Formulation and evaluation of itraconazole liposomes for hedgehog pathway inhibition

Abstract

Itraconazole (ITZ) is an FDA-approved antifungal agent that has recently been explored for novel biological properties. In particular, ITZ was identified as a potent inhibitor of the hedgehog (Hh) pathway, a cell signalling pathway that has been linked to a variety of cancers and accounts for ~ 25 % of pediatric medulloblastoma (MB) cases. To date, there is not a targeted therapeutic option for pediatric MB, resulting in long term side effects such as hormone deficiency, organ damage, and secondary cancers. A primary obstacle for developing targeted therapy for brain ailments is the presence of the blood brain barrier (BBB), which protects the brain from potentially harmful substances. Due to its size and hydrophobicity, ITZ does not penetrate the BBB. Alternatively, liposomes are being increasingly used within the clinic to increase drug bioavailability, target specificity, and BBB permeability. With this in mind, we have successfully developed ITZ-containing liposomes with an optimal size for BBB penetration (<100 nm) and encapsulation efficiency (~95 %) by utilizing a continuous manufacturing approach—turbulent coaxial jet in co-flow. Our preliminary in vitro data demonstrate that these liposomes inhibit the Hh pathway, albeit at a reduced level in comparison to free ITZ.

Introduction

Identifying novel biological activities of FDA-approved drugs has become a viable strategy to expedite the drug development process (Strittmatter 2014). A high-throughput screen of FDA-approved compounds identified itraconazole (ITZ), a clinically efficacious antifungal agent, as an inhibitor of the hedgehog (Hh) pathway (IC50 = 690 nM) (Kim et al. 2010). The Hh pathway is an embryonic developmental signalling pathway that plays a key role in cell differentiation and tissue growth (Amakye et al. 2013, Briscoe and Thérond 2013, Hadden 2014). Not surprisingly, aberrant Hh pathway activity results in abnormal cell proliferation and tumor growth and is primarily associated with basal cell carcinoma (BCC) and medulloblastoma (MB) (Ling et al. 2001, Reifenberger et al. 2005, Gibson et al. 2010, Hadden 2014). For this reason, Hh inhibition has become an attractive chemotherapeutic target. Most Hh pathway inhibitors target Smoothened (Smo), a key regulatory protein within the pathway (Jacoby et al. 2006, Robarge et al. 2009, Pan et al. 2010, Munchhof et al. 2012, Maschinot et al. 2015); however, there has been an emergence of resistance towards FDA-approved Smo inhibitors due to point mutations in Smo (D473H) (Rudin et al. 2009, Yauch et al. 2009, Dijkgraaf et al. 2011, Das et al. 2013; Atwood et al. 2015, Sharpe et al. 2015). This occurrence of Smo resistance highlights the therapeutic need for novel Hh inhibitory agents. ITZ is capable of inhibiting the Hh pathway in the presence of both wild type Smo and mutant Smo making it a promising therapeutic candidate for Hh-dependent malignancies (Kim et al. 2010, 2013).

MB is the most common malignant pediatric brain tumor accounting for ~ 20 % of all childhood brain cancers (Medulloblastoma | American Brain Tumor Association 2017, Medulloblastoma 2017). While there are varying subsets of MB, dysregulation of the Hh pathway is responsible for ~ 25 % of MB cases. Current treatment for MB requires invasive surgery to remove the tumor followed by radiation and high-dose chemotherapy to kill remaining cancer cells and prevent metastasis (Medulloblastoma | American Brain Tumor Association 2017, Medulloblastoma 2017). While survival rates are generally between 70 and 80%, this harsh treatment regimen leaves survivors with life-long side effects including, but not limited to, early onset secondary cancers, hormone deficiency, and organ damage (Medulloblastoma | American Brain Tumor Association 2017, Medulloblastoma 2017). For these reasons, it remains necessary to identify novel and targeted chemotherapeutic treatments for pediatric MB patients.

Hh pathway inhibitors, such as ITZ, provide an alternative approach towards effectively treating Hh-dependent MB. ITZ is a plausible pediatric anticancer chemotherapeutic agent as it has been successfully administered to children for the treatment of various fungal infections (Gupta et al. 2003). While ITZ has a well-known safety profile, the major obstacle in treating brain disorders is the presence of the blood brain barrier (BBB) (Lin and Yamazaki 2003, Pardridge 2003, Staud and Pavek 2005, Vasiliou et al. 2009, Oberoi et al. 2016). Often, drugs that could be advantageous chemotherapeutic agents are not considered due to physicochemical properties that prevent them from penetrating the BBB. While ITZ is already FDA-approved, it still exhibits properties that are not considered to be “drug-like” and may hinder effective BBB penetration (Ogu and Maxa 2000). For instance, ITZ is considered a “large” small molecule (MW = 705.64 g/mol) and it is poorly soluble in water (1 ng/mL) (Domínguez-Gil Hurlé et al. 2006, Savjani et al. 2012, Pubchem 2017). There have been extensive efforts to aid in improving the physicochemical properties of ITZ via rational design and medicinal chemistry (Pace et al. 2016, 2019). Additional efforts to improve ITZ solubility include utilizing formulations such as nano-amorphous powders and nanocrystalline suspensions of ITZ (Kumar et al. 2014, 2015).

Liposomal drug formulations have been FDA-approved for a wide variety of indications including cancer, fungal infections, viral infections, and pain management (Bulbake et al. 2017). There have been significant efforts to formulate ITZ into liposomes to improve solubility and provide an intravenous formulation for immunocompromised patients (Table 1) (Le Conte et al. 1992, Conte et al. 1994, Tang et al. 2010, Wang and Huang2011, Ćurić et al. 2013, Leal et al. 2015, Lankalapalli et al. 2017). These liposome formulations improved bioavailability, retained anti-fungal activity, and in one instance resulted in BBB penetration (Le Conte et al. 1992, Conte et al. 1994, Tang et al. 2010, Wang and Huang2011, Ćurić et al. 2013, Leal et al. 2015, Lankalapalli et al. 2017). Despite the lack of clinical stage ITZ liposome formulations, encapsulating ITZ into liposomes proves promising for the ultimate treatment of Hh-dependent MB (Home -ClinicalTrials.gov 2017). With this in mind, we set out to determine if ITZ liposomes could be utilized to effectively inhibit the Hh pathway in vitro.

Table 1.

Experimental ITZ-liposome formulations.

	Lipids	Solvent	Aqueous Phase	Ref
1	Soy phosphatidyl choline, cholesterol, stearylamine	CHCl3	Phosphate buffer (pH = 7.4)	(Leal et al. 2015)
2	Lecithin, cholesterol,	CHCl3	Phosphate buffer (pH = 5.8)	(Tang et al. 2010)
3	Lecithin, cholesterol, cyclodextrin	CHCl3: MeOH	N/A	(Lankalapalli et al. 2017)
4	Soy lecithin, cholesterol, carboxymethyl chitosan	CHCl3	Phosphate buffer (pH = 7.4)	(Wang and Huang 2011)
5	DPPC, cortisone acetate, polyethylene glycol (PEG200)	CHCl3	Phosphate buffer (pH = 7.4)	(Le Conte et al. 1992)
6	DPPC	CHCl3	10 mM PBS (pH = 7.4)	(Conte et al. 1994)
7	Egg PC, MPEG-2000 DSPE, cholesterol	CHCl3	6.67 mM KH2PO4 (pH = 6)	(Ćurić et al. 2013)

Dipalmitoylphosphotidylcholine (DPPC), potassium dihydrogen phosphate (KH₂PO₄), 1,2-distearoyl-phosphitdylehtanolamine-methyl-polyethylene glycol-2000 (DSPE)

Materials and Methods

Materials.

All chemicals and reagents were purchased from Fisher unless noted otherwise. Itraconazole (98% purity) was purchased from TCI. Hydrogenated soy phosphatidylcholine (HSPC), 1,2-distearoyl-phosphatidylethanolamine-methylpolyethylene glycol-2000 (mPEG-2000 DSPE), and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).

Liposome preparation.

ITZ-containing liposomes were formulated via a continuous manufacturing approach utilizing the coaxial turbulent jet in a co-flow instrument developed at the University of Connecticut (Costa et al. 2016). HSPC (900 mg), DSPC mPEG-2000 (300 mg), cholesterol (300 mg), and ITZ (60 mg) were dissolved in EtOH (100 mL) (organic phase) by heating for 20 minutes at 50 °C and sonication. In parallel, 4 L of a 6.67 mM KH₂PO₄ solution (aqueous phase) was prepared and adjusted to pH 6.0. The lipid solution was added to a stainless-steel tank within the coaxial turbulent jet instrument and this solution was incubated at 50 °C to ensure the lipids and ITZ remain in solution. The aqueous phase was attached to the apparatus within its preparation container. The lipid concentration and lipid formulation were added via computer control using a program designed and developed in our laboratory (National Instruments (NI) LabVIEW™) and the flow rates were set as desired. Liposome size and polydispersity index (PDI) were measured via an at-line Malvern Zetasizer Nano S during processing, upon equilibration of flow rates (organic: aqueous phase, mL/min) and liposomes were collected in 50 mL falcon centrifuge tubes. Liposome samples were stored at 4 °C until use. To date, ITZ liposomes have not been manufactured using the coaxial jet in a co-flow instrument, and as a result, this preliminary formulation (formulation-A) of ITZ loaded liposomes was prepared using a flow rate optimization approach. The system was equilibrated at each flow rate ratio (organic phase: aqueous phase, 10:90, 10:100, 10:300 and 30:300 mL/min) and the particle size and PDI were measured. Following the flow-rate study, an optimized ITZ loaded liposome formulation (formulation-B) was prepared at 30:300 mL/min (organic phase: aqueous phase) and analyzed for encapsulation efficiency and in vitro Hh pathway inhibitory activity.

Free drug separation.

Free ITZ was separated from liposomes by centrifugation (Torchilin and Weissig 2003). Liposomes were processed in a Beckman J2-HC centrifuge at 13,000 rpm for 90 minutes at 4 °C. Liposomes were decanted leaving behind a pellet containing the free drug.

Determination of encapsulation efficiency.

After removal of free drug, the percentage of drug encapsulated in the liposomes was determined using a UV spectrophotometer (BioTek Synergy H1 Hybrid Reader) (Piacentini 2016). Liposomes were lysed with 5 % Triton X-100 at 60 °C for 2 h. The absorbance of ITZ was determined at 254 nm in triplicate. The concentration of ITZ was obtained from a calibration curve of the drug (Panwar et al. 2010).

$$(\mathit{\text{Encapsulation Efficiency}}=\frac{\mathit{\text{Total drug}}-\mathit{\text{free drug}}}{\mathit{\text{Total drug}}}\times 100\%)$$ Eq 1:

Gene expression assay protocol (ASZ-001).

Cells were seeded (10,000 cells per well; 100 μL total volume per well) in a 96-well tissue culture plate. After 24 h, (37 °C, 5% CO₂), growth media was removed and replaced with low FBS media. Cells were incubated for an additional 24 h. DMSO and water (vehicle controls), liposomes (aqueous solution), and free drug (1% DMSO solution) were added to the wells. Cells were incubated (37 °C, 5% CO₂) for 48 h and mRNA was isolated and evaluated by qRT-PCR.

Gene expression assay protocol (C3H10T1/2).

Cells were seeded (50,000 cells per well; 500 μL total volume per well) in a 24-well tissue culture plate. After 24 h, (37 °C, 5 % CO₂), growth media was removed and replaced with low FBS media. Cells were incubated for an additional 24 h. Water (vehicle control) and liposomes (aqueous solution) were added to the wells. Cells were incubated (37 °C, 5% CO₂) for 24 h and mRNA was isolated and evaluated by qRT-PCR.

RT-PCR protocol.

Following treatment and incubation periods, both mRNA extraction and cDNA synthesis were performed using a TaqMan Cells-to-CT (fast) kit. cDNA synthesis utilized a BioRad MyCycler and was programmed according to the manufacturer’s instructions. Quantitative RT-PCR was performed on an ABI 7500 system and made use of the following TaqMan Gene Expression Probes: mouse ActB (Mm00607939_s1) and mouse Gli1 (Mm00494654_m1). Relative gene expression levels were computed via the ΔΔCT method using GraphPad Prism. Corresponding IC₅₀ values were calculated as mean ± SEM for at least three separate experiments performed in triplicate.

Results

ITZ liposome preparation and characterization

Our liposome formulation was based on previous ITZ formulations with a few modifications (Le Conte et al. 1992, Conte et al. 1994, Tang et al. 2010, Wang and Huang 2011, Ćurić et al. 2013, Leal et al. 2015, Lankalapalli et al. 2017) (Table 1). The lipids used in the present study were HSPC, mPEG-2000 DSPE, and cholesterol (Ćurić et al. 2013). This liposome formulation consists of synthetic lipid (HSPC), PEG and cholesterol, while other ITZ liposome formulations contain natural lipid and either PEG or cholesterol. Synthetic lipid is present in all FDA-approved parenteral liposome formulations while natural lipid is utilized in FDA-approved liposome formulations for a variety of administration routes (van Hoogevest and Wendel 2014, Li et al. 2015).

Our ITZ liposomes were prepared via a novel and automated continuous manufacturing approach developed at the University of Connecticut (Costa et al. 2016). This method, turbulent coaxial jet in co-flow, is capable of mass-producing homogenous monodispersed liposomes in a highly efficient and sterile manner. This is a rapid and customizable method that allows the organic phase containing lipid-drug mixture dissolved in a suitable organic solvent and an aqueous phase containing buffer flow rates to be monitored and adjusted in real time to obtain homogenous liposomes with monodispersed particle size and polydispersity index (PDI) for the desired application. Since the liposomes were being manufactured using the coaxial jet in the co-flow system, lipids and ITZ could not be dissolved in a water-immiscible solvent such as chloroform. With this in mind, the solubility of ITZ was determined in a variety of water-miscible solvents such as ethanol (EtOH), methanol (MeOH), and isopropanol (IPA). ITZ was most soluble in EtOH (0.6 mg/mL) after heating and sonication. The flow rates of both the organic phase and the aqueous phase were adjusted until the optimal sized liposomes (ITZ liposomes = 66.82 nm) with PDI value (≤0.1) were reproducibly attained. It was determined that faster flow rates for both the lipid solution (30 mL/min) and aqueous solution (300 mL/min) resulted in small, monodispersed particle size and optimal PDI values. It is important to note that we were targeting a liposome size of less than 100 nm based on previous studies that this is an optimal sized liposome to achieve BBB penetration (Saraiva et al. 2016). We were also targeting a PDI value of less than 0.1 since this indicates a homogenously monodisperse population (Soema et al. 2015). Using our continuous manufacturing system, we were able to obtain monodisperse liposomes with a particle size of 66.82 nm and a PDI of 0.086 at 30:300 mL/min – organic phase:aqueous phase (#4, Table 2).

Table 2.

ITZ-Liposome Formulation-A: Flow Rate Optimization

	Lipid Solution (mL/min)	Aqueous Solution (mL/min)	Particle Size (nm)	PDI
#1	10	90	174.8	0.446
#2	10	100	204.3	0.676
#3	10	300	170.1	0.362
#4	30	300	66.82	0.086

Encapsulation efficiency of ITZ liposomes

In order to determine the encapsulation efficiency, free ITZ must be removed from the solution. With hydrophilic drugs, this is often done by filtration as the liposomes can be rinsed on a filter with water. Liposomes containing hydrophobic drug can be rinsed with organic solvent; however, ITZ is soluble only in harsh solvents (dichloromethane and chloroform) that may disrupt the liposome membrane. Therefore, a centrifugation method was utilized to separate the free hydrophobic drug from the ITZ liposomes. This method relies on the size and density of free drug (Torchilin and Weissig 2003). As ITZ is large, hydrophobic, dense (d = 1.41 g/cm³), and poorly water soluble it was anticipated that free ITZ would form a pellet upon centrifugation (Domínguez-Gil Hurlé et al. 2006, Saraiva et al. 2016, Pubchem 2017). Liposomes remained suspended in the aqueous solution and upon decanting, were separated from the free drug. The pellet had a single R_f value on silica gel TLC (30% acetone: 70% hexanes) comparable to commercially available ITZ.

Encapsulation efficiency of liposomes is the concentration of the incorporated material [either in the aqueous core (hydrophilic drugs) or in the lipid bilayer (hydrophobic drugs)]. ITZ is a hydrophobic drug which is incorporated in the lipid bilayer of the liposome formulation (Piacentini 2016). The concentration of the drug in the liposomes can be determined by lysing the liposomes and measuring the total drug content via UV or fluorescence spectrophotometry (comparing against the drug’s calibration curve). We generated a calibration curve for ITZ by measuring the absorbance of the lysed solution at 254 nm. After removal of the free drug via centrifugation, the liposomes were lysed with Triton X-100 (5%) and heat (2 h at 60 °C). The concentration of drug released from the lysed liposomes was determined using the slope equation of the calibration curve. The encapsulation efficiency of the optimized ITZ liposomes (formulation-B, particle size 67.72 ± 0.89 nm, PDI 0.083 ± 0.004) was 94.8 ± 0.51% with a drug concentration of 162.25 μg/mL.

In vitro evaluation of ITZ liposomes

After determining the encapsulation efficiency and concentration of ITZ in the liposomes, they were used in several cellular assays to evaluate their anti-Hh activity compared to free ITZ. Empty liposomes were also utilized during these studies as a secondary control. ITZ liposomes, empty liposomes, and free ITZ were evaluated for their ability to decrease Gli1 expression in a murine Hh-dependent BCC cell line (ASZ-001). Gli1 is a known target gene of the Hh pathway and the ability of a small molecule to decrease Gli1 expression correlates well with inhibition of Hh signalling. Before testing liposomes in vitro, the concentration of encapsulated ITZ in the liposomes was calculated using the method described to determine the encapsulation efficiency (Appendix A). It is important to note that the optimized ITZ liposome formulation (formulation-B) was used throughout all in vitro assays. Liposomes were analyzed via UV (254 nm) prior to each experiment to confirm that they were not degrading or leaking ITZ while stored at 4 °C.

Initially, we explored down-regulation of Gli1 expression at various time points 24 h, 48 h, 72 h, and 96 h) as a precursor to determine the optimal incubation time for the ITZ liposomes to enter the cells, release the ITZ, and inhibit Hh signalling. Clear dose-response effects were seen for free ITZ when evaluated at 48 h (our standard incubation time in these assays); however, only cells treated with a high concentration of ITZ liposomes (195 μM) demonstrated a significant reduction in Gli1 expression (~50%) (Figure 1A). Lower concentrations of ITZ liposomes (10 and 1 μM), as well as empty liposomes, did not reduce Gli1 expression. In fact, this initial assay suggested that low concentrations of ITZ liposomes and empty liposomes might be upregulating the Hh pathway as several Gli1 mRNA expression values exceeded 100%. Based on these results, the next assay was performed utilizing slightly different concentrations (195, 75, and 10 μM) (Figure 2B). The addition of the 75 μm concentration resulted in more of a dose-response trend within the ITZ liposomes at all the observed time points; however, empty liposomes at the 48 h and 72 h time points still appeared to upregulate Gli1 mRNA expression. It was interesting to note that in both assays, treatment with empty liposomes resulted in cell death at 96 h (data not shown), which prevented us from determining Gli1 mRNA expression in these samples. These results may have been from the prolonged incubation with the liposomes or an impurity in our empty liposomes sample that needed the extended incubation to promote cell death. However, since we were not planning on evaluating empty liposomes at 96 h for any future assays, we did not perform further experiments to clarify this result.

Figure 1.

Time Dependent Gli1 mRNA Down-Regulation in ASZ Cells. Cells were treated with free ITZ, ITZ liposomes or empty liposomes and were incubated for various time periods (24 h, 48 h, 72 h, and 96 h). At each time point, cells were lysed and Gli1 mRNA expression was determined via qRT-PCR. (a) Treatment with 0.195 μM, 0.01 μM, and 0.001 μM of drug/liposome; (b) Treatment with 0.195 μM, 0.075 μM, and 0.01 μM of drug/liposome.

Figure 2.

Relative Gli1 mRNA Expression in C3H10T1/2 Cells. Cells were treated with water or empty liposomes for 24 h at different volumes. Water (vehicle control) was set to 100% and Gli1 mRNA expressions for cells treated with empty liposomes were calculated accordingly.

Based on the preliminary results in the ASZ-001 cells, it was hypothesized that the cholesterol component of the liposome formulation may be upregulating the Hh pathway. Cholesterol and oxysterols (products of cholesterol oxidation), are well-characterized Hh pathway agonists and are known to upregulate Gli1 mRNA expression in mouse embryonic fibroblasts (MEFs) (Corcoran and Scott 2006, Aghaloo et al. 2007, Dwyer et al. 2007, Johnson et al. 2011, Olkkonen et al. 2012). Empty liposomes, centrifuged and not centrifuged, were evaluated for their ability to up-regulate Gli1 expression in the Hh-dependent MEF cell line C3H10T1/2. Cells were treated with various volumes of empty liposomes equal to (5 μL) and greater than (10 μL) those used for in the ASZ-001 cells; however, none of these solutions up-regulated Gli1 expression higher than the control (water) (Figure 3).

Figure 3.

Dose-dependent inhibition of Gli1 mRNA Expression in ASZ Cells. Cells were treated with various concentrations of free ITZ, ITZ liposomes, or non-centrifuged ITZ liposomes and incubated for 48 (A) or 96 h (B). Cells were lysed and Gli1 mRNA expression was determined via qRT-PCR.

After determining that the liposomes were not activating Hh signalling, the ITZ liposomes were tested in a dose-dependent manner alongside free ITZ following a 48 or 72 h incubation (Figure 3). Non-centrifuged ITZ liposomes were also included in these experiments (ITZ + liposomes) to determine the difference in activity between the centrifuged and non-centrifuged samples. Not surprisingly, free ITZ induced Gli1 down-regulation more potently than either liposome sample with IC₅₀ values comparable to those previously determined in these cells (Table 3) (Pace et al. 2016, 2019). Non-centrifuged ITZ liposomes also demonstrated potent inhibition of Hh signalling at both 48 and 96 h (IC₅₀ values = 0.49 and 0.45 μM, respectively), albeit at a slightly reduced level compared to free ITZ. Of the three conditions evaluated, ITZ liposomes were least active in the ASZ cells with IC₅₀ values in the low micromolar range. The difference in anti-Hh activity between the non-centrifuged and centrifuged ITZ liposomes provided further confirmation that free ITZ was removed via the centrifugation method.

Table 3.

In Vitro Analysis of Liposomes

	IC50 (μM)a
Treatment	48 h	96 h
Free ITZ	0.11 ± 0.025	0.14 ± 0.012
ITZ-Liposomes	2.5 ± 0.37	1.04 ± 0.17
Non-Centrifuged ITZ-Liposomes	0.49 ± 0.04	0.45 ± 0.26

^aIC₅₀ and GI₅₀ values represent the Mean ± SEM of at least two separate experiments performed in triplicate.

Discussion

Despite our increasing knowledge of MB and its various molecular subtypes, a targeted chemotherapeutic treatment for this form of cancer has not been successfully developed. Hh pathway inhibitors have been previously explored for their ability to inhibit Hh-dependent MB and several have entered clinical trials for this purpose. ITZ is a promising anti-Hh therapy for the treatment of MB as it (1) maintains potent Hh inhibition in the presence of wild-type and mutant Smo and (2) it is an FDA-approved drug with a well-characterized safety profile (Kim et al. 2010, 2013). In addition, it has been well-tolerated by children when used for a variety of fungal infections, highlighting its potential in the primary MB patient base (Gupta et al. 2003).

A primary challenge for developing any targeted MB drug is the presence of the BBB, which prevents many small molecules, regardless of their physicochemical properties, from entering the central nervous system (Lin and Yamazaki 2003, Pardridge 2003, Staud and Pavek 2005, Oberoi et al. 2016). One efficient way to promote BBB penetration is through the use of nanoparticles, such as liposomes, to ensure targeted drug delivery (Wei et al. 2014). Liposomal formulations can be specifically generated to promote BBB penetration by targeting a particle size that is less than 100 nm. Specific proteins that aid in BBB targeting and penetration can also be incorporated into liposomal formulations. (Wei et al. 2014). These proteins include but are not limited to the transferrin receptor (TfR), lipoprotein receptor-related protein (LRP), and nicotinic acetylcholine receptor (nAChRs) (Wei et al. 2014). In addition, liposomal formulations of cancer medications have been shown to limit off-target toxicities (Federman and Denny 2010, Wei et al. 2014).

With this in mind, we generated a preliminary liposome model that has an optimal size for penetrating the BBB with good encapsulation efficiency (~ 94.8 %). While our ITZ-containing liposomes were not as potent in reducing the Hh signalling activity in Hh-dependent cell culture as free ITZ, our formulation offers promise for the continued development of a targeted MB treatment. Additional studies are needed to understand how ITZ liposomes interact with cells and how rapidly they release ITZ. This information will be crucial in determining what modifications should be made to the liposome formulation to aid in their cell penetration or to manipulate the speed at which they release their contents.

Conclusions

ITZ was successfully encapsulated into a novel liposome formulation utilizing coaxial turbulent jet in co-flow processing. This ITZ liposome formulation had a uniform particle size (< 100 nm) that has previously correlated with promising BBB penetration for liposomal formulations of other drugs, while also demonstrating a good encapsulation efficiency (~95 %). While these ITZ liposomes demonstrated moderate Hh pathway inhibitory activity in comparison to free ITZ, they serve as a promising starting point for achieving targeted drug delivery of ITZ using nanotechnology.