Folate-conjugated near-infrared fluorescent perfluorocarbon nanoemulsions as theranostics for activated macrophage COX-2 inhibition.

Activated macrophages play a critical role in the orchestration of inflammation and inflammatory pain in several chronic diseases. We present here the first perfluorocarbon nanoemulsion (PFC NE) that is designed to preferentially target activated macrophages and can deliver up to three payloads (two fluorescent dyes and a COX-2 inhibitor). Folate receptors are overexpressed on activated macrophages. Therefore, we introduced a folate-PEG-cholesterol conjugate into the formulation. The incorporation of folate conjugate did not require changes in processing parameters and did not change the droplet size or fluorescent properties of the PFC NE. The uptake of folate-conjugated PFC NE was higher in activated macrophages than in resting macrophages. Flow cytometry showed that the uptake of folate-conjugated PFC NE occurred by both phagocytosis and receptor-mediated endocytosis. Furthermore, folate-conjugated PFC NE inhibited the release of proinflammatory cytokines (TNF-α and IL-6) more effectively than nonmodified PFC NE, while drug loading and COX-2 inhibition were comparable. The PFC NEs reported here were successfully produced on multiple scales, from 25 to 200 mL, and by using two distinct processors (microfluidizers: M110S and LM20). Therefore, folate-conjugated PFC NEs are viable anti-inflammatory theranostic nanosystems for macrophage drug delivery and imaging.

Multiple Linear Regression Predictive Modeling of Colloidal and Fluorescence Stability of Theranostic Perfluorocarbon Nanoemulsions.

Perfluorocarbon nanoemulsions (PFC-NEs) are widely used as theranostic nanoformulations with fluorescent dyes commonly incorporated for tracking PFC-NEs in tissues and in cells. Here, we demonstrate that PFC-NE fluorescence can be fully stabilized by controlling their composition and colloidal properties. A quality-by-design (QbD) approach was implemented to evaluate the impact of nanoemulsion composition on colloidal and fluorescence stability. A full factorial, 12-run design of experiments was used to study the impact of hydrocarbon concentration and perfluorocarbon type on nanoemulsion colloidal and fluorescence stability. PFC-NEs were produced with four unique PFCs: perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), perfluoro(polyethylene glycol dimethyl ether) oxide (PFPE), and perfluoro-15-crown-5-ether (PCE). Multiple linear regression modeling (MLR) was used to predict nanoemulsion percent diameter change, polydispersity index (PDI), and percent fluorescence signal loss as a function of PFC type and hydrocarbon content. The optimized PFC-NE was loaded with curcumin, a known natural product with wide therapeutic potential. Through MLR-supported optimization, we identified a fluorescent PFC-NE with stable fluorescence that is unaffected by curcumin, which is known to interfere with fluorescent dyes. The presented work demonstrates the utility of MLR in the development and optimization of fluorescent and theranostic PFC nanoemulsions.

Quality by Design Methodology Applied to Process Optimization and Scale up of Curcumin Nanoemulsions Produced by Catastrophic Phase Inversion.

In the presented study, we report development of a stable, scalable, and high-quality curcumin-loaded oil/water (o/w) nanoemulsion manufactured by concentration-mediated catastrophic phase inversion as a low energy nanoemulsification strategy. A design of experiments (DoE) was constructed to determine the effects of process parameters on the mechanical input required to facilitate the transition from the gel phase to the final o/w nanoemulsion and the long-term effects of the process parameters on product quality. A multiple linear regression (MLR) model was constructed to predict nanoemulsion diameter as a function of nanoemulsion processing parameters. The DoE and subsequent MLR model results showed that the manufacturing process with the lowest temperature (25 °C), highest titration rate (9 g/minute), and lowest stir rate (100 rpm) produced the highest quality nanoemulsion. Both scales of CUR-loaded nanoemulsions (100 g and 500 g) were comparable to the drug-free optimal formulation with 148.7 nm and 155.1 nm diameter, 0.22 and 0.25 PDI, and 96.29 ± 0.76% and 95.60 ± 0.88% drug loading for the 100 g and 500 g scales, respectively. Photostability assessments indicated modest loss of drug (<10%) upon UV exposure of 24 h, which is appropriate for intended transdermal applications, with expected reapplication of every 6-8 h.

Design of Thermoresponsive Polyamine Cross-Linked Perfluoropolyether Hydrogels for Imaging and Delivery Applications.

Perfluorocarbons are versatile compounds with applications in 19F magnetic resonance imaging (MRI) and chemical conjugation to drugs and pH sensors. We present a novel thermoresponsive perfluorocarbon emulsion hydrogel that can be detected by 19F MRI. The developed hydrogel contains perfluoro(polyethylene glycol dimethyl ether) (PFPE) emulsion droplets that are stabilized through ionic cross-linking with polyethylenimine (PEI). Specifically, PFPE ester undergoes hydrolysis upon contact with aqueous PEI solution, resulting in an ionic bond between the PFPE acid and charged PEI amino groups. Due to the ionic nature of the PFPE/PEI bond, potassium buffer is required to preserve the hydrogel's pH and rheological and emulsion droplet stability. The presence of the surface cross-linked PFPE droplets does not affect the hydrogel's rheological behavior, drug loading, or drug release, and the hydrogel is nontoxic. We propose that the presented hydrogel can be adapted to a broad range of biomedical imaging and delivery applications.

Sex Differences Revealed in a Mouse CFA Inflammation Model with Macrophage Targeted Nanotheranostics.

Monocyte derived macrophages (MDMs) infiltrate sites of infection or injury and upregulate cyclooxygenase-2 (COX-2), an enzyme that stimulates prostaglandin-E2 (PgE2). Nanotheranostics combine therapeutic and diagnostic agents into a single nanosystem. In previous studies, we demonstrated that a nanotheranostic strategy, based on theranostic nanoemulsions (NE) loaded with a COX-2 inhibitor (celecoxib, CXB) and equipped with near-infrared fluorescent (NIRF) reporters, can specifically target circulating monocytes and MDMs. The anti-inflammatory and anti-nociceptive effects of such cell-specific COX-2 inhibition lasted several days following Complete Freund's Adjuvant (CFA) or nerve injury in male mice. The overall goal of this study was to investigate the extended (up to 40 days) impact of MDM-targeted COX-2 inhibition and any sex-based differences in treatment response; both of which remain unknown. Our study also evaluates the feasibility and efficacy of a preclinical nanotheranostic strategy for mechanistic investigation of the impact of such sex differences on clinical outcomes. Methods: CFA was administered into the right hind paws of male and female mice. All mice received a single intravenous dose of NIRF labeled CXB loaded NE twelve hours prior to CFA injection. In vivo whole body NIRF imaging and mechanical hypersensitivity assays were performed sequentially and ex vivo NIRF imaging and immunohistopathology of foot pad tissues were performed at the end point of 40 days. Results: Targeted COX-2 inhibition of MDMs in male and female mice successfully improved mechanical hypersensitivity after CFA injury. However, we observed distinct sex-specific differences in the intensity or longevity of the nociceptive responses. In males, a single dose of CXB-NE administered via tail vein injection produced significant improved mechanical hypersensitivity for 32 days as compared to the drug free NE (DF-NE) (untreated) control group. In females, CXB-NE produced similar, though less prominent and shorter-lived effects, lasting up to 11 days. NIRF imaging confirmed that CXB-NE can be detected up to day 40 in the CFA injected foot pad tissues of both sexes. There were distinct signal distribution trends between males and females, suggesting differences in macrophage infiltration dynamics between the sexes. This may also relate to differences in macrophage turnover rate between the sexes, a possibility that requires further investigation in this model. Conclusions: For the first time, this study provides unique insight into MDM dynamics and the early as well as longer-term targeted effects and efficacy of a clinically translatable nanotheranostic agent on MDM mediated inflammation. Our data supports the potential of nanotheranostics as presented in elucidating the kinetics, dynamics and sex-based differences in the adaptive or innate immune responses to inflammatory triggers. Taken together, our study findings lead us closer to true personalized, sex-specific pain nanomedicine for a wide range of inflammatory diseases.

Quality by Design Approach Using Multiple Linear and Logistic Regression Modeling Enables Microemulsion Scale Up.

The development of pharmaceutical nanoformulations has accelerated over the past decade. However, the nano-sized drug carriers continue to meet substantial regulatory and clinical translation challenges. In order to address some of these key challenges in early development, we adopted a quality by design approach to develop robust predictive mathematical models for microemulsion formulation, manufacturing, and scale-up. The presented approach combined risk management, design of experiments, multiple linear regression (MLR), and logistic regression to identify a design space in which microemulsion colloidal properties were dependent solely upon microemulsion composition, thus facilitating scale-up operations. Developed MLR models predicted microemulsion diameter, polydispersity index (PDI), and diameter change over 30 days storage, while logistic regression models predicted the probability of a microemulsion passing quality control testing. A stable microemulsion formulation was identified and successfully scaled up tenfold to 1L without impacting droplet diameter, PDI, or stability.

Development of Theranostic Perfluorocarbon Nanoemulsions as a Model Non-Opioid Pain Nanomedicine Using a Quality by Design (QbD) Approach.

Pain nanomedicine is an emerging field in response to current needs of addressing the opioid crisis in the USA and around the world. Our group has focused on the development of macrophage-targeted perfluorocarbon nanoemulsions as inflammatory pain nanomedicines over the past several years. We present here, for the first time, a quality by design approach used to design pain nanomedicine. Specifically, we used failure mode, effects, and criticality analysis (FMECA) which identified the process and composition parameters that were most likely to impact nanoemulsion critical quality attributes (CQAs). From here, we applied a unique combination approach that compared multiple linear regression, boosted decision tree regression, and partial least squares regression methods in combination with correlation plots. The presented combination approach allowed for in-depth analyses of which formulation steps in the nanoemulsification processes control nanoemulsion droplet diameter, stability, and drug loading. We identified that increase in solubilizer (transcutol) content increased drug loading and decreased nanoemulsion stability. This was mitigated by inclusion of perfluorocarbon oil in the internal phase. We observed negative correlation (R2 = 0.4357, p value 0.0054) between the amount of PCE and the percent diameter increase (destabilization), and no correlation between processing parameters and percent diameter increase over time. Further, we identified that increased sonication time decreases nanoemulsion drug loading but does not significantly impact droplet diameter or stability. We believe the methods presented here can be useful in the development of various nanomedicines to produce higher-quality products with enhanced manufacturing and design control.

Development and characterization of resveratrol nanoemulsions carrying dual-imaging agents.

AIM: Delivery of the natural anti-inflammatory compound resveratrol with nanoemulsions can dramatically improve its tissue targeting, bioavailability and efficacy. Current assessment of resveratrol delivery efficacy is limited to indirect pharmacological measures. Molecular imaging solves this problem. Results/methodology: Nanoemulsions containing two complementary imaging agents, near-infrared dye and perfluoropolyether (PFPE), were developed and evaluated. Nanoemulsion effects on macrophage uptake, toxicity and NO production were also evaluated. The presence of PFPE did not affect nanoemulsion size, zeta potential, colloidal stability, drug loading or drug release. CONCLUSION: PFPE nanoemulsions can be used in future studies to evaluate nanoemulsion biodistribution without interfering with resveratrol delivery and pharmacological outcomes. Developed nanoemulsions show promise as a versatile treatment strategy for cancer and other inflammatory diseases. [Formula: see text].