Antisolvent Precipitative Immobilization of Micro and Nanostructured Griseofulvin on Laboratory Cultured Diatom Frustules for Enhanced Aqueous Dissolution

Abstract

We report for the first time an antisolvent synthesis of nanostructured hydrophobic drug formulation onto a natural diatom. The jewel of the sea, a marine diatom, which is enriched in silicon, was cultured and grown in the laboratory. Its frustules were isolated and purified. The polar functional group on its surface provided unique physical and chemical properties. Griseofulvin (GF), an antifungal drug was used as a model compound was precipitated onto and adsorbed onto hydrophilic diatom surface, while stabilizer hydroxypropyl methyl cellulose (HPMC) was used for restricting particle growth during the composite synthesis. This work demonstrates that the fine drug crystals incorporated onto the diatom silica surface. The structural and morphological properties of the drug was characterized by various techniques. The drug loading of the formulation was estimated to be 41% by weight. The incorporation of micro/nano crystals on the diatom surface dramatically enhanced the dissolution rate, and lowered the time required for 50% dissolution for pure drug from 240 to 58 minutes for the drug composite, and the time required for 80% dissolution or T₈₀ was found to be 180 minutes for the composite while the pure drug reached a maximum of 65% in 300 min.

Graphical Abstract

1.0. Introduction

The development of pharmaceutical drugs has been an increasingly challenging endeavor. Many of the developed drugs are hydrophobic and exhibit poor aqueous solubility. The hydrophobic drugs face several therapeutic inefficacies because of their systematic toxicity, low bioavailability and short half-life circulation [1,2]. The low dissolution of these molecules in gastrointestinal tract limit their overall invitro/in vivo performance [3]. Therefore, their formulations are aimed at increasing their dissolution rate. Some published approaches to dissolution enhancement are nano and micro structuring through top down milling and high-pressure homogenization [4,5], polymer and nanoparticle formulations such as cyclodextrin complexation, microemulsion formation, polymer film encapsulation, use of deformable membrane vesicles, xerogel/silica hybrids covalent functionalization, direct incorporation and covalent attachment of graphene oxide [6–12].

Antisolvent precipitation has emerged as an effective technique to produce nano and micron size drug crystals. Ultrasonication is directly integrated with antisolvent technique to generate small, uniform crystals and provide colloidal stability [13–17]. Different natural and synthetic nanomaterials have been used in the development of new materials and targeting higher therapeutic delivery performance. These include polymeric micelles, liposomes, dendrimers, hydrogel, quantum dots, gold nanoparticles, nanocarbons (CNT) and mesoporous silica [18–24]. These have been considered to be smart nanocarriers for efficient transport and release of drug molecules [25,26].

Silica particles are FDA approved, inexpensive and biocompatible drug delivery materials. They have been used for sustained and controlled drug delivery formulation [27,28]. They have specific porous structures that can enhance diffusion characteristics [29–31], and both mesoporous (2–50 nm) [29–32] and nanoporous (up to 20 nm) [33,34] silica have been used for drug delivery, especially for the slow release of water soluble drugs [35]. Silica functionalization has been carried out by organosilane and phosphonic acid modification, and surface grafting of different silane groups has been done for the loading of hydrophobic and hydrophilic drugs [36]. Synthesis of porous silica has been carried out by various methods such as sol-gel chemistry, evaporation and spray drying process [37–39]. These involve the use of several toxic chemicals whose residues can remain in the particles [40–43]. On the other hand natural diatomaceous earth is a natural biosilica that contains silica frustules [29–32] that persist after decay of the organic matter in algae, while other minerals are geochemically incorporated into the diatomaceous earth [36]. They have well-organized silica structure along with impurities such as rocks and fossils that are often washed by chemical processes [44]. Cultured diatoms typically contains pure algae species thus providing uniform particle size frustules that have a large number of free silanol groups that can be used for surface modifications [45]. Compared to the synthetic alternatives, biogenic diatom silica is considered to be a highly sustainable porous material for biomedical applications. These nano/microporous particles also have much potential in drug delivery due to their hydrophilic surface functionalities [57]. Moreover, diatomaceous earth contains particles that vary in size, and there is significant batch to batch variation in these materials which make drug delivery a challenge [46]. This is not a problem in cultured diatoms. While drugs have been immobilized in the silica pore structure, the immobilization of drugs on the silica surface is yet to be studied; this may be advantageous for hydrophobic drugs.

The objective of this research was to utilize the frustules of cultured diatom Phaeodactylum tricornutum grown in seawater environment to generate micro silica particles for drug delivery. Yet another objective is to incorporate hydrophobic drug Griseofulvin on to the frustule surface via anti solvent precipitation to develop drug composites with high dissolution rate.

2.0. Materials and Methods

2.1. Materials

Griseofulvin (95% pure), hydroxypropyl methyl cellulose form (HPMC, mol. wt. 10,000, viscosity of 5 cP, 2 wt.% in H₂O) were purchased from Sigma Aldrich, and Diatom Phaeodactylum was purchased from marine bieglow. Methanol and sulfuric acid were purchased from Fisher Scientific. Sodium hydroxide was purchased from Sigma Aldrich. Water used for this experiment was milli-Q water purified with Milli-Q plus system.

2.2. Preparation of purified diatom and diatom-drug composite

Diatom culture was grown for 10 days. The cells were collected, and the organic matter was successfully removed via acid and heat treatment. The diatom was cultured and maintained in Aquil medium by using a method described before [47]. Artificial sea water was maintained in diurnal chamber with 12-hour day/night cycles at 19°C ± 1.0 °C. Exponential phase diatom culture was flocculated with 1.0 M NaOH solution. The culture was separated by gravitational settling and membrane filtration and washed with Milli-Q water. The organic matter was decomposed by treating with concentrated H₂SO₄ followed by heating at 200°C for 2 hours [45]. This was followed by vacuum filtration, washed with Milli-Q water to bring back to neutral pH and then dried at 200°C in a vacuum oven. The acid treatment also surface functionalized the diatoms with free hydroxyl group [48]. The diatom silica thus obtained was utilized to prepare diatom drug composites (DDC). The details on the diatom frustules have been presented before [45,49].

Antisolvent precipitation was used to generate Griseofulvin (GF) crystals that were deposited on the diatom surface. The presence of stabilizer HPMC helped inhibit the growth of crystals size, and this was in line with what has been reported before [50,51]. The ratio of Griseofulvin: Diatom: HPMC was selected to be 3:3:1. Sonication was used to facilitate rapid and uniform nucleation that helped the formation of small and uniform size particles. Figure-1 shows the physical appearance of bottles containing the drug, diatoms and DDC in water dispersions.

Figure-1:

Water dispersibility observed for (A) pure drug, (B) diatom, (C) DDC

Griseofulvin was loaded onto the diatom silica via antisolvent precipitation at room temperature. Griseofulvin was dissolved in methanol to form a clear solution. Diatom silica was wetted with milli-Q water and mixed with HPMC and the mixture was sonicated for 1 hour. The drug solution was added to the aqueous phase slowly and dropwise while undergoing bath sonication. After complete addition, the suspension was bath sonicated for another 1 hour, filtered with 10 μm membrane filter and vacuum dried at 100^oC for complete water removal. The final solid containing the diatom silica/HPMC and drug was referred to as the DDC.

2.3. Characterization of Drug-Diatom Composites

Different analytical instruments were used for the characterization of the prepared composite materials. Scanning Electron Microscope JEOL JSM 7900F, Japan was used for studying the morphology of drug, diatom, HPMC and DDC respectively. A Nicolet Almege XR Dispersive Raman with Olympus BX51 Confocal Microscope (Thermo Electron Corp.) using a 532nm laser was used for Raman imaging. Raman spectra was analyzed by Bruker Scientific DXR Raman Microscope with 532 nm wavelength laser and filter. The samples were placed on glass slides for the detection using 100 × optical lenses. A PANalytical EMPYREAN XRD with Cu Kα radiation source under scanning conditions of 5–70 degrees was utilized for structural study of the crystal. The IR Affinity −1, Shimadzu was used for FTIR analysis, and the sample was pressed with KBr to form a pellet. The particle size and zeta potential analysis was carried out using Malvern Zetasizer using a 173° backscattering angle. All the measurements were performed at 25 °C.

Dissolution test was carried out with a Distek Dissolution Tester (North Brunswick, NJ) according to the USP-II paddle method, and Griseofulvin content was measured by Agilent 8453 modelled UV-Visible spectrophotometer. Two different pH medium solution containing 1000 mL of de-ionized water (pH=7.0) and 0.1N HCl (pH=1.40) were used as the dissolution medium. Measurements were done at 37°C and a paddle speed of 50 rpm. The DDC was weighed and an equivalent to a Griseofulvin dose of 8.9 mg was added to the dissolution medium. Aliquots of 4.0 mL were taken manually at intervals of 2, 5, 10, 20, 30, 60, 120, 180, 240 and 300 minutes respectively and filtered using 0.2 μm membrane filter. Then they were analyzed for Griseofulvin content using UV-Visible spectrophotometer at 295nm wavelength. The physical mixture of drug and HPMC was also used separately in the dissolution test for comparison. To determine the amount of drug adsorbed in the DDC, methanol was added to it and sonicated for 30 minutes. The solution was filtered, diluted with milli-Q water and measured for drug content using a UV-Vis spectrophotometer. All the experiments were carried out in triplicates for accuracy measurement.

3.0. Results and Discussion

3.1. Particle analysis

Griseofulvin, an antifungal BCS class II drug that is highly hydrophobic has water solubility of 8.64 ± 1.0 mg/L [49]. The work of this methodology provided an increase in overall solubility by a factor of 1.65 times with 4 times lowered time required for 50% dissolution. Particle size measurements using dynamic light scattering (DLS) showed diatom average particle size of several micrometer ranges and 2 to 3 ± 0.5 μm for diatom drug composites (Figure-2-b) with polydispersity index of 0.675. Additionally, zeta potential calculated −13 ± 2 mV in aqueous solution predicts the stability of DDC composites. The porosity properties of diatom illustrated in figure-2(a) postulates its mesoporous and nano porous architecture [52]. We have also investigated the nitrogen adsorption BET isotherm and found cumulative pore volume to be 0.07 cc/g and surface area of 24.5 ± 0.9 m²/g that was significantly higher than the traditional nano/micro scale particulate drug delivery systems [40].

Figure-2:

a) Histogram data for diatom porosity characterization, and b) DDC particle size analysis by DLS.

3.2. Morphological Characterization

Scanning Electron Microscope illustrated in Figure-3 is for pure Griseofulvin, HPMC and DDC. It is clearly evident that griseofulvin crystals were formed on the diatom frustule surface (Fig-3b). Figure-3d also illustrates HPMC deposition on the drug crystals which helped in inhibiting crystal size growth.

Figure-3:

a) Pure Griseofulvin b) Griseofulvin and Diatom/HPMC composites (3:3:1) c) Pure HPMC and d) Griseofulvin HPMC composite mixture.

3.3. Structural Characterization

Figure-4(a) shows FTIR spectra of diatom, pure griseofulvin, HPMC and DDC. The spectral bands observed at 3600 cm ⁻¹ and 1630 cm⁻¹ in the diatom spectra were attributed to the isolated silanol (Si-OH) and bending mode of physiosorbed water (H₂O) respectively [53]. Diatom also showed Si-O-Si band observed at 1060 cm⁻¹. The characteristic HPMC peaks were observed at 3450 and 2930 cm⁻¹ corresponding to -OH and -CH₃ stretching_. Pure Griseofulvin displayed two distinct peaks at 1704 and 1658 cm⁻¹ which were attributed to the stretching of carbonyl group of benzofuran and cyclohexanone respectively [54]. The spectral frequency at 1658 cm⁻¹ and 1704 cm⁻¹ were broadened in the DDC composite spectra. The broadening of these two peaks can be attributed to the hydrogen bonding between isolated silanols located on the surface of diatom and carbonyl group of benzofuran and cyclohexanone of griseofulvin. Moreover, peaks from griseofulvin in the range of 1617–890 cm⁻¹ could also be seen in DDC indicating the presence of Griseofulvin. All the peaks for diatom and HPMC overlapped onto the DDC structure showing the presence of these components in drug crystals.

Figure-4:

a) FTIR spectra of Drug, Diatom, HPMC and its DDC composite mixture and b) Raman Spectra of drug, diatom, HPMC and DDC.

Next, Raman spectroscopy was used to investigate the structure of DDC. Raman allowed the determination of intra and intermolecular stretching and bending modes in solid state. Figure-4(b) shows the Raman spectra of diatom, Griseofulvin and DDC. Griseofulvin spectra showed strong peaks at 1550–1800 cm⁻¹ and 2800–3200 cm⁻¹ region respectively, which were attributed to the C-O stretching of benzofuran ring and C–H stretching of GF respectively [55]. The same characteristic peaks were also observed in DDC, which indicated no significant alteration of the Griseofulvin structure. Raman chemical mapping was used to image and map the top surface of the drug particle. Two particles embedded in the DDC were mapped and Figure-S-3 present the intensity distribution of Griseofulvin. This was done by plotting the peak area of the selected Raman bands over the entire scanned area. A red color corresponded to a high GF concentration followed by yellow and green, whereas blue color represents the background. The distribution of drug crystals was seen throughout the scanned area. These observations agreed with the SEM measurements suggesting that drugs existed in the form of nano to semi micron particles dispersed in the porous diatom surfaces.

XRD based structural analysis is shown in Figure-5, which shows that immobilization on the diatom did not alter the crystalline structure of drug during the antisolvent precipitation. Therefore, the Griseofulvin crystals were highly pure and the DDC formation did not alter the polymorph.

Figure-5:

XRD analysis Griseofulvin, HPMC, diatom and DDC (Inset: pure Griseofulvin spectra)

3.4. Dissolution Studies

Figure-6 demonstrates the dissolution profile of griseofulvin into diatom silica biomaterial, diatom/HPMC mixture and without the biomaterials. Since griseofulvin works better in lower intestine with at neutral pH, water (pH 7.0) used as dissolution medium. Additionally, 0.1N HCl solution was used at pH 1.40 as for dissolution medium to check the enhancement in the DDC formulations.

Figure-6:

Dissolution profile for griseofulvin and its composite for pH value at 7.0 and gastric pH at value 1.40 ranges respectively.

The DDC contained ~41% of griseofulvin by weight. It is evident that DDC showed increased griseofulvin release. The Table-1 highlights the overall scenario of dissolution enhancement based on T₅₀ (time taken for 50% dissolution) and T₈₀ (time taken for 80% dissolution) for the DDC. Pure griseofulvin showed a T₅₀ of 240 minutes at neutral pH while DDC showed a T₅₀ of 47 minutes. Meanwhile for T₈₀ for pure griseofulvin was more than 5 hours while for DDC it was only 200 minutes. The dissolution profile for DDC at acidic pH (1.40) was slightly less enhanced compared to neutral pH due to the dissolution medium interaction. As there were not significant differences in dissolution of griseofulvin composites in both pH media, it could be postulates that this formulation will work both in gastro and intestinal fluids. The increased dissolution rate demonstrates that the diatom frustules are good candidates to serve as a carrier for hydrophobic drugs. The enhanced dissolution rates are attributed to the formation of very small crystal on the diatom surface and surface attachment via physical adsorption and weak van der Waals interaction of drug molecules onto the diatom frustules [56]. The higher release rate of drug molecules could be attributed to the high surface area of the diatom frustules. Additionally, the presence of the free hydroxyl groups on diatom frustules also facilitated strong interactions between water molecules and the griseofulvin crystals.

Table-1:

Dissolution parameters for Griseofulvin and DDC

Drugs	Dissolution Enhancement @ T(50) minutes	Dissolution Enhancement @ T(80) minutes
Griseofulvin (GF)	240	Infinity
GF/HPMC	114	Infinity
GF/Diato m/HPMC @ pH 7.0	47	200
GF/Diato m/HPMC @ pH 1.40	58	180

4.0. Conclusion

The immobilizing of hydrophobic drug Griseofulvin on functionalized diatom frustules was accomplished via antisolvent precipitation. HPMC was used to control the crystal size of the drug. In vitro dissolution profile showed a dramatic increase in release rate based on T₅₀ and T₈₀ measurements. Compared to pure GF, T₅₀ decreased by 52.5 percent in presence of HPMC, and 80.41% in DDC at pH 7 and 75.8 percent at pH 1.40. Pure drug as well as those in presence of HPMC never reached 80% release. However, T₈₀ at pH 7.0 and 1.4 were 200 and 180 minutes respectively. As a result of increased hydrophilic dissolution of DDC, it is expected that the bioavailability will also be improved. Therefore, diatoms offer unique architecture on which hydrophobic drugs may be immobilized to improve drug delivery.

Highlights.

• Antisolvent crystallization successfully immobilized hydrophobic drug Griseofulvin onto diatom frustule surface.

• Nano and mesoporous silica architecture of frustules facilitated for encapsulation of higher drug loadings.

• Free hydroxyl group generated onto frustule surface accelerated faster Griseofulvin release mechanism.

• This approach is a novel approach to drug delivery using a natural product.