Over 600 neurological disorders can occur throughout a person’s lifetime. Alone in US, the annual cost of neurological disorders is $800 billion. Newborns with neurological disorders are in particular need of therapeutic intervention, due to the immaturity and the rapid development of their brains around the perinatal period, which are associated with long-term neurodevelopmental disability. In many neurological disorders, including neonatal or perinatal brain injury, inflammation results in sustained activation of pro-inflammatory microglia that persists up to weeks after birth in the newborn brains. My doctoral research has focused on using anti-inflammatory small molecules as potential neuroprotective agents for neonatal or perinatal brain disease and injury. One example is curcumin, an anti-inflammatory and antioxidant chemical, which is widely used to treat oxidative and inflammatory conditions, metabolic syndrome, arthritis, anxiety, and hyperlipidemia, and has been demonstrated to be neuroprotective in adult neurological disease. However, the bioavailability of curcumin is greatly affected by its low solubility. Nanoparticles can encapsulate curcumin and improve its solubility, yet the encapsulation efficiency is limited and often less than 10% by weight of the drug-polymer formulation. Building on our prior work that shows curcumin encapsulated in poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles and result in neuroprotection in neonatal brain injury, we sought to improve the formulation to optimize the curcumin encapsulation efficiency, increase reproducibility, and improve long-term stability. We investigated the effects of PLGA length, stabilizer concentration, polymer functionalization, and formulation method on curcumin loading. Upon successful optimization of a PLGA-PEG nanoparticle with high curcumin encapsulation efficiency and drug loading we also sought to assess particle pharmacokinetics (PK) and biodistribution in term-equivalent rats and piglets. With these results, we used the nano-formulated curcumin (NanoCurc) with optimized curcumin loading and systemic circulation half-life to evaluate the neuroprotection effects in neonatal rats at different developmental stages. To move into a clinical-trial ready model, we have also assessed particle accumulation in the fetal growth restriction (FGR) piglet brain and early-stage efficacy outcomes to evaluate NanoCurc’s potential for FGR treatment. Here, I highlight the most critical results from my doctoral research.
1. NanoCurc is reproducible and tunable with high curcumin loading and long-term stability.
With standard nanoprecipitation technique, we found that the size of curcumin nanoparticles increased with the increase of PLGA length and the decrease of the F127 concentration. The curcumin loading reached the highest when formulated with 45k PLGA-PEG. Also, curcumin loading increased when F127 concentration decreased, although the stability and reproducibility of curcumin-loaded nanoparticles were limited when F127 concentration was 0.1%. PLGA-PEG functionalization could partially improve curcumin loading, but undermined the lyophilization stability and caused issue for long-term storage. Curcumin-loaded nanoparticles formulated with standard nanoprecipitation had less than 10% curcumin loading because of the different precipitation rates. We identified that sequential nanoprecipitation resulted in higher drug loading compared to nanoprecipitation. Our optimal NanoCurc formulation was 50 to 60 nm hydrodynamic size, a near neutral surface charge, high drug loading (>39%) and drug encapsulation efficiency (>95%) (Table 1), had a sustained release profile (Fig 1B), and could maintain the colloidal stability at room temperature for at least 1 month and at -20 for at least 6 months.
2. Nanoparticles formulated using F127 stabilizer have longer systemic circulation half-life.
Term-equivalent rats (postnatal day 10 (P10)) rats were used to quantify the PK and biodistribution of PLGA-PEG nanoparticles formulated with different surfactants. Following i.p. injection, serum concentration of PLGA-PEG nanoparticles was present at 1h and continued to increase and reach the maximum concentration by 4 h, with a half-life (t1/2) of 5.89 h for PLGA-PEG/F127 (Fig 2A). The accumulation of PLGA-PEG nanoparticles in brain, heart and lung also reached the peak at around 4 h. Liver, spleen and kidney had a delayed particle accumulation in the first 4 h after injection and then reached peak concentrations at 24 h – no to minimal concentrations were detected at 72 h (Fig 2B). Among all organs, liver accumulated the most nanoparticles, whereas heart, lung and kidney had relatively low accumulation (less than 5% injected dose), and spleen was intermediate. Additionally, PLGA-PEG nanoparticles formulated with different surfactants had different biodistributions, either at the tissue-level or organ-level. t1/2 for PLGA-PEG/P80 (t1/2 = 1.49 h) was shorter than that of PLGA-PEG/F127. The significantly longer circulation half-life of F127 formulated nanoparticle suggests it is more beneficial for drug delivery.
3. Neuroprotection effects of NanoCurc decreases with the increase of age. Neonatal rats at three different developmental stages, preterm-equivalent (P7), term-equivalent (P10), and late-term equivalent (P13), were i.p. administered with 25 mg/kg NanoCurc 30 min after hypoxic-ischemic injury. At the same dose, NanoCurc had significant neuroprotection effects on preterm-equivalent HI rats, had neuroprotection trend for term-equivalent HI rats, showed no neuroprotection effects on late term-equivalent HI rats (Fig 3). NanoCurc has effects on HI brain injury treatment, but the efficacy decreases with the increase of rat developmental ages.
4. Intranasally administered NanoCurc can rapidly accumulate in neonatal pig brain and has a long retention.
The i.n. (Fig. 4B) administration resulted in 2-fold higher particle % injected dose (ID) in the brain parietal cortex and cerebellum at 4 h after administration compared to particles administered i.v. (Fig 4A). Particles were colocalized in microglia as early as 4 h after i.n. administration (Fig. 4C). At 48 hours post treatment particle accumulation decreased to almost undetectable levels, but still a minimal amount of particles can be observed colocalized in microglia. Off-target accumulation in the liver, spleen, and kidney were quantified, with under 10% ID accumulated in these organs at 4 h, and undetectable NanoCurc at 48 h after administration.
Summary and Impact: With sequential nanoprecipitation, a tunable and reproducible NanoCurc formulation with high drug loading and long-term stability can be formulated. This proved that sequential nanoprecipitation technique can successfully load small hydrophobic molecules and has potential for encapsulating big hydrophobic complexes. Therefore, this formulation technique can be used with a variety of polymers and hydrophobic drugs, which can improve the bioavailability, tunability, reproducibility, and storage stability of nano-formulation. PLGA-PEG nanoparticles are very promising drug delivery vehicles for the neonatal population, especially in the field of brain disease and injury, where the dosage determination depends on the development of PK profiles of nanoparticles in neonates. Understanding the PK profiles and biodistribution, and associated residence time, of PLGA-PEG nanoparticles in neonates will help design therapeutic dosing regimens with maximum efficiency and minimum toxicity, and thus help improve the speed of clinical translation and drug safety of nanodrugs into this underserved population. Therapeutic efficacy will be affected by the developmental stages of animals, so the therapeutic dose determination should consider both the weight and age. NanoCurc formulation reflects a promising polymeric platform to deliver a neuroprotective substance to the neonatal brain. The biodistribution study in piglets also demonstrates the validity of intranasal administration of nanoparticles to rapidly reach the newborn brain, which indicates i.n. administration may be a more favorable route for brain delivery compared to i.v. administration.
Research Interests: During my graduate study, I formulated both small molecular and biomolecular neuroprotective substances loaded polymeric nanoparticles. To optimize the formulation, polymer functionalization and drug complexation were conducted. Following the formulation, I characterized their drug loading and physicochemical properties via UV-Vis spectroscopy, spectrophotometer, HPLC, and DLS. The nanotherapeutic effects were analyzed using immunofluorescence staining following confocal imaging and RT-qPCR. Following that, different models from ex vivo organotypic oxygen-glucose deprivation brain slice model to in vivo Vannucci model of hypoxic-ischemic injury and fetal growth restricted pig model were used to screen therapeutic candidates and assess their translational potential. Meanwhile, I was scientific lead on a multi-disciplinary team of clinicians, neuroscientists, and engineers. In the future, I am interested in conducting research in the field of drug delivery, particularly enthusiastic about nanomedicine development and clinical translation.
