Introduction
Temozolomide (TMZ) is an anticancer agent with alkylating properties, included in the group of imidazotetrazine derivatives, developed in Aston University in the 1980s. TMZ was found to have antitumor activity in intracranial and extracranial tumors. It has been used for glioblastoma multiforme treatment (GBM) (1). However, treatment of GBM remains a challenge, largely due to the fast degradation of TMZ, inability to deliver an effective dose of TMZ to tumors, and lack of target specificity which may cause systemic toxicity. Nanoparticles can be a solution for the rapid degredation of TMZ and can specifically deliver TMZ to GBM cells.
Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) have been used as efficient delivery vehicles for therapeutic agents to the brain. PLGA NPs can be produced in any desired shape and size and can trap molecules of any size (2). PLGA has been evaluated for encapsulating small anticancer agents used in nanoparticular cancer chemotherapy (3). Although NPs offer a number of positive properties for drug delivery, NPs have often been combined with hydrogels to further improve the therapeutic index, particularly in localized administration.
Hydrogels, like Pluronic®, excel in controlled release applications because of their tissue compatibility and ease of dispersion in the matrix (4). Hence, we hypothesized that a hydrogel, containing NPs loaded with TMZ, would provide a sustained and local delivery of the drug and destroy cancer cells that might remain in the areas that could not be reached by tumor resection during surgical intervention in the treatment of GBM.
Pluronic F-127 is a hydrophilic polymer which is capable of holding a large amount of water and showing sol-gel transition near 37 °C and the unique thermo-responsive property of the polymer is directed towards a wide area of drug delivery applications (5). In addition to Pluronics inertness and good biocompatibility, its ability to release entrapped drug in aqueous media makes Pluronics particularly suitable as drug carriers in the controlled release of pharmaceuticals (6).
The first part of the study was conducted to formulate TMZ loaded NP formulations with PLGA by emulsion-solvent evaporation method. The PLGA NP formulations were evaluated for their particle size and size distrubution, entrapment efficiency, zeta potential and in vitro drug release studies. Then, the optimum NP formulation was chosen to develop a thermo-responsive hydrogel formulation with Pluronic F-127. Also, hydrogel formulations were tested for their rheological and drug release properties (7).
Many manufacturing process variables affect drug release from dosage forms. To contemplate the TMZ release mechanism from Pluronic F-127 hydrogel and PLGA NPs, different kinetic models were considered to fit the experimental data (8).
After giving a brief information about the aim of the study, it was planned to focus on the kinetics of TMZ release from NPs from a Pluronic F-127 hydrogel matrice. Caccavo reviewed the trend in mathematical models used in the field of hydrogel-based drug delivery system and found that the main fit model equations used were: Zero order kinetics, first order kinetics, Korsmeyer-Peppas kinetics, Weibull and The Higuchi kinetics (9).
Methods
Materials
Temozolomide, PLGA (acid terminated; Mw =24,000-38,000; copolymer ratio 50:50), Mowiol 18-88 (Mw =130,000), and Pluronic® F-127 were supplied by Sigma-Aldrich. Ultrapurified water was obtained from the Milli-Q system. All other reagents and solvents used in this study were of analytical grade.
Methods
Formulation of TMZ Loaded PLGA Nanoparticles
The formulation method of TMZ NPs was modified from the study of Ananta et al. (7,9,10). NPs were prepared using emulsion (w/o) solvent evaporation method. TMZ and PLGA were dissolved in 1 mL dimethylformamide. The resulting solution was added in drops to 5 mL of PVA 5% solution under homogenization. The organic solvent used was evaporated. NPs were collected by centrifugation and washed with deionized water to remove free TMZ. And then NPs were collected from the resulting emulsion.
Particle Size Measurement of Nanoparticles
The mean particle size of the NPs was measured by Malvern Zeta Sizer (Malvern Instrument Ltd.). The dispersions were diluted with nanopure water and the sample was placed in a disposable cuvette at a count rate for 20 seconds. The particle size measurment data are reported in Table 1.
Determination of Encapsulation Efficiency (%)
Entrapped TMZ amount in the NPs was determined with our validated HPLC assay and calculated as equation below (7).
Formulation Design of Nanoparticle Loaded Pluronic F-127 Thermoreversible Gel
The “cold method” was used in the preparation of hydrogels. Pluronic® F-127 was weighed, added to 50 mL of ultrapure water, and mixed on magnetic stirrer at 900 rpm in an ice bath until Pluronic® F-127 was homogeneously dissolved (7). Gel formulations containing 18-20 and 25% (w/v) Pluronic® F-127 were prepared.
Physicochemical Evaluation of Nanoparticle Loaded Pluronic® F-127 Hydrogels
pH Levels of Produced Hydrogels
The pH levels of the hydrogel formulations were measured with Eutech Instruments pH 700 (n=3) (9).
Sol-gel Transition Temperature
The gelation temperatures of developed formulations were determined by a modified version of the reverse-tube method (n=3) (9).
Viscosity
Sol and gel viscosities of the formulations produced were measured by running the viscometer at speeds of 5-100-5 rpm. Viscosities in gel form were measured with T96 spindle in Brookfield DVII device. Measurements were repeated three times and graphs were plotted as viscosity (cP) vs. shear rate (rpm) (9).
Mechanical Properties
Texture profile analysis (TPA) was performed in the TPA mode of the TA-XT2 Texture Analyzer (Surrey, UK). The formulations were transferred in gel form into 25 mL beakers. Measurements were recorded by puncturing the samples (11).
In vitro TMZ Release Studies
In vitro drug release experiments for all formulations were performed in a shaker bath at a temperature of 37 °C ±1 °C and pH 5 buffer containing 0.1% ascorbic acid. The amount calculated according to the data obtained from the loading capacity and which would provide the target dose was weighed and suspended in buffer with 0.1% ascorbic acid, and for hydrogel formulations, they were suspended in Pluronic® F-127 hydrogel and attached with two-end weighted clips in the dialysis membrane tube placed in a beaker. Twenty mL of buffer solution was placed in each beaker. Samples were taken at various time intervals and the release medium was replaced with new medium to prevent degradation of the active substance after 24 hours.
Kinetic Evaluation of In Vitro Drug Release Studies
Dissolution rate profiles were obtained by determining the amount of active substance released by conducting an in vitro release study from the formulations. In order to determine which mathematical model the active substance release profiles fit into, the equations of the zero-order, first-order, Higuchi, Weibull and Korsmeyer-Peppas kinetic models were applied in Excel®. Statistical evaluations were made using GraphPad Prism 6® and the findings obtained were evaluated (12).
Results and Discussion
NPs were prepared by emulsion solvent diffusion method. All formulations were produced with two different TMZ amounts, respectively 5 and 10 mg, so as to observe the effect of TMZ amount on the size and encapsulation efficiency of nanoparticles. NPs were discrete, nearly spherical with a size range of 164.4-235.5nm. NPs of 10-200 nm size are suitable for a nano-carrier systems designed for local application that will cross the blood-brain barrier in the brain and reach the tumor site. Because, nanoparticles smaller than 10 nm are excreted renally and NPs larger than 300 nm are removed from the body by RES (13,14). A2-10 formulation with least particle size had the maximum entrapment efficiency (Table 1). It was determined that increasing the amount of TMZ correlated to a higher encapsulation efficiency in all the formulations (15). This effect is explained by the fact that an excess of drug leads in a more viscous dispersed phase, making mutual dispersion of the phases problematic and resulting in the formation of large particles (16). Based on these findings A2-10 formulation was selected in the preparation of hydrogel formulations.
The Zeta potential indicates that it can generate enough repulsion to overcome the gravitational attraction between NPs and suspension dispersion has better stability (17). If formulations were observed to evaluate the effect of the amount of TMZ on the particle properties in Table 1, it was seen that particle size, polydispersity index (PDI) and zeta potential increased where the active substance was adsorbed to the terminal carboxyl groups of PLGA NPs. This is because terminal carboxyl groups are located on the surface of NPs (14). In addition, this phenomenon can be explained by the production of larger particles by causing more active substances to a more viscous oil phase and making it difficult for phases to disperse into each other (16).
The PDI value of polymeric NPs is required to be less than 0.3. According to the PDI data obtained in our study (Table 1), it was seen that the PDI value of all formulations was within the acceptable range.
Smart hydrogels are attractive because of their unique sol-gel phase transitions at body temperature, biocompatibility, safety, and injectability as a solution in the body before transforming into gel matrices. In formulations containing 18%, 19% and 20% Pluronic® F-127, which showed sol-gel transition, it was observed that the pH level increased the with increasing concentration (18). According to statistical calculations, the difference between pH levels was found to be significant (p<0.0001), but since the pH level of the tumor microenvironment was close to the pH level, the obtained pH level was suitable for local drug administration to the tumor site (19). In our study, increased concentration resulted with a lower transition temperature (Table 2). Sol-gel phase transition temperatures of 18%, 19%, 20% and 25% Pluronic F127 containing solutions were 32 °C, 30 °C, 26 °C and 20 °C, respectively. Elasticity and cohesiveness of gels were close for all formulations.
The importance of the sol-gel transition temperature is that the formulation remains in fluid sol state at room temperature, leaving the package or injector and transforming into an in situ gel and maintaining its shape. It was observed that the gelation temperature decreased with the increase of Pluronic® F-127 concentration in the formulation, and this was confirmed by the literature (18).
It was observed that the formulations were at the bottom of the tubes in the left state at room temperature (25 °C). Concentrations of 18% and above appeared to be a non-flowing gel when the gels were turned upside and incubated (37 °C). It was observed that the formulation containing 17% Pluronic® F-127 returned to its sol form, and formulations containing 15 to 16% Pluronic® F-127 remained in its left form. According to the results which was shown in Table 2, H18 was chosen as a proper injectable system with a gelation temperature of 32 °C which led to be a solution in the room and transited into a gel in the body (5). pH values of gels rangingbetween 6.80-6.94 are suitable for brain application. Also, Persi revealed that acidic pH disabled hypoxia adaptations of cancer cells and compromised tumor cell growth which indicated that pH values of formulations between 6.80-6.94 were suitable for brain cancer treatment with less damage to brain tissue (20).
The A2, A2-5, and A2-10 formulations showed a triphasic profile. In all formulations, a burst effect was observed in the first 4 hours and a plateau was detected in the following 72 hours (Figure 1). The reason for the difference in the percentage of active substance released from the formulations is thought to be proportional to the decrease in the percentage of active substance released per unit time as the encapsulation rate of the active substance increases (21). In Figure 2, the drug release of NPs from the hydrogel formulations reached approximately 25% of the total TMZ in 60 days. The in vitro release profile of the active substance in the hydrogel from the NP system showed an average of 10% of immediate release in the first 12 hours, then reaching a plateau in the next 60 days.
The in vitro release profile of TMZ in hydrogel showed an immediate release of 46% in the first 6 hours and reached a plateau in the next 18 hours. The release profile of the active substance from the NP system in the hydrogel allowed the release of TMZ at lower doses over a long period of time.
Dissolution rate profiles were obtained by determining the amount of active substance released by conducting an in vitro release study from the formulations. In order to determine which mathematical model the active substance release profiles fit into, the equations of the zero-order, first-order, Higuchi, Weibull and Korsmeyer-Peppas kinetic models were applied in Microsoft Office Excel and the findings obtained were evaluated (22). The applied mathematical equations used to describe release characteristics of TMZ NP from Pluronic F-127 gels are given in Table 3.
The mathematical models include: (a) zero-order model, Q=Q0+k0 t, where Q is the cumulative release percentage, k0 zero release rate constant, and t time; (b) first-order model, Log Ct=Log C0-k1t/2.303, where Log C is the cumulative release percentage, k1 first release rate constant, t time (c) Higuchi model, Ft=kHt 1/2, where Ft is the cumulative release percentage, kH Higuchi release rate constant, and t time (11). (d) The formula for Weibull function is F=1-exp (-atb). Where F is the drug fraction released at time t, and a and b are constants. b, as a shape parameter (18). (e) Korsmeyer - Peppas model= Mt/M∞ = kp tn where Mt/Ma is the proportion of drug released at time t, k is the rate constant (Table 3) (11).
As can be seen in Table 4, in vitro release kinetics of the active substance show compatibility with the Weibull kinetic model in A2, A2-5, and A2-10 formulations. The fact that all of the calculated b values were less than 1 indicated a kinetic profile where the active substance release rate occured faster at first, then resembled the 1st degree kinetic profile and reached a plateau (23).
The release of active substance by diffusion and/or relaxation in the polymeric system is explained by the Korsmeyer-Peppas kinetics. According to the R2 values obtained by the calculation made from the A2, A2-5 and A-10 formulations in the hydrogel, the release kinetics of the active substance were in accordance with the Korsmeyer-Peppas kinetics. This finding confirms the fact that Pluronic® F-127 hydrogel swells by absorbing water and the active substance is released as diffuse within the hydrogel system (24). The fact that the calculated n values were less than 0.45 indicated the Fickian diffusion profile (25).
Since the formulations showed compatibility with the Higuchi and 1st order kinetic model at the same time, a graph of the amount of active substance released (Q) versus the amount of active substance released (dQ/dt) in a certain time period was plotted to determine which one was compatible.
The release of water-soluble drugs from anhydrous hydrogel matrices involves simultaneous absorption of water and desorption of drug via a swelling-controlled diffusion mechanism (26). The fact that the calculated n values were less than 0.45 indicated the Fickian diffusion profile (25).
Study Limitations
Since the formulations showed compatibility with the Higuchi and First order kinetic model at the same time, a graph of the amount of active substance released (Q) versus the amount of active substance released (dQ/dt) in a certain time period was plotted to determine which one was compatible. For graphs showing biphasic characteristics, both phases and the entire profile were applied to the graph, and it was observed that the dQ/dt and Q values in all three graphs showed inverse proportion (Figure 3). This showed that the release was in accordance with the Higuchi kinetics (27).
Conclusion
A modified release drug delivery system of TMZ developed as a NP hydrogel served as a depot for sustained drug release and provided a rate-limiting barrier for modulation of drug release. Drug release from the hydrogel system was evaluated by means of mathematical modelling. The use of mathematical modeling turned out to be very useful for estimating the release kinetics before the release systems were implemented. The release mechanism was found to be diffusion controlled and not accompanied by dissolution of matrix. The release kinetics in H18 followed Korsmeyer-Peppas model.
Ethics
Ethics Committee Approval: Ethics committee approval is not required.
Peer-review: Externally peer reviewed.
Financial Disclosure: This study was supported by Ankara University BAP projects with the project number of 18L0237003.