In this work, we sought to test how surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with peptide ligand alters the brain specific delivery of encapsulated molecules. For biodistribution studies, nanoparticles modified with rabies virus glycoprotein (RVG29) were loaded with small molecule drug surrogates and administered to healthy mice by lateral tail vein injection. Mice were perfused two hours after injection and major anatomical regions of the CNS were dissected (striatum, midbrain, cerebellum, hippocampus, cortex, olfactory bulb, brainstem, and cervical, thoracic, lumbar and sacral spinal cord). For functional studies, surface modified nanoparticles were loaded with the chemotherapeutic camptothecin (CPT) and administered to mice bearing intracranial GL261-Luc2 gliomas. Outcome measures included tumor growth, as measured by bioluminescent imaging, and median survival time. We observed that small molecule delivery from PLGA nanoparticles varied by as much as 150% for different tissue regions within the CNS. These differences were directly correlated to regional differences in cerebral blood volume. Although the presence of RVG29 enhanced apparent brain delivery for multiple small molecule payloads, we observed minimal evidence for targeting to muscle or spinal cord, which are the known sites for rabies virus entry into the CNS, and enhancements in brain delivery were not prolonged due to an apparent aqueous instability of the RVG29 ligand. Furthermore, we have identified concerning differences in apparent delivery kinetics as measured by different payloads: nanoparticle encapsulated DiR was observed to accumulate in the brain, whereas encapsulated Nile red was rapidly cleared. Although systemically administered CPT loaded nanoparticles slowed the growth of orthotopic brain tumors to prolong survival, the presence of RVG29 did not enhance therapeutic efficacy compared to control nanoparticles. These data are consistent with a model of delivery of hydrophobic small molecules to the brain that does not rely on internalization of polymer nanoparticles in target tissue. We discuss an important risk for discordance between biodistribution, as typically measured by drug surrogate, and therapeutic outcome, as determined by clinically relevant measurement of drug function in a disease model. These results pose critical considerations for the methods used to design and evaluate targeted drug delivery systems in vivo.

Alternative routes of administration are one approach that could be used to bypass the blood–brain barrier (BBB) for effective drug delivery to the central nervous system (CNS). Here, we focused on intranasal delivery of polymer nanoparticles. We hypothesized that surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with rabies virus glycoprotein (RVG29) would increase residence time and exposure of encapsulated payload to the CNS compared to non-targeted nanoparticles. Delivery kinetics and biodistribution were analyzed by administering nanoparticles loaded with the carbocyanine dye 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide (DiR) to healthy mice. Intranasal administration yielded minimal exposure of nanoparticle payload to most peripheral organs and rapid, effective delivery to whole brain. Regional analysis of payload delivery within the CNS revealed higher delivery to tissues closest to the trigeminal nerve, including the olfactory bulb, striatum, midbrain, brainstem, and cervical spinal cord. RVG29 surface modifications presented modest targeting benefits to the striatum, midbrain, and brainstem 2 h after administration, although targeting was not observed 30 min or 6 h after administration. Payload delivery to the trigeminal nerve was 3.5× higher for targeted nanoparticles compared to control nanoparticles 2 h after nanoparticle administration. These data support a nose-to-brain mechanism of drug delivery that closely implicates the trigeminal nerve for payload delivery from nanoparticles via transport of intact nanoparticles and eventual diffusion of payload. Olfactory and CSF routes are also observed to play a role. These data advance the utility of targeted nanoparticles for nose-to-brain drug delivery of lipophilic payloads and provide mechanistic insight to engineer effective delivery vectors to treat disease in the CNS.

Surveys based on nonrandom site selection, or convenience samples, are often a necessary part of large-scale monitoring programs to help minimize costs. The reliability of convenience samples to inform managers about distributions or population status of imperiled species is questionable, however, because the samples may not be representative of the whole population. We compared fish community data from 20 nonrandom, long-term monitoring sites for Rio Grande Silvery Minnow Hybognathus amarus with those from 20 randomly chosen samples collected during two surveys (one in summer, one in autumn) in the Rio Grande, New Mexico. We compared the species richness, community composition, and the catch per unit effort (CPUE). Fish species compositions, which were similar between both sets of summer and autumn surveys, were nearly identical in the autumn surveys. Similarly, we found consistent Rio Grande Silvery Minnow CPUE between surveys; summer random surveys estimated 0.32 fish/100 m 2 sampled, whereas summer nonrandom surveys estimated 0.37 fish/100 m 2 sampled. In autumn, both surveys showed a marked decline in Rio Grande Silvery Minnow; random surveys found 0.08 fish/100 m 2 sampled (95% confidence interval 0.04-0.18), whereas the nonrandom surveys failed to collect any Rio Grande Silvery Minnow. Both surveys showed a reduction in species richness between summer and autumn with a corresponding increase in dominance by Red Shiner Cyprinella lutrensis and a decline in Rio Grande Silvery Minnow. We failed to find any meaningful differences in either fish community or Rio Grande Silvery Minnow CPUE between random and nonrandom sites, suggesting that the long-term, nonrandom locations currently used to monitor the Rio Grande Silvery Minnow population are representative of the middle Rio Grande. We believe our results are applicable to many monitoring programs in systems with a homogeneous distribution of mesohabitats; nonetheless, we recommend that managers assess potential bias in monitoring programs based on convenience samples.

The prognosis for patients with glioblastoma (GBM), a deadly form of brain tumor with a median survival of <15 months, has not substantially improved since the 1970s. To overcome the myriad of treatment obstacles for GBM, we sought to design a new therapeutic capable of selectively enhancing T cell recognition and killing of all GBM cells while preserving healthy tissue. For this purpose, we have designed, expressed, and performed initial testing of the novel bispecific fusion protein, anti-CD3/chlorotoxin (ACDClx). ACDClx is a fusion of anti-CD3 heavy and light variable regions with a non-toxic, GBM-selective scorpion-derived peptide, chlorotoxin, which has been shown to bind to 100% of GBM cells with no selectivity for any healthy tissue types, including brain. ACDClx was expressed in leaves of N. benthamiana, purified, and tested for the capacity to selectively activate T cells. Initial experiments showed that ACDClx activates T cells, measured via calcium flux and CD69 expression, only if T cells, GBM cells, and ACDClx were all present. Mock purified protein in the presence GBM cells and T cells did not result in T cell activation, nor did ACDClx alone. Before further testing in vitro and in vivo, we sought to increase the production of ACDClx, the expression of which is significantly hampered due to its six disulfide bonds. To this end, we have produced 7 new constructs (5 for N. benthamiana, 2 for E. coli), 5 of which include a unique fusion partner for ACDClx to enhance solubility. Ongoing work includes evaluation of the most efficient construct, followed by evaluation in an immunocompetent tumor model of GBM.