(M1530-12-79) Development of Antibody Conjugated Nanoparticles for Delivery of Nucleic Acid for Stimulation of Perivascular Macrophages

Purpose: Cerebral Amyloid Angiopathy (CAA) is characterized by deposition of amyloid beta (Aβ) in cerebral blood vessels. Blood vessels that are infiltrated by Aβ usually pass from the leptomeninges through the superficial cortex ¹ . The deposition of Aβ weakens the blood vessels and can lead to major lobar hemorrhages and cerebrovascular inflammation¹ . Studies have shown that more than 80% Alzheimer’s disease patients have CAA pathology ² .The appearance of Aβ deposits is observed before the onset of clinical symptoms³ , thus making it a critical target for therapeutic intervention. Chitin, a natural polymer, has been shown to improve the clearance of Aβ by increasing turnover of perivascular macrophages⁴ . Chitosan, derivative of chitin is a biodegradable, nontoxic polymer and we have previously developed theranostic nanovehicles containing chitosan-TPP (pentasodium tripolyphospate) core for detection and treatment of CAA⁵ . In this study, we formulated and evaluated chitosan-polycarbophil nanoparticles for the delivery of RNA or DNA. The nanoparticles were conjugated with bovine serum albumin (BSA) as a control or anti-amyloid antibody IgG 4.1 to selectively target Aβ. We aim to use the nanoparticles to stimulate perivascular macrophages and target noncoding RNA to cerebral vasculature to ameliorate toxic consequences of amyloid beta exposure.

Methods: Chitosan-Polycarbophil nanoparticles were prepared using ionic gelation technique. A solution of low and medium molecular weight chitosan was prepared in purified water containing 1% glacial acetic acid. Polycarbophil-RNA or Polycarbophil-DNA solution was prepared separately and then added to chitosan solution described above. The nanoparticles were allowed to equilibrate at room temperature for 20 minutes. Nanoparticles were then conjugated with either BSA or IgG 4.1 using carbodiimide crosslinking. After conjugation they were purified with spin filtration using 1000 Kd MWCO filters to remove unconjugated proteins and undergo buffer exchange. The size, polydispersity index and zeta potential of the nanoparticles was characterized using Malvern Zetasizer and NanoSight. The encapsulation efficiency of RNA nanoparticles was calculated using Ribogreen assay. The nanoparticles were diluted with 1X Tris-EDTA buffer to measure unencapsulated RNA concentration. Total RNA was calculated by extracting the encapsulated RNA using Heparin (1,000 USP Units/mL) displacement. The samples were compared against standards to obtain unknown concentrations using Ribogreen dye and the florescence was measured using microplate reader with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. BCA Assay was used to calculate the amount of conjugated proteins on the nanoparticles. The samples were diluted 2X with PBS. The diluted samples were treated with BCA reagent mix and the plate was analyzed at 562 nm. The unknown sample concentrations were calculated by comparing against prepared albumin standards. The morphology of the nanoparticles was analyzed using Transmission electron microscopy. In vitro studies were carried out on immortalized human cerebral microvascular endothelial cell (hCMEC/D3) monolayers. Cytotoxicity study was carried out using Live/Dead cell assay, where cells were treated with 50 and 100 microliters of nanoparticles for 24 hours and analyzed using flow cytometry. The uptake of nanoparticles was assessed by conjugating them with fluorescently labelled FITC-BSA or AF647-IgG 4.1 and treating the cells with nanoparticles for 1 hour, followed by flow cytometry analysis.

Results: Average particle sizes before and after conjugation was 92.6 nm and 236.7 nm, respectively for RNA nanoparticles, whereas for DNA nanoparticles they were 85.52 nm and 278.4 nm, respectively. Zeta potential before and after conjugation was 29.1 mV and 40.1 mV, respectively for RNA nanoparticles; for DNA nanoparticles it was 41.7 mV and 32.4 mV, respectively (Figure1A). These nanoparticles demonstrated ~100 % encapsulation.. The Live/Dead cell assay after treating cells with nanoparticles at 50 and 100 microliters indicated that over 95 % cells were viable after exposure to both concentrations, which indicate that nanoparticles show low toxicity. Lastly, the cell monolayers incubated with nanoparticles conjugated with FITC-BSA showed 10 times more intracellular fluorescence compared to control cell monolayers.

Conclusion: Chitosan-Polycarbophil nanoparticles were developed to encapsulate DNA/RNA. They were smaller than 300 nm and had a positive overall charge. The particles are nontoxic and demonstrated excellent encapsulation of RNA. The particles are cross-linked with BSA or IgG 4.1and, which were shown to be readily endocytosed by BBB endothelial cells and are able to increase the cellular uptake of the cargo.

References: 1. Vinters, H. V. Cerebral amyloid angiopathy. A critical review. Stroke 18, 311–324 (1987).
2. Jellinger, K. A. Alzheimer disease and cerebrovascular pathology: an update. Journal of Neural Transmission 109, 813–836 (2002).
3. Sperling, R. A. et al. Toward defining the preclinical stages of Alzheimer’s disease: Recommendations from the National Institute on Aging‐Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimer’s & Dementia 7, 280–292 (2011).
4. Hawkes, C. A. & McLaurin, J. Selective targeting of perivascular macrophages for clearance of β-amyloid in cerebral amyloid angiopathy. Proc. Natl. Acad. Sci. U.S.A. 106, 1261–1266 (2009).
5. Agyare, E. K. et al. Engineering theranostic nanovehicles capable of targeting cerebrovascular amyloid deposits. Journal of Controlled Release 185, 121–129 (2014).

Acknowledgements: This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (MNP#15.31) and National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS125437).

Figure 1: A Particle size, PDI and Zeta potential measured by Zetasizer for unconjugated and conjugated nanoparticles.
B- Snapshot of the video of conjugated RNA nanoparticles from Nanosight.
C- Snapshot of the video of conjugated DNA nanoparticles from Nanosight.

Figure 2: Uptake in hCMEC/D3 cells analyzed by Flow Cytometry. For both the graphs (from left) Grey- Untreated cells, Green- Cells treated with FITC-BSA, Blue- Cells treated with 100 microliters of FITC -BSA conjugated DNA nanoparticles(top) and RNA nanoparticles(bottom), Red- Cells treated with 200 microliters of FITC -BSA conjugated DNA nanoparticles (top) and RNA nanoparticles (bottom).

Figure 3: Live/Dead Cell assay A – Live/Dead assay in hCMEC/D3 cells analyzed by Flow Cytometry, cells were treated with 50 and 100 microliters of conjugated RNA nanoparticles and conjugated DNA nanoparticles.
B- % Live cells for different concentrations of nanoparticles.