Solid Lipid Nanoparticles for Pulmonary Delivery of Biopharmaceuticals: A Review of Opportunities, Challenges, and Delivery Applications.

Biopharmaceuticals such as nucleic acids, proteins, and peptides constitute a new array of treatment modalities for chronic ailments. Invasive routes remain the mainstay of administering biopharmaceuticals due to their labile nature in the biological environment. However, it is not preferred for long-term therapy due to the lack of patient adherence and clinical suitability. Therefore, alternative routes of administration are sought to utilize novel biopharmaceutical therapies to their utmost potential. Nanoparticle-mediated pulmonary delivery of biologics can facilitate both local and systemic disorders. Solid lipid nanoparticles (SLNs) afford many opportunities as pulmonary carriers due to their physicochemical stability and ability to incorporate both hydrophilic and hydrophobic moieties, thus allowing novel combinatorial drug/gene therapies. These applications include pulmonary infections, lung cancer, and cystic fibrosis, while systemic delivery of biomolecules, like insulin, is also attractive for the treatment of chronic ailments. This Review explores physiological and particle-associated factors affecting pulmonary delivery of biopharmaceuticals. It compares the advantages and limitations of SLNs as pulmonary nanocarriers along with design improvements underway to overcome these limitations. Current research illustrating various SLN designs to deliver proteins, peptides, plasmids, oligonucleotides, siRNA, and mRNA is also summarized.

Recent Developments in Inertial and Centrifugal Microfluidic Systems along with the Involved Forces for Cancer Cell Separation: A Review.

The treatment of cancers is a significant challenge in the healthcare context today. Spreading circulating tumor cells (CTCs) throughout the body will eventually lead to cancer metastasis and produce new tumors near the healthy tissues. Therefore, separating these invading cells and extracting cues from them is extremely important for determining the rate of cancer progression inside the body and for the development of individualized treatments, especially at the beginning of the metastasis process. The continuous and fast separation of CTCs has recently been achieved using numerous separation techniques, some of which involve multiple high-level operational protocols. Although a simple blood test can detect the presence of CTCs in the blood circulation system, the detection is still restricted due to the scarcity and heterogeneity of CTCs. The development of more reliable and effective techniques is thus highly desired. The technology of microfluidic devices is promising among many other bio-chemical and bio-physical technologies. This paper reviews recent developments in the two types of microfluidic devices, which are based on the size and/or density of cells, for separating cancer cells. The goal of this review is to identify knowledge or technology gaps and to suggest future works.

Optimized Chitosan-Based Nanoemulsion Improves Luteolin Release.

Luteolin (LUT) is a flavonoid found in several edible and medicinal plants. It is recognized for its biological activities such as antioxidant, anti-inflammatory, neuroprotective, and antitumor effects. However, the limited water solubility of LUT leads to poor absorption after oral administration. Nanoencapsulation may improve the solubility of LUT. Nanoemulsions (NE) were selected for the encapsulation of LUT due to their biodegradability, stability, and ability to control drug release. In this work, chitosan (Ch)-based NE was developed to encapsulate luteolin (NECh-LUT). A 23 factorial design was built to obtain a formulation with optimized amounts of oil, water, and surfactants. NECh-LUT showed a mean diameter of 67.5 nm, polydispersity index 0.174, zeta potential of +12.8 mV, and encapsulation efficiency of 85.49%. Transmission electron microscopy revealed spherical shape and rheological analysis verified the Newtonian behavior of NECh-LUT. SAXS technique confirmed the bimodal characteristic of NECh-LUT, while stability analysis confirmed NECh-LUT stability when stored at room temperature for up to 30 days. Finally, in vitro release studies showed LUT controlled release up to 72 h, indicating the promising potential of NECh-LUT to be used as novel therapeutic option to treat several disorders.

Mucoadhesive nanoemulsion enhances brain bioavailability of luteolin after intranasal administration and induces apoptosis to SH-SY5Y neuroblastoma cells.

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children and accounts for 7 % of all childhood malignancies and 15 % cancer mortality in children. Luteolin (LUT) is recognized by its anticancer activity against several types of cancer. The aim of this study was to prepare chitosan-coated nanoemulsion containing luteolin (NECh-LUT), investigate its potential for brain delivery following intranasal administration, and to evaluate its cytotoxicity against neuroblastoma cells. NECh-LUT was developed by cavitation process and characterized for its size, surface charge, encapsulation efficiency, and mucoadhesion. The developed formulation presented size 68 ± 1 nm, zeta potential + 13 ± 1 mV, and encapsulation efficiency of 85.5 ± 0.3 %. The NECh-LUT presented nearly 6-fold higher permeation through the nasal mucosa ex vivo and prolonged LUT release up to 72 h in vitro, following Baker-Lonsdale kinetic model. The pharmacokinetic evaluation of NECh-LUT revealed a 10-fold increase in drug half-life and a 4.4 times enhancement in LUT biodistribution in brain tissue after intranasal administration of single-dose. In addition, NECh-LUT inhibited the growth of neuroblastoma cells after 24, 48 and 72 h in concentrations starting from 2 µM. The NECh-LUT developed for intranasal administration proved to be a promising alternative for brain delivery of LUT, and a viable option for the treatment of neuroblastoma.

Determination of phytosterol oxidation products in pharmaceutical liposomal formulations and plant vegetable oil extracts using novel fast liquid chromatography - Tandem mass spectrometric methods.

Phytosterol oxidation products (POPs) formed by the auto-oxidation of phytosterols can lead to negative health consequences. New liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative and qualitative approaches were developed. For quantification, sixteen phytosterol oxidation products (POPs) in liposomal formulations; namely 7-keto, 7-hydroxy, 5,6-epoxy, and 5,6-dihydroxy derivatives of brassicasterol, campesterol, stigmasterol, and ß-sitosterol were quantified. The method has a short run time of 5 min, achieved on a poroshell C18 column, using isocratic elution. To the best of our knowledge, this is the shortest run time among reported methods for the quantitative analysis of POPs. Atmospheric pressure chemical ionization (APCI) was used, and the mobile phase was composed of acetonitrile/methanol (99:1 v/v). The quantitative method was validated as per the FDA guidelines for linearity, accuracy, precision, selectivity, sensitivity, matrix effect, dilution integrity, and stability. The method was applied for the quantification of POPs in liposomal phytosterol formulations prepared with and without tocopherols, as antioxidants. The formulation process had little impact on the formation of POPs as only 7-ketobrassicasterol was quantified in tested samples. The quantified value of POPs in liposomal samples was insignificant to impart any toxicological effects. Other degradation products such as 7-hydroxy, 5,6-epoxy and 5,6-dihydroxy derivatives of brassicasterol, campesterol and ß-sitosterol were below the lower limit of quantification. Phytosterol-containing formulations were then assessed for their oxidative stability after microwave exposure for 5 min. The incorporation of tocopherols significantly increased the stability of phytosterols in the liposomal formulations. Finally, LC-MS/MS qualitative identification of phytosterols obtained from extra virgin olive oil was performed. New POPs, namely 7-ketoavenasterol, and 7-ketomethylenecycloartenol were putatively identified, illustrating the applicability of the method to identify POPs with varying structures present in various phytosterol sources. In fact, it is the first time that 7-ketomethylenecycloartenol is reported as a POP.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Drug Delivery Technology Development in Canada.

Canada has a long and rich history of ground-breaking research in drug delivery within academic institutions, pharmaceutical industry and the biotechnology community. Drug delivery refers to approaches, formulations, technologies, and systems for transporting a pharmaceutical compound in the body as needed to safely achieve its desired therapeutic effect. It may involve rational site-targeting, or facilitating systemic pharmacokinetics; in any case, it is typically concerned with both quantity and duration of the presence of the drug in the body. Drug delivery is often approached through a drug's chemical formulation, medical devices or drug-device combination products. Drug delivery is a concept heavily integrated with dosage form development and selection of route of administration; the latter sometimes even being considered part of the definition. Drug delivery technologies modify drug release profile, absorption, distribution and elimination for the benefit of improving product efficacy and safety, as well as patient convenience and adherence. Over the past 30 years, numerous Canadian-based biotechnology companies have been formed stemming from the inventions conceived and developed within academic institutions. Many have led to the development of important drug delivery products that have enhanced the landscape of drug therapy in the treatment of cancer to infectious diseases.  This Special Issue serves to highlight the progress of drug delivery within Canada. We invited articles on all aspects of drug delivery sciences from pre-clinical formulation development to human clinical trials that bring to light the world-class research currently undertaken in Canada for this Special Issue.

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

ß-cyclodextrin (ßCD) 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, ßCD was chemically conjugated to an 18-carbon chain cationic gemini surfactant which undergoes self-assembly to form nanoscale complexes. The novel gemini surfactant-modified ßCD carrier host (hereafter referred to as 18:1ßCDg) was designed to combine the solubilization and encapsulation capacity of the ßCD 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 18:1ßCDg-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 18:1ßCDg-Mel inclusion complexes at the 2:1 host-guest mole ratio; whereas, host-drug interaction was attenuated at greater 18:1ßCDg 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 18:1ßCDg-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 18:1ßCDg delivery system as a safe and efficient carrier for a poorly soluble chemotherapeutic in melanoma therapy.

Peptide-Modified Gemini Surfactants: Preparation and Characterization for Gene Delivery.

Diquaternary ammonium-based gemini surfactants have been investigated widely as nonviral gene delivery systems. These unique cationic lipids have versatility in their chemical structure, show relatively low toxicity, are able to compact genetic material (pDNA, RNA) into nano-sized lipoplexes, and can be easily produced. In addition, the gemini surfactants show significant improvement in the transfection activity and biocompatibility compared to other cationic lipids used as nonviral gene delivery agents. The successful applications of gemini surfactant-based lipoplexes as topical gene delivery systems in animal models indicate their potential as noninvasive carriers for genetic immunization, theranostic agents, and in other gene therapy treatments. Detailed physicochemical characterization of gemini surfactant lipoplexes is a key factor in terms of formulation optimization and elucidation of the cellular uptake and stability of the lipoplexes system. In this chapter, we describe in detail different formulation methods to prepare gemini surfactant lipoplexes and comprehensive physicochemical characterization. In addition, we illustrate general protocols for in vitro evaluations.

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and ß-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.

111In-Labeled Glycoprotein Nonmetastatic b (GPNMB) Targeted Gemini Surfactant-Based Nanoparticles against Melanoma: In Vitro Characterization and in Vivo Evaluation in Melanoma Mouse Xenograft Model.

Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.

The determination of gemini surfactants used as gene delivery agents in cellular matrix using validated tandem mass spectrometric method.

A simple, reliable flow injection analysis (FIA)-tandem mass spectrometric (MS/MS) method was developed for the determination of gemini surfactants, designated as 16-3-16, 16(Py)-S-2-S-(Py)16 and 16-7N(GK)-16, as gene delivery agents in cellular matrix. 16-3-16 is a conventional gemini surfactant bearing two quaternary amines, linked by a 3-carbon spacer region, 16(Py)-S-2-S-(Py)16 contains two pyridinium head groups, while 16-7N(GK)-16 bears a glycine-lysine di-peptide in the space region. The method was fully validated according to USFDA guidelines. It is the first time that FIA-MS/MS method was developed for the quantification of gemini surfactants, belonging to different structural families. The method was superior to existing liquid chromatographic (LC)-MS/MS methods in terms of sensitivity and time of analysis. Positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode were used on a triple quadrupole-linear ion trap (4000 QTRAP®) instrument. Deuterated internal standards were used to correct for matrix effects and variations in ionization within the ESI source. Isotope dilution standard curves were established in cellular matrix, with a linear range of 10 nM-1000 nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20 nM-2000 nM for 16-7N(GK)-16. The precision, accuracy, recovery and stability were all within the acceptable ranges as per the USFDA guidelines. The method was successfully applied for the quantification of target gemini surfactants in the nuclear fraction of PAM 212 keratinocyte cells treated with nanoparticles, which varied significantly and may explain differences in the observed efficiency and/or toxicity of these gemini surfactants in gene delivery.

A 89Zr-labeled lipoplex nanosystem for image-guided gene delivery: design, evaluation of stability and in vivo behavior.

BACKGROUND: With the advances in radiopharmaceutical research, the development of image-guided therapy has become a major interest. While the development of theranostic nanotherapeutics is frequently associated with cancer chemotherapy, phototherapy and radiotherapy, there is little information available on the in vivo monitoring of gene delivery systems and the application of image-guided approach in gene therapy. The goal of this work was to determine the in vivo behavior of DNA delivery nanosystems - based on cationic gemini surfactants - designed for image-guided gene therapy. We tested the feasibility of monitoring tumor accumulation of gene delivery nanoparticles by positron emission tomography. METHODS: To be able to conjugate radiotracers to the nanoparticles, a deferoxamine-modified gemini surfactant was synthesized, DNA-containing lipoplex nanoparticles were formulated, and radiolabeled with Zirconium-89 (89Zr). The pharmacokinetics and biodistribution of 89Zr labeled surfactant and 89Zr labeled nanoparticles were monitored in mice by microPET/CT imaging and ex vivo gamma counting. RESULTS: Modification of the nanoparticles with deferoxamine did not alter their physicochemical properties. The radiolabeled nanoparticles (labeling efficiency of 95±3%) were stable in PBS and serum. The biological half-life of the 89Zr labeled nanoparticles was significantly higher compared to 89Zr labeled surfactant. As expected, the nanoparticles had significantly higher liver accumulation than the radiolabeled surfactant alone and lower kidney accumulation. Tumor uptake was detected at 2 hours post injection and decreased throughout the 3-day monitoring. CONCLUSION: We propose that radiolabeling DNA delivery lipoplex nanosystems is a promising approach for the design and optimization of image-guided nanomedicines, especially in the context of cancer gene therapy.

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

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.

Molecular Engineering as an Approach To Modulate Gene Delivery Efficiency of Peptide-Modified Gemini Surfactants.

The unique molecular structure confers the diquaternary ammonium gemini surfactants with enhanced nucleic acid complexation ability, bottom-up design flexibility, and relatively low cytotoxicity. To capitalize on their potential as gene delivery vectors, novel structural modifications should be explored. In this work, 22 novel peptide-modified gemini surfactants with various alkyl tails and peptide spacer modifications were evaluated. This work represents the first report of dendrimer-like gemini surfactants and first evaluation of the impact of incorporating a hydrocarbon linker into the peptide chain. Our aim was to establish a structure activity relationship of the peptide-modified gemini surfactants and to identify the fundamental architectural requirements needed for the ultimate gene delivery systems. In vitro assessment revealed that the highest transfection efficiency and lowest cytotoxicity were associated with the glycyl-lysine modified gemini surfactants having the hexadecyl tail, 16-7N(G-K)-16. In fact, it showed an 8-fold increase in secreted protein with 20% increase in cell viability relative to the first-generation unsubstituted gemini surfactants. Further increase in the size of the attached peptides resulted in a decrease in the transfection efficiency and cell viability. Whereas the incorporation of a hydrocarbon linker into the peptide chain decreased the transfection efficiency of compounds with dipeptides, it increased the transfection efficiency of compounds with larger peptide chains. Such an increase was more prominent with the incorporation of a longer hydrocarbon linker. We conclude that a balance between the hydrophilic and hydrophobic characteristics of the compound is necessary since it results in physicochemical parameters conducive to the gene delivery process.

The development of simple flow injection analysis tandem mass spectrometric methods for the cutaneous determination of peptide-modified cationic gemini surfactants used as gene delivery vectors.

Diquaternary ammonium gemini surfactants are a class of non-viral gene delivery vectors, primarily studied for their dermal applications. However, their biological fate has rarely been investigated. In this work, we developed simple flow injection analysis tandem mass spectrometric methods, (FIA)-MS/MS, to understand the fate and biodistribution of topically applied gemini surfactant-based therapeutics in an ex-vivo skin model. Three peptide-modified gemini surfactants with varied structures and transfection efficiencies were evaluated. For each compound, two methods were developed to quantify their presence in skin tissue and in phosphate buffered saline (PBS). The methods were developed using single-point calibration mode. Skin penetration was assessed on CD1 mice dorsal skin tissue mounted in a Franz diffusion cell after extraction. Amongst the five evaluated liquid-liquid extraction protocols, the Folch method provides the highest extraction efficiency for all compounds. Weak cationic exchange solid phase extraction was also used to further isolate gemini surfactants from endogenous skin lipids. FIA-MS/MS analysis of the skin revealed that all compounds were detected in the skin with minimal partition into the PBS compartment, which represents circulation. Interestingly, the detected amounts of gemini lipids in the skin were correlated with their transfection efficiencies.

Rapid and simple flow injection analysis tandem mass spectrometric method for the quantification of melphalan in a lipid-based drug delivery system.

RATIONALE: The use of the anticancer drug melphalan is limited due to its poor water solubility. To address this limitation, it is incorporated within a novel delivery system using ß-cyclodextrin-gemini surfactants (18:1ßCDg). METHODS: Herein, two fast and simple flow injection analysis/tandem mass spectrometric (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. RESULTS: 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 and 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 had not been performed. CONCLUSIONS: The developed methods were applied to determine the encapsulation/solubilization of the [18:1ßCDg/Mel] delivery system. 18:1ßCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at a melphalan18:1ßCDg/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 RGD-Modified Gemini Surfactant-Based Lipoplexes for Targeted Gene Therapy in Melanoma Model.

PURPOSE: We have developed and evaluated novel peptide-targeted gemini surfactant-based lipoplexes designed for melanoma gene therapy. METHODS: Integrin receptor targeting peptide, cyclic-arginylglycylaspartic acid (cRGD), was either chemically coupled to a gemini surfactant backbone or physically co-formulated with lipoplexes. Several formulations and transfection techniques were developed. Transfection efficiency and cellular toxicity of the lipoplexes were evaluated in an in vitro human melanoma model. Physicochemical properties were examined using dynamic light scattering, zeta-potential, and small-angle X-ray scattering measurements. RESULTS: RGD-modified gemini surfactant based lipoplexes showed significant enhancement in gene transfection activity in A375 cell lines compared to the standard non-targeted formulation, especially when RGD was chemically conjugated to the gemini surfactant (RGD-G). The RGD had no effect on the cell toxicity profile of the lipoplex systems. Targeting specificity was confirmed by using an excess of free RGD and negative control peptide (RAD) and was demonstrated by using normal human epidermal keratinocytes. Physicochemical characterization showed that all nanoparticles were in the optimal size range for cellular uptake and there were no significant differences between RGD-modified and standard lipoplexes. CONCLUSIONS: These findings indicate the potential of RGD-modified gemini surfactant-based lipoplexes for use in melanoma gene therapy as an alternative to conventional chemotherapy.

Evaluation of ß-cyclodextrin-modified gemini surfactant-based delivery systems in melanoma models.

Novel drug delivery systems are developed to improve the biological behavior of poorly soluble drugs and to improve therapeutic outcomes. In melanoma therapy, the goal is efficient drug delivery and mitigation of drug resistance. Melphalan (Mel), a currently used therapeutic agent for melanoma, requires solvent system for solubilization, leading to poor chemical stability. Moreover, drug resistance often renders the drug inefficient in clinical setting. A novel ß-cyclodextrin-modified gemini surfactant (CDgemini) delivery system was developed to incorporate Mel in order to improve its physicochemical and biological behavior. Melphalan nanoparticles (Mel-NP) showed optimal particle size in the 200-250 nm range for endocytosis and induced significantly higher cell death compared with Mel (50% of inhibitory concentration [IC50] of 36 µM for the complexes vs 82 µM for Mel). The CDgemini delivery system did not alter the pathway of the cellular death triggered by Mel and caused no intrinsic toxicity to the cells. The Mel-NP complexes induced significant cell death in melanoma cells that were rendered resistant to Mel. These findings demonstrate in principle the applicability of the CDgemini delivery system as safe and efficient alternative to the current melanoma therapy, especially in chemoresistant cases.