THG@PCL) was ascertained in vitro and in vivo by cell counting and histological examination. Next, we loaded TMZ into such matrices (THG@SiO2-TMZ and THG@PCL-TMZ) and tested their therapeutic potential both in vitro and in vivo, in a glioblastoma resection and recurrence mouse model based on orthotopic growth of human cancer cells. The two newly designed anticancer formulations, consisting in TMZ-silica (SiO2@TMZ) dispersed in the thermogel matrix (THG@SiO2-TMZ) and TMZ, spray-dried on PLC and incorporated into the thermogel (THG@PCL-TMZ), induced cell death in vitro. When applied intracranially to a resected U87-MG-Red-FLuc human GBM model, THG@SiO2-TMZ and THG@PCL-TMZ caused a significant reduction in the growth of tumor recurrences, when compared to untreated controls. THG@SiO2-TMZ and THG@PCL-TMZ are therefore new promising gel-based local therapy candidates for the treatment of GBM.

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HPLC–UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies

Cited by 12 publications

References 20 publications

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide

Dual p38MAPK and MEK inhibition disrupts adaptive chemoresistance in mesenchymal glioblastoma to temozolomide

Using design of experiments (DoE) to optimize performance and stability of biomimetic cell membrane-coated nanostructures for cancer therapy

Loco-regional treatment with temozolomide-loaded thermogels prevents glioblastoma recurrences in orthotopic human xenograft models

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