PHYSIOCHEMICAL STABILITY AND MASS SPECTROMETRIC ANALYSIS OF GEMINI SURFACTANT-BASED LIPOPLEXES

Cationic lipids have been comprehensively studied as non-viral vectors for gene therapy, focusing on improving the gene transfer efficiency and the safety profile. However, clinical applications of cationic lipid/DNA lipoplexes are restricted due to their low physical stability in aqueous formulations. One specific group of cationic lipids that showed efficient transfection activity is the gemini surfactants. Two main objectives were determined in this work. The first was to evaluate the feasibility of lyophilization as a formulation technique for preparing gemini surfactant-based lipoplexes with long-term stability. The second objective was to establish a universal tandem mass spectrometric “fingerprint” of novel amino acid modified gemini surfactants as a pre-requirement for the identification and quantification of gemini surfactants in different pharmaceutical matrices. In order to investigate the influence of lyophilization on the essential physiochemical properties and the in vitro transfection efficiency of gemini surfactant-lipoplexes, a diquaternary ammonium gemini surfactant (12-7NH-12) and plasmid DNA (pDNA) encoding for interferon-γ (IFNγ) were used to prepare pDNA/gemini surfactant [P/G] lipoplexes. Helper lipid DOPE [L] was incorporated in all formulations producing a [P/G/L] system. Several excipients were utilized as stabilizing agents. Lipoplexes formulated with the cryoprotectant were subjected to a lyophilization/rehydration cycle. Transfection activity was assessed by measuring the level of expressed IFNγ and cellular toxicity (MTT assay). The results showed that the physiochemical properties of gemini surfactant-based lipoplexes were dependent on the nature of the stabilizing agents used to prepare the lipoplexes. Disaccharide sugars, sucrose and trehalose, provided the most efficient cryoprotectant effect based on their ability to physically stabilize the lipoplexes during the lyophilization process. The transfection efficiency of the lyophilized lipoplexes increased 2-3 fold compared to fresh formulations upon lyophilization. This effect can be attributed to the improvement of DNA compaction and changes in the lipoplex morphology due to the lyophilization/rehydration cycles. Based on these results, we evaluated the ability of lyophilization to improve the stability of gemini surfactant-based lipoplexes. Four lyophilized formulations were stored at 25˚C for three months. The formulations were analyzed monthly for physical appearance, physiochemical properties (particle size and zeta potential, pDNA compaction, gemini surfactant:pDNA interaction) and in vitro transfection. The physiochemical properties of the lyophilized formulations were maintained throughout the three month study. All lyophilized formulations showed a loss of gene transfection activity after three months of storage. Nevertheless, no significant losses of transfection efficiency were observed for three formulations after two months storage at 25 ˚C. These findings suggest that lyophilization significantly improved the physiochemical stability of gemini surfactant-based lipoplexes compared to liquid formulations. As well, lyophilization improved the transfection efficiency of gemini surfactant-based lipoplexes. The loss of transfection activity upon storage is most probably due to the conformational changes in the supramolecular structure of the lipoplexes as a function of time and temperature, rather than to DNA degradation. To establish a foundation for employing the mass spectrometric methods in the evaluation of the chemical stability of the gemini surfactant, we evaluated the tandem mass spectrometric (MS/MS) behavior of six amino acid/di-peptide modified gemini surfactants that were synthesized based on the precursor compound 12-7NH-12. This was accomplished by using a hybrid quadrupole orthogonal time-of-flight mass spectrometer (QqToF-MS) and a triple quadrupole linear ion trap mass spectrometer (QqQ-LIT MS) equipped with electrospray ionization (ESI) source. The single stage QqToF-MS data obtained in the positive ion mode verified the molecular composition of all tested gemini surfactants. Tandem mass spectrometric (MS/MS) analysis showed common fragmentation behavior among all tested compounds, allowing for the establishment of a universal fragmentation pattern. The fragmentation pathway was confirmed by MS/MS/MS experiments utilizing a Q-TrapTM 4000 LC/MS/MS system and (MS/MS) analysis of the deuterated form of 12-7N(Glycine)-12 gemini surfactant. Unique product ions, originating from the loss of one or both head groups along with the attached tail region(s), confirmed the chemical structure of the tested compounds. In conclusion, different lyophilization strategies and analytical methods have been established to develop and examine the physiochemical stability of gemini surfactant-based lipoplex. A tandem mass spectrometric fragmentation pathway was established to enable the identification and quantification of these compounds in pharmaceutical formulations

Rapid and Simple Flow Injection Analysis-Tandem Mass Spectrometric (FIA-MS/MS) Method for the Quantification of Melphalan in Lipid-Based Drug Delivery System

Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation (CFI)Peer ReviewedThe use of the anticancer drug melphalan is limited due to its poor water solubility. To address such limitation, it is incorporated within a novel delivery system using β-cyclodextrin-gemini surfactants (18:1βCDg). Herein, two fast and simple FIA-MS/MS methods are developed for the quantification of melphalan (Mel) within the drug delivery system so that the solubilization efficiency of the system can be assessed. FIA-MS/MS methods are developed using a triple quadrupole-linear ion trap mass spectrometer, equipped with electrospray ionization (ESI) in the positive ion mode. A deuterated form of melphalan (melphalan-d8) was used as an internal standard (IS). The methods were validated according to the FDA guidance. A linearity in the range of 2–100 ng/mL and accuracy and precision below 15% were observed for all standard points and quality control samples. The intra- and inter-day variations, freeze-thaw stability were within the acceptable range according to the criteria set by regulatory guidelines. On the other hand, other stability measures, such as room temperature stability and long term stability did not meet the required guidelines in some cases, indicating the need for quick sample analysis upon preparation. Such a fact could have been overlooked if full method validation was not performed. The developed methods were applied to determine the encapsulation/solubilisation of [18:1βCDg\Mel] delivery system. 18:1βCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at the 18:1βCDg\Mel complex molar ratio of 2:1. This study demonstrated that a fast analysis for the purpose of quantifying a chemically unstable drug, such as melphalan is feasible and important for the development of commercial dosage forms

Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy

Purpose Achieving successful gene therapy requires delivery of a gene vector specifically to the targeted tissue with efficient expression and a good safety profile. The objective of this work was to develop, characterize and determine if a novel gemini surfactant-based lipoplex systems, modified with a cancer-targeting peptide p18-4, could serve this role. Methods The targeting peptide p18-4 was either chemically coupled to a gemini surfactant backbone or physically co-formulated with the lipoplexes. The influence of targeting ligand and formulation strategies on essential physicochemical properties of the lipoplexes was evaluated by dynamic light scattering and small angle X-ray scattering techniques. In vitro transfection activity and cellular toxicity of lipoplexes were assessed in a model human melanoma cell line. Results All lipoplexes zeta potential and particle size were optimal for cellular uptake and physical stability of the system. The lipoplexes adopted an inverted-hexagonal lipid arrangement. The lipoplexes modified with the peptide showed no significant changes in physicochemical properties or lipoplex assembly. The modification of the lipoplexes with the targeting peptide significantly enhanced protein expression 2-6 fold compared to non-modified lipoplexes. In addition, p18-4 modified lipoplexes significantly improved the safety of the lipoplexes. The ability of the p18-4 modified lipoplexes to selectively express the model protein was confirmed by using healthy human epidermal keratinocytes (HEKa). Conclusion The gemini surfactant-based lipoplexes modified with p18-4 peptide showed significantly higher efficiency and safety compared to the system that did not contain a cancer targeting peptide and provided evidence for their potential application to achieve targeted melanoma gene therapy

Analysis of a Series of Chlorogenic Acid Isomers using Differential Ion Mobility and Tandem Mass Spectrometry

Canada Foundation for InnovationChlorogenic acids are among the most abundant phenolics found in the human diet. Of these, the mono-caffeoylquinic acids are the predominant phenolics found in fruits, such as apples and pears, and products derived from them. In this research, a comprehensive study of the electrospray ionization (ESI) tandem mass spectrometric (MS/MS) dissociation behavior of the three most common mono-caffeoylquinic acids, namely 5-O-caffeoylquinic acid (5-CQA), 3-O-caffeoylquinic acid (3-CQA) and 4-O-caffeoylquinic acid (4-CQA), were determined using both positive and negative ionization. All proposed structures of the observed product ions were confirmed with second-generation MS3 experiments. Similarities and differences between the dissociation pathways in the positive and negative ion modes are discussed, confirming the proposed structures and the established MS/MS fingerprints. MS/MS dissociation was primarily driven via the cleavage of the ester bond linking the quinic acid moiety to the caffeic acid moiety within tested molecules. Despite being structural isomers with the same m/z values and dissociation behaviors, the MS/MS data in the negative ion mode was able to differentiate the three isomers based on ion intensity for the major product ions, observed at m/z 191, 178 and 171. This differentiation was consistent among various MS instruments. In addition, ESI coupled with high-field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) was employed for the separation of these compounds for the first time. By combining MS/MS data and differential ion mobility, a method for the separation and identification of mono-caffeoylquinic in apple/pear juice samples was developed with a run time of less than one minute. It is envisaged that this methodology could be used to identify pure juices based on their chlorogenic acid profile (i.e., metabolomics), and could also be used to detect juice-to-juice adulteration (e.g., apple juice addition to pear juice)

NOVEL GEMINI SURFACTANT-BASED GENE/DRUG DELIVERY SYSTEMS Potential Applications in Melanoma Therapy

Melanoma is the most life-threatening form of skin cancer and world-wide statistics show an alarming increase in melanoma incidence and mortality. The aim of my work was to develop and investigate the potential applications of novel drug/gene delivery systems in melanoma therapy. In the first research theme, I explored the application of targeted-gemini surfactant-based gene delivery system in melanoma therapy. Two cancer targeting-peptides (cyclic-RGD and p18-4]) were used to develop melanoma-targeting lipoplexes. Different formulations and transfection techniques were developed and evaluated. Physicochemical and structural properties of peptide-modified lipoplexes were examined. In vitro transfection efficiency and cellular toxicity of the developed lipoplexes were evaluated in human melanoma cell line. Results showed that peptide-modified gemini surfactant-based lipoplexes significantly enhanced the gene transfection activity in A375 cells compared to the non-targeted formulation. In the second research theme, I investigated the application of a novel β-cyclodextrin (βCD) modified gemini surfactant as a potential drug delivery system in melanoma. βCD was chemically attached to a gemini surfactant, producing a novel βCD-conjugate (18:1βCDg) that can self-assemble and form supramolecular nano-complexes. The 18:1βCDg was designed to combine the solubilizing capacity of the βCD and the cell-penetrating ability of the gemini surfactant. Melphalan (Mel) was selected as a model compound for a poorly soluble drug. To assess the solubilizing efficiency of 18:1βCDg, flow-injection analysis tandem mass spectrometric (FIA-MS/MS) methods were developed. FIA-MS/MS results showed a significant increase (over three-fold) in the aqueous solubility of Mel, at 2:1 host-guest molar ratio. Supporting this finding is the fact that One-/two-dimensional Rotating-frame-Overhauser Spectroscopy-Nuclear Magnetic Resonance (1D/2D ROESY-NMR) results indicated the formation of stable 18:1βCDg/Mel inclusion complex at the same molar ratio. In vitro evaluations showed that the optimized 18:1βCDg/Mel complexes significantly improved the chemotherapeutic efficacy of Mel in monolayer, 3D spheroid and in Mel-resistant melanoma cell lines. Subsequently, in vivo acute toxicity in Sprague-Dawley rats and hemolysis assessment were conducted to determine the carrier’s safety profile. It was shown that 18:1βCDg has low hemolytic effect in comparison to native βCD and basic gemini surfactant molecule. Acute toxicity assessments indicated that 18:1βCDg was well-tolerated without significant toxicity

Inclusion Complexes of Melphalan with Gemini-Conjugated β-Cyclodextrin: Physicochemical Properties and Chemotherapeutic Efficacy in In-Vitro Tumor Models

β-cyclodextrin (βCD) has been widely explored as an excipient for pharmaceuticals and nutraceuticals as it forms stable host–guest inclusion complexes and enhances the solubility of poorly soluble active agents. To enhance intracellular drug delivery, βCD was chemically conjugated to an 18-carbon chain cationic gemini surfactant which undergoes self-assembly to form nanoscale complexes. The novel gemini surfactant-modified βCD carrier host (hereafter referred to as 18:1βCDg) was designed to combine the solubilization and encapsulation capacity of the βCD macrocycle and the cell-penetrating ability of the gemini surfactant conjugate. Melphalan (Mel), a chemotherapeutic agent for melanoma, was selected as a model for a poorly soluble drug. Characterization of the 18:1βCDg-Mel host–guest complex was carried out using 1D/2D 1H NMR spectroscopy and dynamic light scattering (DLS). The 1D/2D NMR spectral results indicated the formation of stable and well-defined 18:1βCDg-Mel inclusion complexes at the 2:1 host–guest mole ratio; whereas, host–drug interaction was attenuated at greater 18:1βCDg mole ratio due to hydrophobic aggregation that accounts for the reduced Mel solubility. The in vitro evaluations were performed using monolayer, 3D spheroid, and Mel-resistant melanoma cell lines. The 18:1βCDg-Mel complex showed significant enhancement in the chemotherapeutic efficacy of Mel with 2–3-fold decrease in Mel half maximal inhibitory concentration (IC50) values. The findings demonstrate the potential applicability of the 18:1βCDg delivery system as a safe and efficient carrier for a poorly soluble chemotherapeutic in melanoma therapy

Zirconium-89 Labeled Gene Delivery Nanoparticles as Theranostic Agents for Melanoma

KAL

Impact of the COVID-19 pandemic on patients with paediatric cancer in low-income, middle-income and high-income countries: a multicentre, international, observational cohort study

OBJECTIVES: Paediatric cancer is a leading cause of death for children. Children in low-income and middle-income countries (LMICs) were four times more likely to die than children in high-income countries (HICs). This study aimed to test the hypothesis that the COVID-19 pandemic had affected the delivery of healthcare services worldwide, and exacerbated the disparity in paediatric cancer outcomes between LMICs and HICs. DESIGN: A multicentre, international, collaborative cohort study. SETTING: 91 hospitals and cancer centres in 39 countries providing cancer treatment to paediatric patients between March and December 2020. PARTICIPANTS: Patients were included if they were under the age of 18 years, and newly diagnosed with or undergoing active cancer treatment for Acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, Hodgkin lymphoma, Wilms' tumour, sarcoma, retinoblastoma, gliomas, medulloblastomas or neuroblastomas, in keeping with the WHO Global Initiative for Childhood Cancer. MAIN OUTCOME MEASURE: All-cause mortality at 30 days and 90 days. RESULTS: 1660 patients were recruited. 219 children had changes to their treatment due to the pandemic. Patients in LMICs were primarily affected (n=182/219, 83.1%). Relative to patients with paediatric cancer in HICs, patients with paediatric cancer in LMICs had 12.1 (95% CI 2.93 to 50.3) and 7.9 (95% CI 3.2 to 19.7) times the odds of death at 30 days and 90 days, respectively, after presentation during the COVID-19 pandemic (p<0.001). After adjusting for confounders, patients with paediatric cancer in LMICs had 15.6 (95% CI 3.7 to 65.8) times the odds of death at 30 days (p<0.001). CONCLUSIONS: The COVID-19 pandemic has affected paediatric oncology service provision. It has disproportionately affected patients in LMICs, highlighting and compounding existing disparities in healthcare systems globally that need addressing urgently. However, many patients with paediatric cancer continued to receive their normal standard of care. This speaks to the adaptability and resilience of healthcare systems and healthcare workers globally