Next, I would like to thank my advisor Mark Davis for his academic support and mentorship. I would like to thank Yashodan Bhawe, my officemate of four years, and Jonathan Zuckerman for their friendship, all the laughs we shared, and making the workday enjoyable. I would like to thank John Zuckerman for all our stimulating discussions and for his moral support as I tried to step out of my chemical engineering comfort zone.
I would like to thank the other professors and administration at Caltech who have been so encouraging throughout my Ph.D. I would like to thank Martha Hepworth, Kathy Bubash, Karen Baumgartner, Anne Hormann and Yvette Grant for their administrative help and Suresh Gupta for his immense help with IT.
Introduction
However, these large molecule therapies may be ineffective in treating brain diseases because they cannot enter the brain due to the blood-brain barrier (see below). Among the strategies currently being explored to deliver therapy to the brain include disruption of the blood-brain barrier (mannitol [21], ultrasound [22], adenosine [23]), antibody-drug conjugate systems [24], and various types of targeted and non-targeted nanoparticle systems (liposomes [25], PBCA [26] and targeted exosomes [27]). Receptor-mediated transcytosis of antibodies [35] and nanoparticles [26] across the blood brain barrier has been reported.
Pluronic block copolymers as modulators of drug efflux transporter activity in the blood-brain barrier. Human serum albumin nanoparticles modified with apolipoprotein A-I cross the blood-brain barrier and enter the rodent brain.
![Figure 1.1: Statistics on Alzheimer’s Disease – figure from [3]. (Left Panel) Rates of mortality due to cancers, cardiovascular diseases, and infectious diseases have decreased over the last decade](https://thumb-ap.123doks.com/thumbv2/123dok/11220188.0/15.918.171.804.118.401/statistics-alzheimer-disease-mortality-cardiovascular-diseases-infectious-decreased.webp)
E. coli Glycoprotein 96
Further experimentation with additional imaging modalities was chosen to confirm that Ecgp96 was not located at the mouse blood-brain barrier and that antibodies against Ecgp96 did not accumulate in the brain. After injection and imaging, the bulk signal of the Ecgp96 antibody-treated brain was not different from the non-injected control brain, indicating no detectable accumulation of the antibody in the brain. No fluorescent signal was seen by confocal microscopy in the brain from the systemically injected anti-Ecgp96 antibody.
Tissues in the body that contain blood-accessible Ecgp96 will be bound by antibodies, and the accumulation of antibodies in the tissues can be observed by PET. Therefore, this analysis gave negative results that the antibody significantly accumulated in the brain due to the presence of Ecgp96. Although the PET scans showed that Ecgp96 was probably not accessible to antibodies from the blood at the blood-brain barrier, there was an interesting finding in the eyes of mice tested with Ecgp96.
That is, there was a significantly higher positron signal in the eyes of the mouse tested with Ecgp96 than the control mouse (Figure 2.10). The increased signal in the eye of the Ecgp96 antibody was also observed after 22 hours. PBS perfusion was used to remove any residual antibody in the blood that had not adhered to the blood-brain barrier.
There was no increase in the fluorescence signal in the brain of the injected mouse over the autofluorescence measured from the brain of the control mouse. This is consistent with the PET/CT ROI analysis which showed that there was no increased antibody signal in the brain over the signal that was present in the blood. There was a significant increase in the fluorescent signal associated with liver antibodies in the injected mouse - consistent with the PET/CT scan.
These results show that in normal neonatal mice there is no accumulation of antibody against Ecgp96 in the brain. Ecgp96 is therefore found in the blood vessels, but it is not available from the blood.
Formulation and Characterization of Transferrin Containing Gold Nanoparticles *Transferrin Containing Gold Nanoparticles*
These authors showed that in the animal body the biodistribution of transferrin containing gold nanoparticles of ca. Thus, the targeting ligand acts as a facilitator of cell entry rather than altering nanoparticle biodistribution. Furthermore, we found that gold nanoparticles are suitable for studies of the blood-brain barrier because they can be visualized inside and outside the vasculature by light microscopy through silver enhancement.
These data would be consistent with a similar Poisson-type distribution of transferrin over the population of nanoparticles for each formulation. The nanoparticles were formulated to have a wide range of transferrin on the surface of the nanoparticles; each size has a formulation with very little transferrin and a formulation that approaches the maximum. The theoretical maximum density of transferrin on the nanoparticle surface was estimated based on the total surface area of the particle (from the surface of a sphere) and an estimate of the amount of surface that each transferrin molecule could cover (hydrodynamic radius of approximately 4 nm for transferrin [20] ).
More critical than the exact number of transferrin in each nanoparticle formulation is the avidity of the nanoparticles and how it varies with transferrin content. The iron content of transferrin was measured by UV-VIS via the A465/A280 ratio and compared to the same ratio of the original unprocessed holotransferrin. 64Cu labeling of transferrin was tested and confirmed by instant thin layer chromatography (ITLC) (Biodex, Tec-Control).
The concentration of radiolabeled Tf-PEG-OPSS was determined using a Nanodrop 2000 (Thermo), and Tf-PEG-OPSS was stirred at room temperature with the indicated ratio of gold nanoparticles for at least one hour. This is important to ensure that the zeta potentials and transferrin content of the nanoparticle population are homogeneous and that the nanoparticles within the population will act similarly in crossing the blood-brain barrier. This provided the first direct measurement of transferrin content in gold nanoparticles as previous measurements used methods such as ELISA that only indirectly measured the transferrin content of nanoparticles through mass balances [12].
In-vivo Blood-Brain Barrier Study of Transferrin Containing Nanoparticles *Transferrin Containing Nanoparticles*
Nanoparticles in the parenchyma of each image were quantified and plotted in square plots in Figure 4.1a. These data illustrate how nanoparticle accumulation in the brain parenchyma was modified by nanoparticle size and transferrin content. Nanoparticles in the 45 nm and 80 nm size range were observed in the brain parenchyma, and statistically significant maxima were achieved in the formulations studied for both sizes.
20 nm as well as all mPEG-only formulations were not clearly seen in the parenchyma. These non-specific events due to silver growth are clearly distinguished from the signal in the parenchyma due to gold nanoparticles. 20 nm formulations that remain predominantly in the blood vessels (vessels stained black with a lack of clearly visible nanoparticles outside the vasculature).
Images of the 45 nm, 30 Tf and 80 nm, 20 Tf formulations are representative of the majority of images captured with a clear nanoparticle signal in the parenchyma. Furthermore, the 80 nm silver nanoparticle enhancement signal appeared discretely in blood vessels (unlike the continuous 20 nm and 45 nm nanoparticle signal in vessels) and this vessel-associated nanoparticle signal was quantified for each 80 nm formulation and data compiled in Figure 4.1b. Furthermore, no nanoparticles were observed in the vessel lumen after vascular perfusion, although transferrin-coated nanoparticles were again localized in endothelial cells and parenchyma (Figure 4.5h-i).
Nanoparticles visualized in the parenchyma and 80 nm nanoparticles visualized in the vessels were manually counted and the data were saved in Matlab. Nanoparticles with the highest avidities bound to the blood-brain barrier but had reduced accumulation in the brain parenchyma compared to nanoparticles with reduced avidities. Epithelial and endothelial barriers in the olfactory region of the rat nasal cavity.
Summary and Conclusion
Additionally, nanoparticle designs and understanding how to optimize these designs to facilitate receptor-mediated transcytosis across the blood-brain barrier are investigated here. The nanoparticles were systemically injected into mice to determine their interactions and transcytosis behaviors at the blood-brain barrier. These studies provided information on how to properly design nanoparticles that safely and efficiently cross the blood-brain barrier.
A strategy was proposed to target nanoparticles to the same receptors in the blood-brain barrier that these pathogens reportedly used to enter the brain. The collected results from the PET/CT, SPECT/CT, Fluorescence Xenogen imaging, MRI, and confocal microscopy studies indicated that Ecgp96 is located in the endothelial cells of the blood-brain barrier; However, Ecgp96 is not accessible to. Confocal microscopy studies of excised brain tissue showed that Ecgp96 was located in endothelial cells at the blood-brain barrier, but not on the surface of endothelial cells.
These SPECT/CT studies showed that Ecgp96 was also not available from the blood on the blood-brain barrier endothelial cells of three-day-old mice. To move forward in determining how to design nanoparticles that can cross the blood-brain barrier, a receptor known to exist on the blood-brain barrier and facilitate the transport of its ligand to the brain was chosen. The transferrin receptor has been used for several blood–brain barrier transcytosis studies of antibodies and nanoparticles [20–22] .
These antibodies, when engineered to have reduced receptor affinity, have been shown to engage the receptor on the blood side of the blood-brain barrier and be released from the receptor on the brain side of the blood-brain barrier [19, 23]. ]. In addition, TEM imaging studies of 80 nm nanoparticles showed that transferrin-containing nanoparticles entered the endothelial cells of the blood-brain barrier and entered the brain parenchyma, whereas non-targeted nanoparticles did not. Very little of the injected dose of the optimized nanoparticle formulation reached the brain parenchyma.
