Preparation and Characterization of SN-38-Encapsulated Phytantriol Cubosomes Containing α-Monoglyceride Additives

Abstract

SN-38 is a potent active metabolite of irinotecan that has been considered as an anticancer candidate. However, the clinical development of this compound has been hampered by its poor aqueous solubility and chemical instability. In this study, we developed SN-38-encapsulated cubosomes to resolve these problems. Six α-monoglyceride additives, comprising monocaprylin, monocaprin, monolaurin, monomyristin, monopalmitin, and monostearin, were used to prepare phytantriol (PHYT) cubosomes by probe sonication. The mean particle size, polydispersity index, and zeta potential values of these systems were around 190–230 nm, 0.19–0.25 and −17 to −22 mV, respectively. Small-angle X-ray scattering analyses confirmed that the SN-38-encapsulated cubosomes existed in the Pn̄3m space group both with and without the additives. The monoglyceride additives led to around a two-fold increase in the solubility of SN-38 compared with the PHYT cubosome. The drug entrapment efficiency of PHYT cubosomes with additives was greater than 97%. The results of a stability study at 25°C showed no dramatic changes in the particle size or polydispersity index characteristics, with at least 85% of the SN-38 existing in its active lactone form after 10 d, demonstrating the high stability of the cubosome nanoparticles. Furthermore, approximately 55% of SN-38 was slowly released from the cubosomes with additives over 96 h in vitro under physiological conditions. Taken together, these results show that the SN-38-encapsulated PHYT cubosome particles are promising drug carriers that should be considered for further in vivo experiments, including drug delivery to tumor cells using the enhanced permeability and retention effect.

7-Ethyl-10-hydroxyl camptothecin (SN-38) is an active metabolite of the anticancer agent irinotecan (CPT-11, a derivative of camptothecin) which has been approved as an anticancer agent in its own right.¹⁾ It has been reported that only 1–9% of each dose of irinotecan injected into a human is converted into the active metabolite SN-38 in the liver by carboxylesterase.²⁾ The antitumor activity of SN-38 is based on its inhibition of DNA topoisomerase I, which is involved in controlling the replication and transcription of DNA.³⁾ SN-38 has been reported to be approximately 100- to 1000-fold more potent than CPT-11 as a topoisomerase I inhibitor in a variety of tumor cell lines.⁴⁾ The high potency of SN-38 makes it a potential candidate for the treatment of several types of cancer, including colorectal, lung, ovarian and breast cancers.^5,6) However, there are two problems associated with the formulation of SN-38. First, SN-38 cannot be directly administered to humans because of its extremely low water solubility. Second, SN-38 shows poor chemical stability in solution. Under physiological conditions (pH 7.4), the lactone form of SN-38 is rapidly hydrolyzed to give the corresponding carboxylate form (Fig. 1), which is devoid of any antitumor activity, as is the case with several other camptothecin compounds.^7–9)

Fig. 1. pH Dependent Conversion of Lactone and Carboxylate Forms of SN-38

It was envisaged that the solubility and bioavailability of SN-38 could be improved by formulating this as nanoparticles. Nanoparticles have been used to encapsulate a wide variety of poorly water-soluble drugs, leading to dramatic improvements in their solubility, stability and drug-targeting properties, as well as reducing any adverse effects.^10–13) Furthermore, nanoparticles-based drug delivery systems can preferentially target tumors by taking advantage of the enhanced permeability and retention (EPR) effect.¹⁴⁾ A broad range of drug delivery systems, including PEGylated conjugates, polymer micelles, liposome formulations and dendrimers, have been extensively investigated for the formulation of SN-38 to improve its water solubility and bioavailability.^3,15–17) However, oligo(ethylene glycol)-conjugated micelles of SN-38 with diameters in the range of 25–30 nm required esterase activation.¹⁸⁾ Furthermore, there are several limitations associated with the use of liposomal formulations such as poor stability under physiological conditions and low entrapment efficiency, as well as difficulties in controlling the liposome size.¹⁹⁾ Liposomal formulations can also result in the entrapment of the inactive form of SN-38.²⁰⁾ Oral polyamidoamine (PAMAM) dendrimer formulation of SN-38 are not stable under the acidic conditions found in the stomach, resulting in the rapid release of the drug from the dendrimer complex.²¹⁾

Cubosomes are inverse bicontinuous cubic lyotropic liquid crystalline nanoparticles that have attracted considerable interest from researchers working in drug delivery applications because of their compartmentalized and highly ordered internal structure, high lipid content and large surface area. Lipid molecules, such as monoolein (GMO) and phytantriol (PHYT), are common examples of the amphiphilic building blocks that have been used to prepare cubosomes in excess water.²²⁾ PHYT is a chemically stable amphiphile that can be used to generate cubic lipid bilayers because its structure does not contain any unsaturated double bonds or ester linkages. A comparison of the chemical stability, and purity of PHYT and GMO suggested that the former of these two compounds would be the best candidate for drug delivery applications.^23,24) Cubosome dispersions are only stable for extended periods as colloids when they are prepared in the presence of a steric stabilizer capable of preventing particle aggregation. Pluronic F127 is currently the most popular of all of the reported steric stabilizers and is generally regarded as the gold standard for this type of work.²⁵⁾

A limited number of studies have been conducted pertaining to the effects of additives on the physicochemical properties and stability of cubosome nanoparticles. Notably, anionic and cationic amphiphilic additives such as anionic sodium bis(2-ethylhexyl)sulfosuccinate and cationic didodecyldimethylammonium bromide (DDAB) have been reported to induce phase transitions,^26,27) and could therefore affect the stability of PHYT cubosome particles. A PHYT cubosome containing ionic amphiphilic additives such as cationic DDAB was recently shown to enhance the solubility of SN-38, with the resulting cubosomes remaining stable for approximately 30 h.²⁸⁾ These ionic additives widened the scope of cubosomes in terms of their potential application as drug carriers for improving the solubility of hydrophobic drugs. However, the inclusion of such additives could also lead to an increased risk of cytotoxicity or hemolysis, which would prevent them from being used in vivo. The effects of non-ionic additives on the physicochemical properties of PHYT cubosome therefore remain unknown. In this study, we have prepared SN-38-encapsulated PHYT cubosomes containing non-ionic and biocompatible additives, including six monoglycerides of saturated fatty acids with carbon chain lengths in the range of C₈ to C₁₈. The resulting systems were subsequently evaluated in terms of their effects on the particle properties of the cubosomes, as well as the solubility and chemical stability of the encapsulated SN-38.

Experimental

Materials

PHYT, SN-38 and the six monoglycerides; monocaprylin, monocaprin, monolaurin, monomyristin, monopalmitin and monostearin (Table 1) were purchased from Tokyo Chemical Co., Ltd. (Tokyo, Japan). Pluronic F127 was purchased from BASF (Ludwigshafen, Germany). Dimethyl sulfoxide (DMSO), chloroform, acetonitrile, hydrochloric acid and sodium hydroxide were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All of the reagents used in this study were purchased as the highest grades available from commercial sources.

Table 1. List of the α-Monoglycerides with Saturated Fatty Acids Used in This Study

Name	Aliphatic carbon chain length	Chemical structure
Monocaprylin	C8	CH3(CH2)6COO–CH2CH(OH)CH2OH
Monocaprin	C10	CH3(CH2)8COO–CH2CH(OH)CH2OH
Monolaurin	C12	CH3(CH2)10COO–CH2CH(OH)CH2OH
Monomyristin	C14	CH3(CH2)12COO–CH2CH(OH)CH2OH
Monopalmitin	C16	CH3(CH2)14COO–CH2CH(OH)CH2OH
Monostearin	C18	CH3(CH2)16COO–CH2CH(OH)CH2OH

Preparation of SN-38-Encapsulated PHYT Cubosomes

PHYT cubosomes were prepared by probe-sonication according to the modified method of Hartnett et al.²⁹⁾ Briefly, 200 mg of PHYT was dissolved in chloroform, and the resulting solution was mixed with 550 µg of SN-38, which was prepared as a solution in DMSO (1 mg/mL). The mixture was then treated with one of the six different monoglyceride additives (20 mg), as shown in Table 2. The amounts of additives were fixed to 10% of PHTY weight, because it had been reported that the cubic phase of PHYT could tolerate up to 10% additives but not to 15%²⁷⁾ and that PHYT dispersion became highly viscous when the quantity of additives over 10%.²⁶⁾ Twenty-two milligrams of pluronic F127 was then added to each formulation, and the resulting mixture was manually agitated to give a solution. The volume of chloroform in each formulation was around 3 mL. The solvents in the formulation were subsequently evaporated under a stream of nitrogen gas to give a residue, which was dried under vacuum for 24 h to allow for the complete removal of the organic solvents.

Table 2. Formulation of the SN-38-Encapsulated PHYT Cubosomes

Name	PHYT (mg)	α-Monoglyceride (mg)	Pluronic F127 (mg)	SN-38 (µg)	Water (mL)
Cubosome
with additives	200	20	22	550	2
without additives	200	—	22	550	2

Two milliliters of water was added to each sample and the samples were sonicated using a probe sonicator (Branson Sonifier 250, Danbury, CT, U.S.A.) for 15 min (30 s sonication/3 s break cycle) to obtain a milky-white homogenous dispersion. The resulting cubosome dispersions were centrifuged at 1000×g for 5 min to remove any un-encapsulated drug agglomerates. When PHYT formulations containing SN-38 were centrifuged, agglomerates of pale yellow color were found at the bottom of centrifugation tubes. However, no agglomerate was found when PHYT formulations containing additives and SN-38 were centrifuged. These indicated that the agglomerates were SN-38 and this centrifugation would not affect the ratio of PHYT to monoglycerides in cubosomes. The supernatant was vortexed for 15 min at 2500 rpm. The final cubosome samples were stored at 25°C until use.

When myristic acid and lauric acid additives were incorporated into the preparation, the pH of the cubosome dispersions (or hexosomes) were 5.4 and 4.0, respectively. The pH values of these dispersions were lower than that of PHYT cubosomes without any additives (pH 5.7).

Particle Size Distribution and Zeta Potential Measurement

The mean particle size, polydispersity index (PDI) and zeta potential characteristics of the cubosomes prepared in the current study were characterized by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 system (Malvern Instruments, Malvern, U.K.). The samples were diluted 100-fold with Milli-Q water and the measurements were conducted at 25°C. All of the experiments were performed in triplicate.

Small Angle X-Ray Scattering (SAXS) Measurement

SAXS measurements were performed to characterize the liquid crystalline phase of the cubosomes by using a NANO-Viewer system (Rigaku, Tokyo, Japan) equipped with rotating anode X-ray generator (CuKα). The SAXS profiles were recorded using a two-dimensional detector PILATUS 100K (Dectris, Baden, Switzerland). Twenty-microliter samples were loaded into polyimide tubes with 1 mm in diameter. The sample to detector distance was set to approximately 510 mm, as calibrated using the powder of silver behenate. The temperature and exposure time were set to 25°C and 5 min, respectively. The lattice parameters of the cubic phase, together with their standard errors, were calculated using Grafit 5 (Erithacus Software, Surrey, U.K.).

SN-38 Quantification by HPLC

The cubosome samples were diluted as necessary with acetonitrile and thoroughly mixed before being filtered through 0.45 µm membrane filters. The lactone and carboxylate forms were immediately quantified³⁰⁾ by using a TSKgel ODS-80Tm® column (4.6 mm×150 mm, 5 µm particle size) on a Shimadzu LC-2010C HT® system (Shimadzu Corporation, Kyoto, Japan). The mobile phase used for the elution of the LC system was composed of a 1 : 1 (v/v) mixture of acetonitrile and 25 mM sodium phosphate buffer (pH 7.4). The flow rate was set to 1 mL/min and the injection volume of the analytical sample was set to 10 µL. The column heater and UV-detector were set at 40°C and 265 nm, respectively. All of the quantification experiments were performed in triplicate.

Drug Entrapment Efficiency

Drug entrapment efficiency was determined by ultra-centrifugation method.³⁰⁾ Briefly, 500 µL of cubosome was placed in Amicon Ultra-0.5 Centrifugal Filters-50K device (Amicon®, Millipore Corporation, Ireland) and centrifuged for 15 min at 15000×g. The free drug passed through filter membrane was determined by HPLC as described above. The percentage of the drug entrapped in the cubosome was calculated as follows:   

where, C_total is the total drug concentration in the cubosome, and C_free is the free drug concentration in the filtrate after ultra-filtration.

In Vitro Release of SN-38 from Cubosome

The in vitro release of SN-38 from the different cubosome samples was analyzed using a membrane dialysis method in phosphate buffer saline (PBS) at pH 7.4 and 37°C (the concentration of Na₂HPO₄, KH₂PO₄, NaCl, KCl were 10, 1.8, 137, 2.7 mmol/L, respectively).²⁰⁾ Briefly, a 500 µL cubosome sample was placed into the dialysis membrane (MWCO 14 kDa; Viskase Companies Inc., Darien, IL, U.S.A.), which was subsequently clamped at both ends before being immersed in 200 mL of PBS medium and rotated at 100 rpm and 37.0±0.5°C. Five hundred microliter samples of the medium were collected at time intervals of 2, 10, 16, 24, 36, 48 and 96 h, while the same volume of fresh medium was added to the release medium. The samples were immediately diluted with 0.1 M sodium acetate buffer (pH 4.0) to allow for the conversion of the SN-38 molecule to the lactone form⁷⁾ and filtered through a 0.45 µm membrane filter. The fluorescence intensity of each sample was measured to quantify the SN-38 concentration at excitation and emission wavelengths of 375 and 550 nm, respectively, using a Hitachi F-2000 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Japan). All of these experiments were conducted in triplicate.

Stability Studies of SN-38-Encapsulated Cubosomes

The SN-38-encapsulated cubosomes were stored at 25°C and the particle properties and SN-38 concentration at 0, 6 and 10 d were determined using the method described above.

Statistical Analysis

Statistical analyses were performed using the Student’s t-test. A probability value of p<0.05 was considered to indicate statistical significance.

Results and Discussion

Characterization and Physicochemical Properties of SN-38-Encapsulated Cubosomes

SN-38-encapsulated PHYT cubosomes containing the monoglycerides of six saturated fatty acids were prepared successfully. All of the formulations were milky white in appearance. The characteristics of the SN-38-encapsulated cubosome nanoparticles are shown in Figs. 2a and b. The particle sizes of the SN-38-encapsulated cubosomes containing additives were all in the range of 190–230 nm, making them smaller than the PHYT cubosome particles prepared without additives (242 nm). The PDI values of the SN-38-encapsulated cubosomes containing additives were in the range of 0.19–0.25, making them lower than the PDI of PHYT cubosome (0.30). Furthermore, the zeta potentials of the SN-38-encapsulated cubosomes with additives were in the range of −17 to −22 mV, while the value of the cubosome without any additives was −22 mV.

Fig. 2. Dynamic Light Scattering Results of SN-38-Encapsulated PHYT Cubosome Particles with and without Additives

(a) Mean particle size in diameter (nm) and polydispersity index (PDI). (b) Zeta potential (mV). Each value represents the mean±S.D. (n=3).

The cubic phases of the SN-38-encapsulated cubosome particles were characterized by SAXS measurements. The SAXS profiles and lattice parameters are shown in Fig. 3 and Table 3, respectively. The SAXS diffraction patterns of the PHYT cubosomes with or without the additives showed at least four Bragg peaks with relative positions at spacing ratios of   

These peak could be indexed according to the Miller indices (hkl)=(110), (111), (200) and (211) for the cubic space group Pn̄3m.³¹⁾ The addition of the monoglycerides to PHYT had no impact on its cubic space group Pn̄3m structure, which remained stable for up to 28 d (see Supplementary Fig. S1). The incorporation of the monoglycerides into PHYT led to an increase in the lattice parameters of the cubic phase by around 3–9%. The hydrophilic headgroup of PHYT and the glycerol moieties of the monoglycerides are similar in size, whereas the hydrophobic groups on these molecules differed considerably in terms of their shape and size. The hydrophobic moiety of PHYT consisted of a branched and bulky aliphatic chain, whereas those of the monoglycerides were linear and slim aliphatic chains. When monoglycerides were included in the PHYT bilayers, there might be a noticeable decrease in the bilayer curvature of the cubic phase,³²⁾ which resulted in the observed increase in the lattice parameters. Cubosomes containing monoglycerides with shorter aliphatic chains showed larger lattice parameters. The aliphatic chains of the monoglycerides in the PHYT bilayers were located beneath the surface of the bilayer, especially for the shorter aliphatic chains. The localization of the aliphatic chains of the monoglycerides in this way led to a decrease in the curvature of the lipid bilayer (the extent of which was dependent on the chain length) and afforded larger lattice parameters.

Fig. 3. SAXS Profiles of SN-38-Encapsulated PHYT Cubosome Particles with and without Additives at 25°C

q=2 sin θ/λ, where θ is Bragg angle, λ is wavelength of X-ray.

Table 3. Effect of the Different Monoglyceride Additives on the Space Group and Lattice Parameter of the SN-38-Encapsulated PHYT Cubosomes

Cubosome type	Phase	Space group	Lattice parameter, a (Å)
PHYT-monocaprylin	Cubic	Pn̄3m	71.1±0.1
PHYT-monocaprin	Cubic	Pn̄3m	69.1±0.1
PHYT-monolaurin	Cubic	Pn̄3m	68.9±0.1
PHYT-monomyristin	Cubic	Pn̄3m	69.8±0.1
PHYT-monopalmitin	Cubic	Pn̄3m	68.9±0.1
PHYT-monostearin	Cubic	Pn̄3m	67.6±0.1
PHYT-no additives	Cubic	Pn̄3m	65.3±0.1

Although the monoglyceride-PHYT systems appeared to form cubic phases in water, very little is known about the phase behavior of the corresponding saturated fatty acids systems. We attempted to characterize the phase behaviors of PHYT systems formed using lauric acid and myristic acid as additives. The addition of 10% lauric acid or myristic acid to PHYT led to the formation of distinct Bragg peaks with relative positions at spacing ratios of   

These peaks could be indexed as Miller indices (hk)=(10), (11) and (20) with two-dimensional hexagonal^31,33) lattice parameters of 45.2 and 45.1 Å for the lauric acid and myristic acid additives, respectively (Fig. 4). This result indicated the presence of particles with a hexagonal phase, such as hexosomes. The addition of lauric acid or myristic acid to PHYT led to an increase in the surface charge density, as shown by the higher zeta potential values of −29 and −30 mV for the lauric acid and myristic acid hexosomes, respectively (Table 4). A transition from the cubic to the hexagonal phase was also observed following the addition of the fatty acid, oleic acid to the cubosomes of monoolein.³³⁾ The particle size and PDI value of these PHYT hexosomes were around 2-fold larger than those of the SN-38-encapsulated PHYT cubosomes containing the monoglyceride additives, implying that they would be unsuitable as drug carriers utilizing the EPR effect.

Fig. 4. SAXS Patterns of PHYT with Lauric Acid and Myristic Acid

Table 4. Particle Properties of PHYT with Lauric Acid and Myristic Acid

Name	Particle size in diameter (nm)	PDI	ζ Potential (mV)
PHYT-lauric acid	401	0.451	−29
PHYT-myristic acid	452	0.503	−30

The drug entrapment efficiency of cubosomes was found to be greater than 97% with monoglyceride additives (Table 5), indicated that most of the SN-38 was entrapped within the cubosomes. The solubility of SN-38 increased in all of the cubosome formulations prepared in the current study both with and without the additives. Around 92–96% of the SN-38 existed in its physiologically active lactone form in all of the PHYT cubosomes both with and without the monoglyceride additives. The remaining SN-38 molecule existed in the inactive carboxylate form. Furthermore, the SN-38 molecule was around 2-fold more soluble in the PHYT cubosomes prepared using a monoglyceride additive compared with the PHYT cubosome prepared without an additive, as shown in Fig. 5. The effects of the different hydrophobic chain length of monoglyceride additives on enhancing the solubility of SN-38 were comparable among them. This increase in the solubility of SN-38 could be attributed to two factors: (i) an increase in the hydrophobic volume of the lipid bilayer of the PHYT cubosomes following the addition of the monoglyceride additive, and (ii) the stabilization of the SN-38 molecules in the hydrophobic region of the cubosome. PHYT consists of a branched and bulky hydrophobic chain, whereas the monoglycerides consist of a flexible, linear hydrophobic chain, as described above. The incorporation of the flexible hydrophobic chains of the monoglyceride additives would therefore lead to the formation of closer and tighter contacts between the hydrophobic chains of the cubosomes and the planar SN-38 molecule. These contacts would decrease the chemical potential of the SN-38 molecules in the hydrophobic region by enhancing the van der Waals interactions. This would increase in the number of SN-38 molecules in the hydrophobic lipid bilayer of the cubosomes prepared with a monoglyceride additive, resulting in the enhanced solubility of SN-38. The fact that the additives enhanced the solubility of this highly insoluble drug while maintaining the cubic phase of the cubosome suggested that this method could widen the applicability of cubosomes for the solubilization of drugs with low solubility.

Table 5. Drug Entrapment Efficiency of PHYT Cubosomes with and without Additives

Cubosome type	Entrapment efficiency (%)
PHYT-monocaprylin	97.9±0.3
PHYT-monocaprin	97.3±0.2
PHYT-monolaurin	97.6±0.2
PHYT-monomyristin	97.6±0.2
PHYT-monopalmitin	97.7±0.3
PHYT-monostearin	97.4±0.2
PHYT-no additives	95.8±0.6

Fig. 5. Effect of the Monoglyceride Additives on the Solubility of SN-38 in PHYT Cubosomes with and without Additives

Each value represents the mean±S.D. (n=3), * p<0.05 compared with no additives or without additives.

In Vitro Release of SN-38 from Cubosomes

Figure 6 shows the in vitro release profiles of SN-38 from the PHYT cubosomes both with and without the different additives. Approximately, 20–30% of the SN-38 was released from the cubosomes with additives into a solution of PBS (pH 7.4) during the first 24 h, followed by slow release rate, and reached up to 60% after 96 h. The SN-38 release profiles of PHYT cubosomes containing monoglyceride additives were comparable to those of polymeric nanoparticles and PEGylated liposomal formulation of SN-38. SN-38 released from polymeric nanoparticles and PEGylated liposomes were around 55 and 40% after 96 h, respectively.^34,35) PHYT cubosomes containing monoglyceride additives were superior in the suppression of burst release than irinotecan-loaded liposomal formulation. PHYT cubosomes containing monoglycerides released only 15% of encapsulated SN-38 during first 12 h and SN-38 released up to 50% after 72 h (Fig. 6). However, irinotecan released 60% from liposomes during first 12 h and released completely within 72 h.³⁶⁾ Notably, all of the cubosomes prepared in the current study allowed for the slow release of SN-38 in PBS solution. Furthermore, none of these systems showed any premature burst release, which is a general problem for most nanoparticle drug carriers.³⁷⁾ The slow release of SN-38 at physiological pH provided some assurance that this molecule would remain entrapped within the hydrophobic lipid bilayer of the cubosome under physiological conditions. These cubosome nanoparticles would allow the most of the drug molecule in its active lactone form to be delivered to the tumor cells by EPR effect. SN-38 released from cubosomes before they reach tumor cells would be hydrolyzed to inactive carboxylate form in plasma. Therefore, the slow release characteristics would be particularly advantageous in terms of maintaining an effective concentration of the drug in the active lactone form to be delivered to tumor cells.³⁸⁾

Fig. 6. In Vitro Release Profiles of SN-38 from PHYT Cubosomes with and without Additives

Each value represents the mean±S.D. (n=3).

Stability Studies

The particle size and PDI characteristics of the cubosomes prepared in the current study remained largely unchanged for up to 10 d (Fig. 7). The cubosomes initially contained 92–96% of the physiologically active lactone form of SN-38, which changed to 85–90% following 10 d of storage at 25°C (Fig. 8). These results therefore demonstrated that the active lactone form of SN-38 was stable in the cubosomes. In contrast, SN-38 encapsulated in liposomes²⁰⁾ were inactive carboxylate form and required to be transformed to active lactone form before administration. Liposomes were only stable at 2–8°C in a lyophilized state and their vesicle sizes increased by about 15% after reconstitution. Moreover, the stability of the lactone form of SN-38 in the cubosomes could be attributed to the hydrophobic nature of this compound, which would allow it to be readily incorporated into the cubic phase of the lipid bilayer of the cubosomes. In this way, SN-38 would be shielded from water molecules, thereby protecting its lactone ring against hydrolysis.

Fig. 7. Changes in Particle Size in Diameter and the PDI of SN-38-Encapsulated PHYT Cubosomes on Storage at 25°C Up to 10 d

Monocaprylin (a), monocaprin (b), monolaurin (c), monomyristin (d), monopalmitin (e), monostearin (f), and no additives (g). Each value represents the mean±S.D. (n=3).

Fig. 8. Changes in the Percentages (%) of SN-38 Lactone Form in PHYT Cubosomes with and without Additives Up to 10 d at 25°C

Each value represents the mean±S.D. (n=3).

Conclusion

We have successfully prepared SN-38-encapsulated PHYT cubosome nanoparticles with the aim of not only overcoming the drawbacks of CPT-11 and SN-38 but also addressing the limitations reported for SN-38-incorporating drug delivery systems (i.e., the stability of the SN-38 lactone form, particle stability and premature burst release). The results of this study have shown that PHYT cubosomes containing SN-38 and a monoglyceride additive led to around 2-fold increase in the solubility of the drug compared with the PHYT cubosome without an additive. SAXS analyses confirmed that these cubosomes existed in the cubic space group Pn̄3m and that they retained their internal structure for up to 28 d. The mean particle sizes of the different cubosome nanoparticles were in the range of 190–230 nm and the cubosomes themselves were monodispersed. The stability studies indicated that the cubosome nanoparticles and SN-38 lactone form were stable for up to 10 d. Furthermore, SN-38 was slowly released from the cubosomes with additives approximately 55% over 96 h, in vitro under physiological conditions. Taken together, these results demonstrate that the SN-38-encapsulated PHYT cubosome particles prepared in the current study are promising drug carriers that should be considered for further in vivo experiments, including drug delivery to tumor cells using the EPR effect.

Acknowledgment

We acknowledge the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan for providing a Doctoral Scholarship to M. A. Ali. We would also like to express our gratitude to Professor Naoto Oku at the Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka for the support of his laboratory in conducting the probe sonication and DLS analysis. This work was partly supported by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 26460224, 26460039 and 26460226).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials. The SAXS profiles of the SN-38-encapsulated PHYT cubosome particles with and without additives at 25°C after 28 d are shown in Fig. S1. q=2 sin θ/λ, where θ is Bragg angle, λ is wavelength of X-ray.

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