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Conclusions from previously reported studies have revealed that many commonly used pharmaceutical excipients, known to be pharmacologically inert, show effects on drug transporters and/or metabolic enzymes. Thus, the pharmacokinetics (absorption, distribution, metabolism, and elimination) of active pharmaceutical ingredients are possibly altered because of their transport and metabolism modulation from the incorporated excipients. Excipients such as surfactants, polymers, fatty acids, and solvents have been reported previously as CYP450 inhibitors. Based on all the stated outcomes, the most potent inhibitors were found to be surfactants and the least effective were organic solvents. However, there are many factors that can influence the inhibition of CYP450, for instance type of excipient, concentration of excipient, type of CYP450 isoenzyme, incubation condition, etc. Such evidence will be very useful in dosage form design, so that the right formulation can be designed to maximize drug availability, especially for poorly bioavailable drugs. The aim of this study is to identify the most potent fatty acids that can inhibit CYP450 enzymes and produce solid-lipid nanoparticles (SLNs) to minimize metabolism and increase drug permeability without harmful side effects. The high-performance Liquid Chromatography-Mass Spectrometric (LCMS) methods were developed for (i) simultaneous quantification of omeprazole, dextromethorphan, verapamil, and propranolol (IS) (ii) individual quantification of testosterone. The separation for both methods were achieved on Poroshell 120 EC-C18 (150 x 4.6 mm) using gradient and isocratic approaches. These analytical methods were successfully used for screening of fatty acids, pre-formulation, and formulation studies. Microsomal assay with rat-liver microsomes was used for screening of fatty acids based on their inhibitory effect on various CYP450s. The most potent inhibitor (stearic acid) was then incorporated in three different solid-lipid nanoparticles formulations (SLNs). The pre-formulation and formulation studies were systematically optimized to produce SLNs. These SLNs were characterized by SEM, zetasizer, DSC, dialysis membrane, and HPLC. Moreover, metabolic assay, permeability assay and cytotoxicity were also studied to investigate the effect of stearic acid, present in SLNs, on CYP450s enzymes and caco-2 cell permeability. The results of the LCMS method validation revealed that the coefficient of determination (R2) values for all probe drugs were greater than 0.99. Furthermore, all the analytes showed highest recovery and lowest interday/intraday values. The effect of fatty acids on CYP450s activity was evaluated using rat-liver microsomes. The percentage of probe drugs recovered in the presence of heptadecanoic acid, myristic acid, pentadecanoic acid, and stearic acid were found to be higher than the control study. Stearic acid was the most potent inhibitor out of all the excipients with an IC50 value ³ 30 µM for CYP3A4, 3A5, 2C8, 2C19, and 2D6. At approximately 200 µM of stearic acid, nearly 100% of the compounds were recovered. Based on the screening phase data, three solid-lipid nanoparticles formulations were produced using stearic acid as an oil phase. Results indicated that testosterone loaded SLNs were spherical in shape with an average particle of 278.29 nm diameter with 55% entrapment efficiency. Moreover, microsomal and permeability assays revealed that the presence of stearic acid in testosterone decreased metabolism by CYP450s and increased permeability via caco-2 layer. Approximately, 54% of testosterone was recovered from the metabolic assay with CLint of 0.04 µL/min/mg and apparent permeability was successfully improved with Papp value of 1.53x10-6 cm/sec. On the other hand, verapamil loaded SLNs were spherical in shape with an average particle size of 300.30 nm but with a better entrapment efficiency of 77%. The formulation demonstrated burst release with nearly 20% drug release in under 0.5 hours, followed by sustained release for 23.5. In contrast to testosterone, metabolic assay revealed poor inhibition for CYPs with net recovery of 2.53% and CLint of 0.014 µL/min/mg for verapamil. However, there was significant improvement in the apparent permeability of verapamil (Papp = 2.39x10-6 cm/sec) when compared to control (Papp = 9.00 x10-6 cm/sec). The third formulation, dextromethorphan loaded SLNs was unsuccessful because of its chemical nature and water solubility. Several strategies were used to prepare dextromethorphan-SLNs such as double emulsion, solvent evaporation, and convention hot emulsion method. The affinity of lipophilic drugs for lipids makes them simple to integrate into SLN. While hydrophilic drugs (such as dextromethorphan) are more readily partitioned in the water phase during the production process, they are more difficult to incorporate into the hydrophobic matrix and therefore, further investigation is required for this formulation. The cell cytotoxicity study further confirms the safety of all the ingredients at different concentrations present in various formulations. Thus, stearic acid based SLNs are a potential formulation approach which can inhibit CYP450 enzymes and improve intestinal permeability of various drugs and therefore, requires further investigation to improve the formulation with various approaches.

Inflammation is a physiological response to tissue injury or infection. It is a coordinated immune reaction intended to protect the host from potentially dangerous toxins or pathogens. Acutely, inflammation is a critical component of innate immunity; however, under some circumstances, inflammation may be detrimental and potentially lethal. Chronic inflammation underlies a litany of debilitating conditions, including many top causes of death worldwide. Over the past decade, tremendous research has been undertaken to develop new treatment strategies for chronic inflammatory related diseases. While much success has been achieved, a need still exists for more targeted and innovative therapies. The objective of the first study was to investigate the ability of nanoparticles to improve the delivery of anti-inflammatory therapies to chronic inflammation sites. Here, nanoparticles, composed of poly (lactic-co-glycolic acid) (PLGA), were surface modified with mannose as a targeting ligand to macrophages, which are abundant in the chronic inflammation site. Furthermore, the nanoparticles were modified with acid-sensitive sheddable PEGylation, which would promote uptake of the nanoparticles by macrophages at the acidic inflammation site. In this work, we encapsulated betamethasone-21-acetate, an anti-inflammatory drug, into the nanoparticles and found that these formulation modifications (i.e. mannose ligand, acid-sensitive sheddable PEGylation) successfully increased the delivery of betamethasone to sites of inflammation and improved the retention of the formulation within the chronic inflammation site. The primary objective of the second study was to reduce the acute immunogenicity of a solid lipid nanoparticle formulation designed in our laboratory to deliver anti-inflammatory TNF-α siRNA to chronic inflammatory sites in a mouse model of arthritis. In previously published work, we reported the therapeutic efficacy of a solid lipid nanoparticle containing TNF-α siRNA; however, upon further investigation, this nanoparticle formulation was found to be acutely immunogenic, inducing pro-inflammatory cytokine production shortly after administration. In the second study, betamethasone-21-acetate was incorporated into the siRNA-containing solid lipid nanoparticles and tested for its ability to reduce activation of the acute immune response by the siRNA and its carrier. In this work, we found that inclusion of betamethasone successfully reduced the immunogenicity of the siRNA nanoparticle formulation, and that betamethasone, together with TNF-α siRNA, increased the ability of the TNF-α siRNA solid lipid nanoparticle formulation to reduce TNF-α production in vitro.

The strategy to improve the lipophilicity of gemcitabine by conjugation with a lipid (e.g. docosahexaenoic acid (DHA) or stearoyl acid) allows to prevent the deamination of gemcitabine by cytidine deaminase, main cause of gemcitabine degradation in vivo. In addition, this strategy may confer gemcitabine a strong antitumor activity compared to parental drug. 4-(N)-docosahexaenoyl 2´, 2´-difluorodeoxycytidine (DHA-dFdC) is a novel compound with strong in vitro and in vivo antitumor activity in several cancer models (e.g. pancreatic, breast, leukemia, and lung cancer). However, the toxicity of DHA-dFdC has not been tested in an animal model. In this dissertation, a preclinical short-term toxicity study to evaluate the tolerability of DHA-dFdC was performed in healthy DBA/2 mice. DHA-dFdC showed a dose-dependent toxicity, affecting mainly the spleen. The repeat-dose maximum tolerated dose (RD-MTD) of DHA-dFdC was 50 mg/kg. DHA-dFdC exhibits a strong efficacy at or below its RD-MTD in mouse models of pancreatic cancer or leukemia. Unfortunately, DHA-dFdC has two main issues, chemical instability and poor solubility in water. In this dissertation, a solid lipid nanoparticle (SLN) formulation of DHA-dFdC was developed to improve its chemical stability and water solubility. The SLN formulation improves the apparent water solubility of DHA-dFdC by 2.6-fold, as compared to a previously developed DHA-dFdC in Tween 80-ethanol-water solution. The SLN formulation as a lyophilized powder also improves the chemical stability of DHA-dFdC. In addition, DHA-dFdC-SLNs showed stronger antitumor activity than DHA-dFdC in Tween-ethanol-water solution in a mouse model when given intravenously. Finally, this dissertation demonstrates in a mouse model that the absolute oral bioavailability of DHA-dFdC in the DHA-dFdC-SLN formulation is 68%. It is concluded that the SLN formulation of the DHA-dFdC (i.e. DHA-dFdC-SLNs) improves the chemical stability of DHA-dFdC and increases its apparent water solubility, and the DHA-dFdC-SLNs can be administered intravenously or orally

Solid lipid nanoparticles (SLNs) are a category of delivery systems applicable to various bioactive compounds in the food industry. Compared to conventional emulsions that have a fluidic oil phase, the mobility and release of bioactive compounds can be controlled by encapsulation in the solid lipid matrix with appropriate properties. Common approaches of preparing SLNs are high energy methods and solvent evaporation methods, which have can lead to degradation of compounds during processing and residues of organic solvent, respectively. In this thesis, a low energy approach based on the phase inversion temperature method has been used to prepare SLNs based on anhydrous milk fat (AMF). Food grade surfactant Tween 80 was used as a surfactant, and beta-carotene was used as a model lipophilic bioactive compound. AMF and surfactant solution with 0-1.0 M NaCl were mixed to form coarse emulsions that were heated at 80-95 °C [Celsius degree] for 30 min to induce phase inversion, followed by a fast cooling process in ice bath. The phase inversion temperature decreased from >95 °C to 73°C when NaCl increased from 0 to 1.0 M in the aqueous phase. Up to 10% w/w of AMF can be encapsulated in the system as transparent dispersions, with particle mean diameter smaller than 25 nm. The SLN dispersions were dilution and dialysis stable, and the particle size and turbidity maintained unchanged during the 90-day storage at room temperature. Compared with beta-carotene encapsulated in soybean oil-based nanoemulsion, degradation of beta-carotene in SLNs was much reduced. The studied SLNs may find unique applications in incorporating lipophilic bioactive compounds in transparent beverages.

Our goal was to develop a mucoadhesive, oral vaccination delivery platform designed to target Peyer’s patches at ileum. In order to achieve this, we prepared poly(methyl methacrylate) (PMMA) particles of various sizes using W/O/W emulsification solvent evaporation and surface polymerization methods. We then coated and employed mucoadhesive polymers into the carrier system to enhance the residence time in the targeted site. Also we developed our own in vitro mucoadhesion testing ramp as an evaluation tool. Finally, nano- and micro-structured particles were manufactured as two different oral vaccine delivery systems (Solid Lipid Nanoparticles, SLNs; and Protein Coated Microcrystals, PCMC). After the model antigen, bovine serum albumin (BSA) was loaded into the SLNs or PCMC; mucoadhesive polymers were then incorporated and formulated the mixture into pellets. The pellets were then layered with an enteric coating, which was composed of a mixture of Eudragit® FS 30 D/Eudragit® L 30 D-55 for ileum targeted delivery. The in vitro mucoadhesion test ramp was capable of investigating the mucoadhesive properties of tablets and pellets, providing a rank order for study. Most important of all, it was anticipated that this might reduce the burden of testing animals for future proposed mucoadhesive studies. Microcapsules/beads of specific size were manufactured reproducibly by solvent evaporation and surface polymerization. Although we could not specify the cut-off size at the pyloric sphincter in mice, we concluded that the cut-off size at the pyloric sphincter in rats was approximately 2.5-3 mm, which was supported by both the biodistribution data and the direct image results from scintigraphy scanning. Moreover, we found that the particle size significantly alters the gastric emptying time in both rodent models. The small microcapsules/beads were hindered in the folds of the stomach (size 50-100μm for mice and size 0.5-1 mm for rats) and emptied the slowest, followed by the large particles, then the medium particles. Finally, PCMC and SLNs we manufactured were suitable carriers for protein API, such as BSA. These particles were of fitting size for M cell uptake, which would possibly induce mucosal immune responses. Therefore, an antigen containing PCMC and SLNs might be suitable platforms for oral vaccination.

Solid lipid nanoparticles (SLNs) consist of spherical solid lipid particles in the nanometer size range, which are dispersed in water or in an aqueous surfactant solution. SLN technology represents a promising new approach to deliver hydrophilic as well as lipophilic drugs. The commercialization of SLN technology remains limited despite numerous efforts from researchers. The purpose of this research was to advance SLN preparation methodology by investigating the feasibility of preparing glyceryl monostearate (GMS) nanoparticles by using three preparation methods namely microemulsion technique, magnetic stirring technique and temperature modulated solidification technique of which the latter two were developed in our laboratory. An anticancer drug 5-fluorouracil was incorporated in the SLNs prepared via the temperature modulated solidification process. Optimization of the magnetic stirring process was performed to evaluate how the physicochemical properties of the SLN was influenced by systematically varying process parameters including concentration of the lipid, concentration of the surfactant, type of surfactant, time of stirring and temperature of storage. The results demonstrated 1:2 GMS to tween 80 ratio, 150 ml dispersion medium and 45 min stirring at 4000 RPM speed provided an optimum formulation via the temperature modulated solidification process. SLN dispersions were lyophilized to stabilize the solid lipid nanoparticles and the lyophilizates exhibited good redispersibility. The SLNs were characterized by particle size analysis via dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM), differential scanning calorimetry (DSC), drug encapsulation efficiency and in vitro drug release studies. Particle size of SLN dispersion prepared via the three preparation techniques was approximately 66 nm and that of redispersed lyophilizates was below 500 nm. TEM images showed spherical to oval particles that were less dense in the core with a well-defined shell and the particle size was in agreement with the particle size analysis data obtained by DLS. DSC thermograms of the lyophilized SLNs indicate a reduction in the crystallinity order of GMS particles. The drug encapsulation efficiency was found to be approximately 30%. In vitro drug release studies from redispersed lyophilized SLNs showed that 17 % of the encapsulated drug was released within 2 h. The SLNs prepared in our lab demonstrated characteristics that can potentially be utilized in an anticancer drug delivery system. Future in vitro cell culture and in vivo animal model studies will delineate compatibility and utility of these formulations in biological systems.

Pre-clinical and clinical trials suggest that pre-treatment with dexamethasone (Dex) may facilitate enhanced uptake of subsequently administered chemotherapeutic agents. To reduce the side effects associated with systemic administration of Dex, solid lipid nanoparticles (SLNs) containing dexamethasone palmitate (Dex-P) were prepared as a means of achieving tumor-targeted drug delivery. These studies were aimed at evaluating the physicochemical properties and both the physiological and storage stability of the SLNs. SLNs were prepared using nanotemplate engineering technology. Stearyl alcohol (SA) was used as the lipid phase with Brij® 78 and Polysorbate 60 as surfactants and PEG6000 monostearate as a long-chain PEGylating agent. Both formulations exhibited a small particle size, ellipsoidal shape, and low polydispersity. 1H-NMR spectroscopy confirmed that SLNs have the expected solid core and PEGylated surface. Analysis of the bulk materials indicated that a number of complex interactions are present among the SLN components, including a eutectic between SA and Brij® 78. Dex-P could be incorporated in SLNs at 10-30% w/w SA with encapsulation efficiencies >85%. A preferential interaction with the SA-Brij® 78 eutectic was identified, indicating a possible interfacial localization. For comparison, SLNs were also prepared with ascorbyl palmitate (AP) and curcumin. Higher drug loads were achieved with both palmitate-containing prodrugs than curcumin, though all appeared to align differently within the SLNs. SLNs undergo a concentration-dependent particle size growth when incubated at physiological temperature. However, they appear to remain intact with over 85% of the added Dex-P retained at 24 h in conditions mimicking human plasma. In the presence of carboxylesterase, SLNs became turbid and showed a reduction in particle size as compared to controls. This instability was shown to be a result of the hydrolysis of PEG6000 monostearate and Polysorbate 60. To enhance storage stability, a lyophilization protocol designed to minimize changes in the physicochemical properties of SLNs was developed. During a 3 month period, lyophilized SLNs stored at 4°C demonstrated the greatest stability, showing a consistent particle size and an encapsulation efficiency >80%. Overall, these results indicate that Dex-P loaded SLNs possess the physicochemical properties and stability desirable for development as a tumor-targeted drug delivery system.